Mirk/Dyrk1B Mediates Survival during the Differentiation of C2C12Myoblasts *

Stephen E. Mercer; Daina Z. Ewton; Xiaobing Deng; Seunghwan Lim; Thomas R. Mazur; Eileen Friedman

doi:10.1074/jbc.m413594200

Mirk/Dyrk1B Mediates Survival during the Differentiation of C2C12Myoblasts *

Mercer, Stephen E.;Ewton, Daina Z.;Deng, Xiaobing;Lim, Seunghwan;Mazur, Thomas R.;Friedman, Eileen 2005-07-08 00:00:00 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 27, Issue of July 8, pp. 25788 –25801, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Mirk/Dyrk1B Mediates Survival during the Differentiation of C2C12 Myoblasts* Received for publication, December 2, 2004, and in revised form, March 29, 2005 Published, JBC Papers in Press, April 25, 2005, DOI 10.1074/jbc.M413594200 Stephen E. Mercer, Daina Z. Ewton, Xiaobing Deng, Seunghwan Lim, Thomas R. Mazur, and Eileen Friedman‡ From the Department of Pathology, Upstate Medical University, State University of New York, Syracuse, New York 13210 in controlling C2C12 myoblast differentiation following the The kinase Mirk/dyrk1B is essential for the differen- tiation of C2C12 myoblasts. Mirk reinforces the G /G commitment stage of myogenesis. Depletion of endogenous 0 1 arrest state in which differentiation occurs by directly Mirk by RNA interference blocked the transcription of myoge- Kip1 phosphorylating and stabilizing p27 and destabiliz- nin and the subsequent myoblast differentiation program (1). ing cyclin D1. We now demonstrate that Mirk is anti- Mirk controls the activation of the myogenin transcription fac- apoptotic in myoblasts. Knockdown of endogenous Mirk tor MEF2 by regulating nuclear accumulation of the MEF2 by RNA interference activated caspase 3 and decreased inhibitors, class II histone deacetylases (2). However, it has myoblast survival by 75%, whereas transient overex- been reported that embryonic knockout of Mirk did not block pression of Mirk increased cell survival. Mirk exerts its skeletal muscle development in the early embryo (3), suggest- anti-apoptotic effects during muscle differentiation at ing that Mirk may have a more significant function in muscle least in part through effects on the cell cycle inhibitor repair than in initial myogenesis. Cip1 and pro-survival molecule p21 . Overexpression and Mirk (Minibrain-related kinase; also known as Dyrk1B) is a RNA interference experiments demonstrated that Mirk member of an evolutionarily conserved family of proteins, the phosphorylates p21 within its nuclear localization do- Dyrk/Minibrain family of arginine-directed serine/threonine main at Ser-153 causing a portion of the typically nu- protein kinases (4 –7), that play roles in controlling the switch clear p21 to localize in the cytoplasm. Phosphomimetic from proliferation to differentiation in a wide variety of orga- GFP-p21-S153D was pancellular in both cycling C2C12 nisms. The roles of Dyrk/Minibrain/Mirk homologues in yeast myoblasts and NIH3T3 cells. Endogenous Mirk in myo- (Yak1) and slime mold (YakA) suggest that this group of ki- tubes and overexpressed Mirk in NIH3T3 cells were able nases helps to regulate the transition from growth to differen- to cause the pancellular localization of wild-type GFP- tiation in response to environmental stresses (8, 9). p21 but not the nonphosphorylatable mutant GFP-p21- Mirk is a unique, multifunctional kinase with primary S153A. Translocation to the cytoplasm enables p21 to block apoptosis through inhibitory interaction with activity in G phase and early differentiation. Specifically, pro-apoptotic molecules. Phosphomimetic p21-S153D Mirk elongates the G phase of the cell cycle in C2C12 myo- was more effective than wild-type p21 in blocking the blasts, Mv1Lu epithelial cells, and NIH3T3 fibroblasts by Kip1 activation of caspase 3. Transient expression of p21- stabilizing the CDK inhibitor p27 (10) and by destabiliz- S153D also increased myoblast viability in colony form- ing the G cyclin, cyclin D1 (11). Mirk has also been shown to ing assays, whereas the p21-S153A mutant had no effect. act as a transcriptional activator (2, 12, 13) and to inhibit cell This Mirk-dependent change in p21 intracellular local- motility (14). Because of these actions of Mirk on cell cycle ization is a natural part of myoblast differentiation. En- regulators active in G /G during myoblast differentiation, 0 1 dogenous p21 localized exclusively to the nuclei of pro- Cip1 we extended our study of Mirk substrates to p21 . The liferating myoblasts but was also found in the cytoplasm CDK inhibitor p21 is highly expressed in differentiating mus- of post-mitotic multinucleated myotubes and adult hu- cle in vivo (15) and has been implicated as playing a central man skeletal myofibers. role in mediating the pre-differentiation growth arrest in- duced by the major muscle regulatory factor MyoD (16). It is believed that this effect of p21 during myogenesis is because Although there is a large body of work describing the func- of inhibition of G cyclin-CDK complexes and subsequent tions of myogenic regulatory factors in skeletal muscle commit- blockade of retinoblastoma protein phosphorylation (16). ment and differentiation, much less is known regarding the Studies using mice deficient in members of the Cip/Kip CDK signaling pathways that control the specific molecular events inhibitor family have demonstrated that in the absence of p21, involved in terminal differentiation and maintenance of skele- mice can develop into normal adults (17). This appears to be tal muscle. We demonstrated recently (1) that a novel gene, due to redundant functions of p57, because mice deficient in which we cloned in 1997, the kinase Mirk, plays a critical role both p21 and p57 demonstrate severely arrested muscle devel- opment (18). Specifically, the double mutant mice displayed * This work was supported by United States Public Health Service Award RO1 CA67405 (to E. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 DM, differentiation medium; GM, growth medium; BrdUrd, bromode- U.S.C. Section 1734 solely to indicate this fact. oxyuridine; WT, wild type; FBS, fetal bovine serum; DMEM, Dulbecco’s ‡ To whom correspondence should be addressed: Pathology Dept., modified Eagle’s medium; DTT, dithiothreitol; PBS, phosphate-buffered Upstate Medical University, 750 East Adams St., Syracuse, NY 13210. saline; BSA, bovine serum albumin; GST, glutathione S-transferase; Tel.: 315-464-7138; Fax: 315-464-8419; E-mail: [email protected]. TUNEL, terminal dUTP nick-end labeling; DAPI, 4,6-diamidino-2- The abbreviations used are: Mirk, minibrain-related kinase; GFP, phenylindole hydrochloride; fmk, fluoromethyl ketone; CDK, cyclin-de- green fluorescent protein; CDK, cyclin-directed kinase; PCNA, prolifer- pendent kinase; hsp, heat shock protein; RNAi, RNA interference; ating cell nuclear antigen; MAPK, mitogen-activated protein kinase; siRNA, short interfering RNA. 25788 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Mirk Blocks Apoptosis through Translocation of p21 25789 g/liter glucose, containing 10% bovine calf serum. Cell cultures were increased proliferation and apoptosis of myoblasts and failed to typically maintained without the use of antibiotics. Cells were used for form myotubes. Significantly, although p21-deficient mice ap- experiments only from passages 3 to 10 from our frozen stocks. pear to develop normally, p21 is essential for normal regener- Transient Transfections—Two strategies were employed for tran- ation through the muscle progenitor (or satellite) cells that are sient transfections. In order to achieve high level short term expression responsible for repairing muscle following injury (19). It has (for GFP localization and in vivo labeling), we used a modification of the been suggested that muscle differentiation during embryogen- manufacturer’s standard protocol for Lipofectamine 2000 (Invitrogen). Specifically, we transfected cells with a 2:1 ratio (l/g) of Lipo- esis and during regeneration are fundamentally different, with fectamine 2000 to plasmid DNA immediately after plating freshly p57 playing a dominant role in embryogenesis and p21 being trypsinized cells. This increased transfection efficiency in the C2C12 the critical CDK inhibitor during regeneration (18). cell line from an average of 20 –30% to nearly 70 – 80%. Lipofectamine In the current study, we demonstrate that in the C2C12 2000 (4 –32 l, generally 16 l) and DNA (2–16 g, generally 8 g) were myoblast model of muscle regeneration, Mirk functions to pro- diluted in separate 500-l portions of Opti-MEM I (Invitrogen) or se- mote cell survival during the initial stages of differentiation, rum-free DMEM and combined after 5 min of incubation at room temperature. The DNA-Lipofectamine 2000 complexes were incubated similar to the known function of Mirk in mediating survival of for an additional 20 min at room temperature and then were added colon carcinoma cells in serum-free conditions (20). Undiffer- directly to 10 freshly plated cells in 3 ml of DMEM supplemented with entiated or partly differentiated cells are removed from cul- 10% FBS in 60-mm culture dishes. Media were typically replaced with tures of fusing myoblasts by apoptosis, causing a loss of 20 – DMEM containing 10% FBS 24 h after transfection. When lower levels 30% of C2C12 myoblasts during the first 48 h in differentiation of transfection were required, we transiently transfected cells using a medium (15). Mirk diminishes the extent of myoblast apoptosis 1:2:3 ratio of plasmid DNA/PLUS reagent/Lipofectamine in serum-free media. To lower the level of expression further for stability experi- during the differentiation process, at least in part by direct Cip1 ments, the p21 plasmids were diluted 1:5–1:10 with carrier plasmid modulation of p21 localization. Mirk phosphorylates p21 DNA. The media were replaced with 10% FBS/DMEM after4hof within its nuclear localization domain. This phosphorylation transfection in serum-free media. Antibiotics were not used at any time maintains a portion of p21 protein in the cytoplasm where p21 with cells used for transfection. has been reported to be unable to mediate cell cycle arrest (21) GST Fusion Proteins—p21 constructs were subcloned into the pGEX- and where p21 blocks caspase 3 activation (22). 4T1 vector (Amersham Biosciences) and expressed and purified as we have described in detail previously (23). Mirk and kinase-inactive Mirk EXPERIMENTAL PROCEDURES (YF) were subcloned into the pGEX-6P1 vector (Amersham Biosciences) Materials—Rabbit polyclonal antibodies were raised to unique se- and expressed in the Escherichia coli strain BL-21 (Amersham Bio- quences at the N terminus (amino acids 1–19) (20) and C terminus sciences). GST-Mirk proved to be a difficult protein to purify using (amino acids 595– 624) of Mirk and affinity-purified. The C-terminal standard techniques as it was rapidly incorporated into inclusion bodies antibody was raised to a slightly longer peptide than the C-terminal and tended to copurify with high quantities of the bacterial hsp70 antibody used in all our previous studies on Mirk (20), but it was only homologue, the dnaK gene product. This protein has been reported to homologous to Mirk/dyrk1B in BLAST analysis. This antibody detected play a role in degradation of abnormally folded proteins in E. coli (24). all three Mirk splice variants found in normal muscle as follows: 69, 70, The following protocol allowed the majority of the heat shock protein and 75 kDa, the 69- and 70-kDa variants found in C2C12 cells (1) and (hsp) to be eliminated from the final preparation. In addition, it appears the inducible 70-kDa Mirk form stably expressed in Mv1Lu cells (14), that the conditions also promoted hsp-dependent refolding of GST-Mirk but no other proteins at the dilutions used in the current study (West- (25, 26), resulting in a highly purified and active enzyme preparation. ern blotting data not shown). The C-terminal antibody was labeled with 300 ml of LB medium was seeded with 10% of an overnight culture of Alexa Fluor 594 using the Zenon rabbit IgG labeling kit (Molecular BL-21 carrying the desired plasmid and incubated at 37 °C for 1–2 h. Probes) for use in direct immunofluorescence experiments. Alexa Fluor Protein expression was induced with 0.1 mM isopropyl -D-thiogalacto- 488 and 594 (highly cross-adsorbed) secondary antibody conjugates and pyranoside for 6 h at 24 °C, and bacteria were collected by centrifuga- Alexa Fluor 594 phalloidin were purchased from Molecular Probes. tion and frozen at 70 °C. Pellets (averaging 2 g each) were resus- Antibodies to p21 (C-19 and F-5), GFP, myogenin, and tubulin were pended in 5 ml of ice-cold sonication buffer (140 mM NaCl, 2.7 mM KCl, from Santa Cruz Biotechnology. Rabbit monoclonal antibody to cleaved 10 mM NaH PO , 1.8 mM KH PO ,10mM MgCl ,5mM ATP, 5 mM DTT, 2 4 2 4 2 caspase 3 was from Cell Signaling. Antibody to the FLAG epitope was 20% glycerol, 2% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, from Sigma. Polyvinylidene difluoride transfer paper (Immobilon-P) 100 g/ml lysozyme, EDTA-free complete protease inhibitor set, pH 7.3; was purchased from Millipore. Lipofectamine, Lipofectamine 2000, and Roche Applied Science). The ATP and Mg are required for the activity PLUS reagent were from Invitrogen. All radioactive materials were of the heat shock protein (25, 26). The resuspended bacteria were purchased from PerkinElmer Life Sciences, and ECL reagents were incubated on ice for 15 min, and the bacteria were then lysed by from Amersham Biosciences. Tissue culture reagents were obtained sonication using a microtip (Misonix, setting 2.5) for 3 min with 5-s from Mediatech. SB203580 was purchased from Calbiochem. All other bursts separated by5sof rest. Lysates were clarified by centrifugation reagents were from Sigma. for 15 min at 15,000 g to remove insoluble material. 13-ml portions of Plasmids—pCDNA3.1-HisA-Mirk and the kinase-inactive forms the cleared lysate were added to 1 ml (bed volume) of glutathione- pCDNA3.1-HisA-Mirk (KR) and pCDNA3.1-HisA-Mirk (YF) had been Sepharose 4B (Amersham Biosciences) in a 15-ml conical tube, and an Cip1 generated previously. pMT-5/p21 was the kind gift of Dr. M.-C. additional 750 l of 100 mM ATP, 150 lof1 M MgCl , and a Complete Cip1 Hung. Wild-type p21 was subcloned into pGEX-4T1 (Amersham Protease Inhibitor Mini-Tab (Roche Applied Science) was then added to Biosciences), and site-directed mutagenesis was performed using the each sample. The beads were then incubated at 4 °C overnight with GeneEditor in vitro site-directed mutagenesis system (Promega). Mu- end-on rotation. Beads were washed four times for 10 min with 10 ml of tants were subsequently subcloned into pCMV-tag2B (Stratagene) to standard wash buffer (290 mM NaCl, 2.7 mM KCl, 10 mM NaH PO , 1.8 2 4 yield FLAG-p21 and pEGFP-C2 (Clontech) to yield GFP-p21 for mam- mM KH PO , 10% glycerol, 0.1% Triton X-100, pH 7.3) to remove the 2 4 malian expression. All mutant p21 constructs were sequenced to con- majority of contaminants from the beads. The beads were then washed firm the mutated sequence. five times for 1 h with 10-ml portions of Mg/ATP wash buffer (290 mM RNA Interference—A sequence within the murine Mirk coding do- NaCl, 2.7 mM KCl, 10 mM NaH PO , 1.8 mM KH PO ,5mM MgCl ,2 2 4 2 4 2 main si1, GACCTACAAGCACATCATT, was used to knock down en- mM ATP, 2 mM DTT, 10% glycerol, 0.1% Triton X-100, pH 7.3) to remove dogenous Mirk, whereas another sequence mutant at two positions to the majority of the heat shock protein contaminant. The beads were murine Mirk si2, GCTCTCTGTGGACCTCA, served as the mutant con- then washed three times for 5 min with standard wash buffer. All trol (1, 10). Each was inserted into the pSilencer plasmid (Ambion). washes and incubations were performed at 4 °C with end-on rotation. Cell Culture—C2C12 mouse myoblasts and NIH3T3 cells were ob- The PreScission enzyme, which is active at 4 °C, was used to cleave tained from the ATCC and cultured according to their recommenda- Mirk from GST. The efficiency of PreScission protease cleavage was tions. C2C12 cells were maintained in growth medium (GM; Dulbecco’s enhanced significantly by first eluting the GST fusion proteins from the modified Eagle’s medium, 4 mML-glutamine, 4.5 g/liter glucose, con- beads by the addition of 1 ml of glutathione elution buffer (75 mM taining 20% fetal bovine serum) and induced to undergo differentiation HEPES, pH 7.4, 150 mM NaCl, 20 mM reduced glutathione, 5 mM DTT, by switching to differentiation medium (DM; Dulbecco’s modified Ea- 0.1% Triton X-100). Elution was typically carried out overnight at 4 °C gle’s medium containing 2% horse serum). NIH3T3 cells were main- with end-on rotation. The eluate was concentrated; the glutathione was tained in Dulbecco’s modified Eagle’s medium, 4 mML-glutamine, 4.5 dialyzed out, and the elution buffer was exchanged for PreScission 25790 Mirk Blocks Apoptosis through Translocation of p21 cleavage buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, Triton in PBS (PBST) for 5 min, and blocked with 3% BSA/PBS for 30 0.01% Triton X-100, pH 7.0) by three concentrations with an Amicon min. Slides were incubated for 20 min with 2 units of Alexa Fluor 594 Ultra-4 30,000 molecular weight cut-off centrifugal filter device (Milli- phalloidin (Molecular Probes) diluted in 3% BSA/PBS, washed twice, pore). Protein concentration was measured using the Bradford protein and then incubated for 5 min with a 2 ng/ml solution of 4,6-diamidino- assay, and 20 units of PreScission enzyme were added per mg of pro- 2-phenylindole hydrochloride (DAPI) in PBST. Following three 5-min tein. Proteolytic cleavage was carried out overnight at 4 °C with end-on washes with PBST, slides were rinsed with distilled water, blotted dry, rotation. The cleaved protein solution was then incubated two times for and mounted with Biomedia Gel/Mount. Preparations were analyzed 4 h at 4 °C with equal volumes (bed volume) of glutathione-Sepharose and photographed as described below. 4B in order to clear the sample of uncleaved protein, GST, and the Statistical Analysis of Localization—Cell counts were obtained at PreScission enzyme (also a GST fusion protein). The resulting sample 400 using a Nikon Eclipse E50i fluorescent microscope. For C2C12 was concentrated, and the cleavage buffer was replaced with PBS with cells, at least 300 cells were observed in 8 –10 random fields in each of 10% glycerol and protease inhibitors using centrifugal filtration. The four separate preparations, and the number of cells with localization of product retained high activity for up to 1 year when stored at 20 °C. green fluorescence in the nucleus only and localization of green fluo- In Vitro Kinase Assays—2–5 g of GST-p21 fusion proteins bound to rescence in the nucleus and cytoplasm was determined. Two separate glutathione-Sepharose beads were washed twice with kinase assay preparations of NIH3T3 fibroblasts were examined in the same way. buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM MgCl , 0.5 mM Combined counts were analyzed by the test (Minitab) to determine dithiothreitol) and then incubated with 100 ng of recombinant Mirk for the significance of differences between the WT and mutant constructs. 20 min at 30 °C in 30 l of kinase assay buffer containing 10 M Immunofluorescence of Cultured Cells—C2C12 myoblasts were 32 5 unlabeled ATP plus 5 Ci of [- P]ATP. Reaction products were ana- plated in Lab-Tek 2-well chamber slides (2 10 cells per well), lyzed by SDS-PAGE and autoradiography. cultured for 1 day in growth media, and then switched to differenti- In Vivo Labeling and Immunoprecipitations—2 10 C2C12 cells ation media. After 24 h of expression in GM or 48 h in DM, cells were were plated in 60-mm dishes and transfected immediately with 12 gof washed twice with PBS, fixed in 4% paraformaldehyde for 5 min, FLAG-p21 constructs and 4 g of HisA-Mirk using 32 l of Lipo- washed twice for 5 min with PBS, permeabilized with 0.1% Triton fectamine 2000 in DMEM containing 10% FBS. Following 24 h of X-100/PBS (wash buffer; used in all subsequent washes), and blocked expression, cells were switched to differentiation media for 19 h, then to with 10% normal goat serum/PBS for 30 min. Dual staining for phosphate-free media for 1 h, and then incubated with 400 Ci of endogenous p21 and Mirk was accomplished by simultaneously incu- [ P]orthophosphate for4hin2mlof reduced phosphate medium (75% bating slides for 30 min with 1:500 dilutions of anti-p21 mouse phosphate-free DMEM, 25% DMEM). Cells were washed twice and then monoclonal antibody (F-5, Santa Cruz Biotechnology) and anti-C- lysed in 0.5 ml of buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM terminal Mirk antibody conjugated to Alexa Fluor 594. As a control NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, Roche Complete for nonspecific antibody interactions, we also performed identical Protease Inhibitor Mixture, and Sigma Phosphatase Inhibitor Mixture experiments with the C-19 polyclonal antibody (Santa Cruz Biotech- I and II (lysis buffer). Lysates were precleared by rocking incubation at nology) and our anti-N-terminal Mirk antibody. Additional controls 4 °C with 50 l of protein A-agarose (Santa Cruz Biotechnology) for 1 h included use of direct immunofluorescence (using the Zenon labeling and then pelleted in a microcentrifuge at 10,000 g for 20 min to system) and substitution of the primary antibody with nonspecific remove insoluble material. FLAG constructs were isolated using 15 l mouse or rabbit IgG as appropriate. All labeling incubations were of pre-washed EZview Anti-FLAG M2 Affinity Gel (Sigma), which was diluted in 10% normal goat serum/PBS. After three 5-min washes, incubated with the cell lysate at 4 °C for 2 h and then washed five times cells were incubated with a 1:1000 dilution of goat anti-mouse anti- with lysis buffer before analysis. Because both endogenous and FLAG- body conjugated to Alexa Fluor 488 (Molecular Probes). Slides were p21 ran extremely close to the IgG light chain on SDS-PAGE, we used washed two times for 5 min. During single-labeling experiments for the TrueBlot anti-mouse IgG secondary detection system (eBioscience; detection of p21, cells were incubated for 20 min with 2 units of Alexa 1:1000) for Western analysis of immunoprecipitated p21. Fluor 594 phalloidin (Molecular Probes), diluted in 3% BSA/PBS, and Two-dimensional Phosphopeptide Analysis—Chymotrypsin or tryp- washed twice. When using direct labeling, cells were fixed a second sin cleavage and two-dimensional analysis of p21 phosphopeptides were time (4% paraformaldehyde for 5 min) to stabilize the Zenon label carried out as described (23). Following an in vitro kinase assay or after the secondary antibody washes. Nuclei were counterstained immunoprecipitation of phospho-labeled proteins, the substrate was with a 5-min incubation bya2ng/mlsolutionof DAPI. Following resolved by SDS-PAGE and transferred to a nitrocellulose membrane, three 5-min washes, slides were rinsed with distilled water, blotted and the protein band was identified by autoradiography and excised dry, and mounted with Biomedia Gel/Mount. Images were obtained from the membrane. Proteins were digested with sequencing grade as described below. chymotrypsin or trypsin (20 gin200 l of freshly prepared 50 mM Immunofluorescence of Human Muscle Frozen Sections—Anony- NH HCO overnight at 37 °C), and the polypeptides were spotted onto mous samples of flash-frozen human muscle tissue on slides were 4 3 20 20-cm thin layer cellulose plates and separated in the first dimen- obtained from the Department of Pathology, State University of New sion by electrophoresis for 45 min at 1000 V on an HTLE-7002 electro- York, Upstate Medical University, in accordance with institutional phoresis system (C.B.S. Scientific) using buffer containing 2.2% formic review procedures for clinical specimen use. Slides were thawed at acid and 7.8% acetic acid, pH 1.9. Peptides were separated in the second room temperature for 15 min, hydrated with PBS for 15 min, and dimension by chromatography for9hin phosphochromatography buffer then fixed in 4% paraformaldehyde for 1 min at room temperature or (38% 1-butanol, 25% pyridine, and 7.5% acetic acid). Dried plates were ice-cold acetone for 10 min at 0 °C. Slides were rinsed three times for exposed to Eastman Kodak Biomax MS film for 2– 8 days. 5 min with PBS. The sections were then demarcated with a PAP pen Immunodetection—Following treatment as indicated and washing (Zymed Laboratories Inc.), permeabilized for 20 min with 0.2% Triton with phosphate-buffered saline, cells were lysed as described previously X-100 in PBS (wash buffer; used in all subsequent steps), and blocked (23). Depending upon the experiment, 20 –50 g of the cell lysate was with 10% normal goat serum, 0.2% Triton, PBS for 30 min. Mirk was resolved by SDS-PAGE and blotted onto polyvinylidene difluoride mem- visualized with polyclonal antibody to either the N or C terminus of branes. The blots were blocked in Tris-buffered saline containing 0.1% Mirk (both at 1:500 dilution for 1 h), whereas p21 was visualized with Tween 20 (TBST) and 5% nonfat dry milk for 1 h and incubated for 1–2 either the polyclonal C-19 antibody (1:250 for 1 h) or the monoclonal h at room temperature with the primary antibody (1:1000 dilution of F-5 antibody (1:50 for 2 h). Nonspecific IgG isolated from the same Cip1 antibodies to p21 , tubulin, and MyoD). Proteins were subsequently species as the primary antibody diluted to an equivalent mass/con- detected by enhanced chemiluminescence. Immunoblots were scanned centration was used as a negative control. All labeling incubations using a Lacie Silverscanner DTP, and densitometry was performed were diluted in 10% normal goat serum, 0.2% Triton, PBS. After three using the IP Lab Gel program (Scanalytics). 5-min washes, cells were incubated for 30 min with a 1:1000 dilution Localization of GFP-p21 Mutant Constructs—C2C12 myoblasts were of goat anti-IgG antibody Alexa Fluor conjugate (Molecular Probes). plated in Lab-Tek 2-well chamber slides (3.5–5.0 10 cells per well) Sections were washed twice for 5 min. In some experiments, sections and then immediately transfected with GFP-p21-WT, nonphosphory- were incubated for 20 min with 2 units of Alexa Fluor 594 phalloidin latable S153A, or the phosphomimetic S153D (3 g of plasmid DNA and (Molecular Probes), diluted in 3% BSA/PBS, and washed twice. Nuclei 6 l of Lipofectamine 2000 per well). NIH3T3 cells were plated at 1.5 were counterstained by a 5-min incubation with a 2 ng/ml solution of 10 cells per well, incubated overnight before transfection, and then DAPI. Following three 5-min washes, slides were rinsed with distilled transfected with a mixture of 3 g of plasmid DNA, 5 l of PLUS water, blotted dry, and mounted with Biomedia Gel/Mount. Images reagent, and 9 l of Lipofectamine per well. After 24 h of expression, were obtained as described below. cells were washed three times with PBS, fixed in 4% paraformaldehyde Imaging—Monochrome fluorescence images were obtained at 400 for 5 min, washed twice for 5 min with PBS, permeabilized with 0.1% or 1000 (as indicated) using a Diagnostic Instruments SPOT RT Mirk Blocks Apoptosis through Translocation of p21 25791 camera mounted on a Nikon Eclipse E50i fluorescent microscope. SPOT the cultures were undergoing apoptosis. In contrast, the RT software version 4.0.9 was used to pseudocolor the images, adjust amount of YF-Mirk decreased far more, about l0-fold. To de- the RGB histogram, and merge the images. Image manipulation con- termine what percentage of Mirk left after 48 h was endoge- sisted of resetting the zero point of the RGB histogram of the green and nous, another set of transfections was performed. Cell lysates red fluorescent channels to stretch the darker areas of the image yield- were analyzed for expression of ectopic YF-Mirk and for endog- ing a uniform black background consistent with the image viewed through the microscope. Final figures were arranged using Adobe Pho- enous Mirk from the cultures transfected with the vector alone toshop version 7.0. (Fig. 1B, bottom). These blots were exposed for a much longer BrdUrd Incorporation Assay—C2C12 myoblasts were plated over- time to visualize endogenous Mirk, which accounted for ⁄3 of night in Labtek 2-well chamber slides (10 cells per well) and then the Mirk detected in cultures transfected with YF-Mirk at the transfected (2 g of plasmid DNA, 4 l of PLUS, and 4 l of Lipo- 48-h point. Only about 8% of the YF-Mirk was left after 48 h, fectamine per well) with GFP vector, GFP-p21-WT, or the phosphomi- metic GFP-p21-S153D. Cells were transfected in serum-free media for whereas about 50% of wild-type Mirk was retained. Half-life 4 h, and an equal volume of 20% FBS/DMEM was then added. BrdUrd measurements showed that this effect was not because of dif- incorporation was measured using the cell proliferation assay kit (Am- ferences in protein stability (data not shown). Thus both bio- ersham Biosciences). After 24 h of expression, cells were incubated for chemical analysis and immunofluorescence studies demon- 1 h with BrdUrd labeling reagent diluted 1:500 in 10% FBS/DMEM. strated that about 4 –5 times as many C2C12 myoblasts Cells were rinsed twice with PBS and fixed for 30 min at room temper- survived apoptosis in cultures undergoing differentiation if ature with acid alcohol (90% absolute ethanol, 5% glacial acetic acid, 5% water). Cells were then rinsed three times with PBS, blocked for 30 min they expressed ectopic wild-type Mirk than if they expressed in 10% normal goat serum/PBST, and then incubated for1hat room ectopic kinase-inactive YF-Mirk. temperature with a DNase I solution containing a 1:100 dilution of In a recent report (1), we showed that depletion of endoge- anti-BrdUrd antibody (Amersham Biosciences). The denatured GFP nous Mirk by RNA interference in C2C12 myoblasts undergo- protein was labeled by a 30-min incubation with anti-GFP antibody ing differentiation blocked the induction of myogenin and con- (Santa Cruz Biotechnology 8334; 1:500). BrdUrd was visualized using anti-mouse Alexa Fluor 594; GFP was visualized with anti-rabbit Alexa tractile proteins and subsequent myoblast fusion. In these Fluor 488. At least 300 GFP-expressing cells were observed in 8 –10 experiments, we also observed a loss of those cells in which random fields in each of three separate preparations, and the number of Mirk had been depleted, visualized by expression of cotrans- cells labeled for both GFP and BrdUrd was determined using a Green/ fected DsRed by fluorescence microscopy. To test directly Orange V2 filter set (Chroma Technology) that allowed simultaneous whether Mirk played some role in myoblast survival, we de- visualization of both fluorophores. As an additional control, the propor- pleted Mirk in C2C12 cells by RNA interference using RNAi to tion of cells incorporating BrdUrd was also assessed in a set of non- transfected slides stained with DAPI. Combined counts were analyzed Mirk and a mutant RNAi as the control, and we measured cell by the test (Minitab) to determine the significance of differences viability by colony formation after Mirk depletion (Fig. 1, C and between the GFP vector and GFP-p21 constructs. D). C2C12 cells were cotransfected with the neomycin resist- ance gene and with the pSilencer vector encoding either RNAi RESULTS to Mirk, a mutant RNAi, or vector alone, and the extent of Mirk Mirk Promotes Myoblast Survival—Undifferentiated or depletion was analyzed by Western blotting (Fig. 1C). The partly differentiated cells are removed from cultures of fusing Mirk-depleted cells and controls were plated at single cell den- myoblasts by apoptosis. Programmed cell death of 20 –30% of sity and selected in growth medium containing G418 for 3 C2C12 myoblasts is seen during the first 48 h after myoblasts weeks to determine the number of cells capable of proliferation are transferred from growth medium to differentiation medium to form a colony. Colony formation was reduced 75% in the (15). We had observed in earlier studies that the serine/threo- C2C12 cells experiencing Mirk knockdown at the time of plat- nine kinase Mirk afforded colon carcinoma cells increased sur- ing compared with cells transfected with the mutant RNAi or vival capabilites, whereas mutant forms of kinase-inactive the vector control (Fig. 1D). Parallel studies with NIH3T3 cells Mirk did not (20). Mirk is highly expressed in skeletal muscle showed that the cells in colonies that did arise after Mirk cells and in myoblasts, so we speculated that one function of knockdown expressed Mirk (data not shown). Thus these colo- Mirk was to enhance the survival of differentiating myoblasts. nies presumably had arisen either from cells that had not been Mirk is enriched in NIH3T3 cells in G /G and down-regulated 0 1 transfected or from cells that had lost the Mirk RNAi plasmid. when cells are treated with mitogens (10). Likewise, Mirk was These data indicate that knockdown of Mirk levels at the time found to be enriched in C2C12 myoblasts undergoing mitosis C2C12 myoblasts were seeded as single cells had reduced their and in early G (Fig. 1A). Levels of Mirk rapidly increase in viability and thus their ability to give rise to colonies, and C2C12 cells arresting in G /G when placed in DM (1, 11). We 0 1 indicate that Mirk functions as a survival factor. hypothesized that if Mirk mediated cell survival, C2C12 cells We next assayed whether depletion of Mirk would render expressing ectopic wild-type Mirk would survive in greater cells more susceptible to apoptosis in short term experiments numbers during myoblast differentiation than cells expressing without chemical selection of transfected cells (Fig. 1E). We kinase-inactive YF-Mirk. C2C12 cells were transfected with depleted Mirk in C2C12 cells by RNA interference, with a either wild-type Mirk, YF mutant Mirk, or vector alone. The mutant RNAi and the empty vector as controls, and we used constructs were allowed to express for 18 h, and the cells were cotransfected DsRed to mark the transfectants. After 24 h to then transferred to differentiation medium for 0 – 48 h. Expres- induce Mirk knockdown, we transferred the cells to differenti- sion of Mirk or YF-Mirk was determined by immunofluores- ation medium for 1 day, and we identified the apoptotic cells cence analysis. Each construct was expressed in roughly the within the DsRed population by incubation with a fluorescent same percentage of C2C12 cells, 30 –34%, after culture for 18 h conjugate of the permeable caspase inhibitor VAD-fmk. 48% of in growth medium. When cells were cultured in DM for 48 h, cells with Mirk knockdown were undergoing apoptosis com- four times as many cells in the differentiating cultures were pared with 22.9 and 20.1%, respectively, of cells treated with found expressing ectopic wild-type Mirk as expressing kinase- mutant RNAi or vector, a highly significant difference (Fig. inactive YF-Mirk. These data supported the hypothesis that Mirk provided some survival advantage to differentiating myo- 1E). A similar knockdown experiment was performed in a time course format by measuring the abundance of Mirk, and the blasts. In parallel transfections, cell lysates were analyzed for expression of ectopic Mirk and YF-Mirk normalized to -tubu- apoptosis effector activated caspase 3 by Western blotting (Fig. lin by Western blotting (Fig. 1B, top). The amount of wild-type 1F). After 1 day, Mirk levels were depleted to about 50% of Mirk declined about 2-fold by 48 h, during which time cells in control values, and caspase 3 was activated to 160% of control 25792 Mirk Blocks Apoptosis through Translocation of p21 FIG.1. Mirk mediates myoblast survival. A, Mirk is expressed in cycling C2C12 myoblasts. Fluorescence microscopy demonstrates that Mirk expression is greatest in C2C12 cells in mitosis and in cells in early G . Affinity-purified anti-peptide antibody to the Mirk C terminus was conjugated to Alexa Fluor 594 and used to detect endogenous Mirk in C2C12 cells in growth medium. Nuclei were stained with DAPI, and identical fields were merged with SPOT RT software. Similar results were obtained with antibody directed to the unique N terminus of Mirk. B, top, ectopic Mirk maintains the survival of differentiating C2C12 cells. Cells were transiently transfected with wild-type Mirk or kinase-inactive mutant YF-Mirk, allowed to express for 18 h, and then transferred to differentiation medium for up to 48 h. Lysates were analyzed for expression of Mirk Mirk Blocks Apoptosis through Translocation of p21 25793 values. After 2 days, there was less Mirk knockdown and less activation of caspase 3. After 3 days, there was no Mirk knock- down and no activation of caspase 3 (Fig. 1F). Thus, depletion of Mirk protein correlated with increased abundance of acti- vated caspase 3 in multiple experiments and led to loss of cells by apoptosis. In another experiment, we measured the effect of the caspase inhibitor benzyloxycarbonyl-VAD-fmk on differen- tiation-induced apoptosis, as assayed by the TUNEL reaction FIG.2. Mirk phosphorylates transfected FLAG-p21 in vivo. Top (Fig. 1G). The caspase inhibitor blocked apoptosis to a similar panel, autoradiogram after SDS-PAGE of in vivo phosphorylation of extent, 40% in control cultures, 50% in mutant RNAi-treated wild-type FLAG-p21 cotransfected into NIH3T3 cells with either wild- cultures, and 33% in cultures where Mirk knockdown occurred. type or kinase-inactive Mirk and allowed to coexpress for 24 h. After 4 h of labeling, FLAG-p21 was immunoprecipitated (IP) with anti-FLAG These studies employing either Mirk knockdown or Mirk over- M2 antibody, and the immunoprecipitates were resolved on SDS-PAGE expression demonstrate that Mirk has anti-apoptotic survival with FLAG-p21 migrating at the expected position. FLAG-p21 was functions in C2C12 myoblasts. phosphorylated only in the cotransfections with wild-type Mirk. The Mirk Phosphorylates p21 in Vivo—The CDK inhibitor nonspecific doublet seen at the top of each lane serves as the loading Cip1 control. Bottom panel, Anti-FLAG Western blot (WB) showed that equal p21 has been implicated in the control of apoptosis during amounts of FLAG-p21 were immunoprecipitated. The upper band rep- myoblast fusion (15), possibly through stabilization by ERKs resents light chain, and the lower band represents FLAG-p21. There Kip1 (27). Mirk phosphorylates another CDK inhibitor p27 in appears to be a slight decrease in mobility in the p21 phosphorylated by vivo (10), so we hypothesized that Mirk might also phosphoryl- Mirk/dyrk1B. Cip1 ate the closely related p21 . A wild-type p21 construct with an N-terminal FLAG epitope tag was cotransfected into ilar data were obtained when Mirk was coexpressed with p21 in NIH3T3 fibroblasts with either wild-type or kinase-inactive C2C12 myoblasts (data not shown). The many functions of p21 Mirk and allowed to coexpress for 24 h. After4hof labeling rely on its association with a variety of proteins, including with [ P]orthophosphate, FLAG-p21 was immunoprecipitated cyclins, CDKs, PCNA, and others, which associate with p21 with anti-FLAG M2 antibody, and the immunoprecipitates within specific domains. It was essential to determine the phos- were resolved by SDS-PAGE and analyzed by autoradiography phorylation site of Mirk in p21 in order to be able to postulate (Fig. 2). FLAG-p21 migrated at the expected molecular weight the biological effect of this phosphorylation. For initial studies, but was phosphorylated only in the cotransfections with wild- we measured the capacity of Mirk to phosphorylate in vitro a type Mirk. FLAG-p21 was not labeled when coexpressed with series of GST-p21 constructs that were mutated to the non- kinase-inactive KR-Mirk or vector (Fig. 2, upper panel). West- phosphorylatable alanine residue at known phosphorylation ern blotting for the FLAG epitope (Fig. 2, lower panel) demon- sites: Ser-130 (the p38 MAPK site (23)), Thr-57 (the GSK3 site strated that equal amounts of FLAG-p21 were immunoprecipi- (28)), and Ser-146 (a protein kinase C site (29) and one of two tated. The lower band of the doublet was FLAG-p21, and the Akt sites (30)). Mirk phosphorylated each of these constructs to upper band was light chain. The FLAG-p21 protein exhibited a similar degree (data not shown), indicating that none of these retarded mobility only when coexpressed with wild-type Mirk sites was the Mirk phosphorylation site. (Fig. 2, center lane), consistent with its phosphorylation. Sim- We then compared human and mouse p21 sequences for and -tubulin by Western blotting. Bottom, to determine whether the amount of Mirk remaining in cells expressing ectopic YF-Mirk after 48 h was either ectopic or endogenous Mirk, cells were transiently transfected with kinase-inactive mutant YF-Mirk, or vector, allowed to express for 18 h, and then transferred to differentiation medium for up to 48 h. Lysates were analyzed for expression of Mirk and -tubulin by Western blotting. The YF-Mirk and vector blots were exposed for the same amount of time to allow direct comparison. One of duplicate experiments with similar results is shown. C, depletion of Mirk in cycling myoblasts by RNA interference reduced C2C12 cell viability in colony formation assays. Cells were plated at 5 10 per 60-mm dish, three dishes per point for 24 h, and then were transfected for4hin serum-free medium with 5 g of pSilencer DNA encoding either RNAi to Mirk (RNAi), a mutant RNAi (Mt), or vector (Vc) together with 0.5 g of neomycin resistance plasmid, using 10 l each of Lipofectamine and PLUS reagent. Fetal bovine serum was then added to 10% to maintain cell viability during expression. Cells were incubated for 20 h before trypsinization and then lysed for Western blotting (WB). Ct, control, cross-reacting band showing similar loading. D, same experiment as in C, but transfected cells were reseeded at the same cell density and selected in 400 g/ml G418 for 3 weeks. Mean colony number S.E. is shown. E, Mirk knockdown increases apoptosis measured by fluorescence microscopy using an activated caspase 3 inhibitor. C2C12 myoblasts were plated overnight in LabTek 2-well chamber slides (2 10 cells per well) and then cotransfected with 0.5 g of pDsRed and 1.5 g of pSilencer vector (Vec), mutant si or RNAi to Mirk (4 l of PLUS and 4 l of Lipofectamine per well). Cells were transfected in serum-free media for 4 h, and an equal volume of 20% FBS/DMEM was then added. Following 24 h of expression, cells were incubated with differentiation media for 24 h. Activated caspase was then labeled by a 30-min incubation with 10 M CaspACE FITC-VAD-fmk in situ marker (Promega) diluted in DM. This is a fluorescent conjugate of the permeable caspase inhibitor VAD-fmk that was used as an in situ marker for apoptosis. Slides were washed and mounted with Biomedia GelMount. At least 200 DsRed expressing cells were observed in each of four separate preparations, and the number of cells labeled for both DsRed and the FITC-VAD-fmk marker was determined using a Green/Orange V2 filter set (Chroma) that allowed simultaneous visualization of both fluorophores. Combined counts were analyzed by the test to determine the significance of differences between the RNAi constructs. Efficiency of cotransfection was determined to be greater than 85% in parallel experiments using a combination of GFP and DsRed. The number of cells scored per assay conditions is shown. F, knockdown of endogenous Mirk by RNA interference inhibits the activation of caspase 3 in differentiating C2C12 cells. C2C12 cells (5 10 per 60-mm dish) were transfected with pSilencer plasmid encoding RNAi to Mirk (RNAi) or mutant RNAi (Mt) for 24 h and then switched to differentiation medium for 1–3 days, as noted. The relative abundance of Mirk/tubulin and of activated caspase 3/tubulin in lysates was determined by Western blotting on the upper and lower sections of same blot that was cut in half and then reunited for the exposure, with tubulin used as the loading control. The relative abundance of either protein in cells treated with RNAi to Mirk was then normalized to their relative abundance in cells treated with mutant RNAi. Mean S.E. (if greater than 5%) is shown from data from three experiments. G, apoptosis induced by differentiation medium or Mirk knockdown blocked to a similar extent by a caspase inhibitor. C2C12 myoblasts were plated, transfected, and cultured as in E, except they were cotransfected with 0.5 g of pEGFP. A parallel set of cultures was incubated with DM containing 20 M of the cell-permeable caspase inhibitor benzyloxycarbonyl-VAD-fmk (Promega). After 24 h in DM, DNA breaks in apoptotic cells were labeled with tetramethylrhodamine-dUTP by terminal deoxynucleotidyltransferase-mediated in situ end labeling (TUNEL) using the in situ cell death detection kit (Roche Applied Science). At least 300 GFP-expressing cells were observed in each of four separate preparations, so that an average of 1250 cells were scored per point. The number of GFP-expressing cells labeled with the TUNEL marker was determined using a Green/Orange V2 filter set (Chroma) that allowed simultaneous visualization of both fluorophores. Combined counts were analyzed by the test. Efficiency of cotransfection was determined to be greater than 85% in parallel experiments using a combination of GFP and DsRed. 25794 Mirk Blocks Apoptosis through Translocation of p21 conserved threonine or serine residues within three or four residues of an arginine, the usual site of phosphorylation by Mirk, and we mutated each of these six amino acids to the nonphosphorylatable alanine residue as follows: S2A, S15A, S27A, T97A, T145A, S153A. Mutation of only one site, serine 153 to alanine, caused a reduction in in vitro phosphorylation by Mirk (data not shown). We confirmed that Mirk phospho- rylated p21 at Ser-153 in vitro by performing two-dimensional phosphopeptide mapping following digestion of the phosphoryl- ated p21 protein by trypsin. Three phosphopeptides were de- rived from wild-type p21 after in vitro phosphorylation by Mirk, but only one phosphopeptide was seen after phosphoryl- ation of the mutant p21-S153A construct (Fig. 3A). The slower migrating peptide on chromatography (Fig. 3A, long arrow) was probably a partial digestion product. The tryptic peptide containing serine 153 consists of amino acids 143–154 and is flanked by two KRR sequences, so trypsin could cleave at multiple points within both of these regions. Alternative cleav- age may also have been promoted by steric hindrance resulting from phosphorylation of Ser-153. Supporting this interpreta- tion was the observation that the relative abundance of the two peptides varied in different experiments. However, the same general pattern was seen in three separate experiments, dem- onstrating that Mirk phosphorylates p21 at serine 153. In Vivo Phosphorylation of FLAG-p21 by Cotransfected Mirk Occurs at Ser-153 in Differentiating Myoblasts—In vivo phos- phorylation is known to be more specific than in vitro phospho- The peptide identified by the longer arrow may be a partial digestion product containing Ser-153, due to steric interference of the enzyme by the phosphate group. Similar data were seen in two additional peptide maps. B, Mirk phosphorylates p21 at Ser-153 in vivo as shown by peptide mapping with chymotrypsin. Wild-type FLAG-p21, mutant p21-S153A, and mutant p21-S130A were each cotransfected with wild- type Mirk into C2C12 cells. Constructs were allowed to express over- night, and cells were then placed in myoblast differentiation medium for24h,thelast4hof which cells were labeled with [ P]orthophos- phate. In one dish transfected with wild-type p21, 10 M SB203580 was added at the same time as the label. FLAG-p21 was immunoprecipi- tated with anti-FLAG M2 antibody; the immunoprecipitates were di- gested with chymotrypsin and then subjected to two-dimensional pep- tide mapping. The p21-S153A mutant incorporated about 40% as much label as wild-type p21 when normalized to the total amount of p21 immunoprecipitated. Chymotrypsin was used because it yields a sim- pler map with a broader distribution of peptides. The peptide indicated by the arrow is lost in the p21-S153A mutant and is the presumed 152–159-amino acid peptide. C, depletion of endogenous Mirk by RNA interference blocks the phosphorylation of transfected wild-type p21 but not p21-S153A mutant at the Mirk phosphorylation site. Cells were plated at 5 10 per 60-mm dish, cultured for 16 h, and then were transfected for4hin serum-free medium with 2.5 g of either FLAG- p21 or FLAG-p21-S153A, and 2.5 g of pSilencer DNA encoding either RNAi to Mirk (RNAi) or vector. As controls, FLAG-p21 was cotrans- fected with either wild-type Mirk (M) or kinase-inactive YF-Mirk (YF). Fetal bovine serum was then added to 10% to maintain cell viability during overnight expression, and cells were switched to Opti-MEM differentiation medium for 24 h, with 325 Ci of [ P]orthophosphate added to each dish for the last 4 h following1hin phosphate-free medium. Anti-FLAG epitope immunoprecipitates were separated by SDS-PAGE, and the FLAG-p21 bands were detected by autoradiogra- phy (top lanes) and then by Western blotting for FLAG. The amounts of p21 and Mirk in the lysates were determined by Western blotting (lower 2 bands). D, SB203580 inhibits myoblast differentiation as measured by the level of expression of the myogenic regulatory factor myogenin that controls the differentiation program. C2C12 cells were placed in differ- entiation medium 10 M SB203580 for 1 and 2 days, and the abun- dance of myogenin was determined by Western blotting. Ct, cross- reacting protein used as a loading control. E, relative stability of FIG.3. Mirk phosphorylates p21 at Ser-153 in vitro and in mutant p21 constructs. C2C12 cells were transfected with either mu- differentiating myoblasts. A, Mirk phosphorylates p21 at Ser-153 in tant FLAG-p21-S153D, FLAG-p21-S153A, or wild-type FLAG-p21, and vitro as shown by peptide mapping with trypsin. Wild-type (Wt) and constructs were allowed to express for 24 h. Cycloheximide at 20 g/ml mutant p21-S153A constructs were phosphorylated in vitro by purified was added to each culture, and FLAG-p21 and tubulin abundances recombinant Mirk, digested with trypsin, and then subjected to two- were determined at the indicated times by Western blotting and nor- dimensional peptide mapping. The peptide containing Ser-153 and con- malized to the 0 time values. Mean S.E. is shown from two separate sisting of amino acids 143–154 is shown by a short arrow in both panels. experiments. Mirk Blocks Apoptosis through Translocation of p21 25795 TABLE I rylation, especially on a substrate such as p21, which exists in BrdUrd incorporation in C2C12 cells expressing GFP-p21 constructs vivo in several different complexes, and is only found as a free None Vector p21(WT) p21(S153D) molecule immediately after synthesis (31). In fact, in our pre- vious study, p38 MAPK phosphorylated p21 strongly at Thr-57 GFP 910 973 922 935 and weakly at Ser-130 in vitro, but in colon carcinoma cells p38 GFP BrdUrd 272 292 23 18 % S phase 29.9 30.0 2.5 1.9 MAPK only phosphorylated p21 at Ser-130 (23). Mirk was (p 0.0001) (p 0.0001) cotransfected into proliferating myoblasts with either wild-type FLAG-p21, the Mirk site mutant FLAG-p21-S153A, or the p38 site mutant FLAG-p21-S130A. The cells were then switched to at least within the time frame of these experiments. Mutation differentiation media for 24 h and labeled with [ P]orthophos- of p21 at the p38 MAPK site of Ser-130 and treatment of cells phate for the last 4 h. The immunoprecipitated p21 constructs with the p38 inhibitor SB203580 did not eliminate any of the were digested with chymotrypsin before phosphopeptide map- three phosphorylated peptides. In addition, the Ser-153-con- ping. Trypsin digestion yields 17 fragments from p21, whereas taining peptide was phosphorylated in the FLAG-p21-S130A chymotrypsin yields 11, and only 6 of these peptide fragments mutant construct to a similar extent as in wild-type p21, dem- contain either serine or threonine residues, resulting in a much onstrating that phosphorylation of p21 at Ser-130 is not an simpler pattern. Additionally, the position of the peptide con- essential prerequisite for phosphorylation by Mirk (Fig. 3B). taining the p38 MAPK phosphorylation site on p21 is readily The SB203580 preparation used in these experiments was ac- identifiable in the chymotrypsin map (23). Mirk was found to tive because exposure of C2C12 cells in DM for 1 or 2 days to phosphorylate p21 at Ser-153 in differentiating myoblasts, in this drug prevented induction of myogenin, an essential myo- accordance with Mirk’s in vitro phosphorylation of p21. Muta- genic regulatory factor, whose transcription is mediated by the tion of p21 to S153A at the Mirk phosphorylation site blocked p38 MAPK substrate MEF2 (Fig. 3D). Treatment of differenti- phosphorylation in vivo. FLAG-p21 was phosphorylated on ating C2 myoblasts with SB203580 at 5 M for 48 h has been three phosphopeptides in vivo, whereas FLAG-p21-S153A was reported to block the kinase activity of p38 on the exogenous phosphorylated on only two phosphopeptides (Fig. 3B). The substrate myelin basic protein but not to block the phosphoryl- peptide containing Ser-153 is indicated by an arrow in each ation of p38 (35). Other investigators as well as ourselves (data panel of Fig. 3B. This peptide consists of amino acids 152–159 not shown) have also noted this discrepancy. However, our and contains only one serine and no threonines. Mutation of studies do not entirely depend on the use of the inhibitor. There this peptide at serine 153 to alanine blocked its in vivo phos- was no loss in phosphorylation of the p38 site mutant, p21- phorylation. Therefore, p21 is phosphorylated at Ser-153 in S130A, during myoblast differentiation because the peptide differentiating myoblasts by Mirk. containing this mutant sequence was as equally phosphoryl- Knockdown of endogenous Mirk by RNAi blocked phospho- ated as the wild-type peptide (Fig. 3B). Therefore, we could rylation of wild-type FLAG-p21, but not FLAG-p21-S153A, mu- detect no phosphorylation of p21 by p38 MAPK during this tant at the Mirk phosphorylation site (Fig. 3C), confirming that initial period of myogenic differentiation. Mirk phosphorylates p21 at Ser-153 in differentiating myo- The phosphorylation of p21 by p38 MAPK at Ser-130 in- blasts in vivo. C2C12 cells were transfected with either wild- creased the stability of p21 in colon carcinoma cells (23). How- type or mutant p21 together with the pSilencer plasmid for ever, phosphorylation of p21 by Mirk did not stabilize p21 in Mirk RNAi or the vector alone. Parallel cultures were trans- C2C12 cells. In fact, p21-S153D was slightly less stable than fected with wild-type p21 and either wild-type Mirk or kinase- wild-type p21, as demonstrated by cycloheximide arrest exper- inactive YF-Mirk. Cells in DM were metabolically labeled with iments (Fig. 3E). Therefore, neither Mirk nor p38 MAPK sta- [ P]orthophosphate. The p21 constructs were immunoprecipi- bilizes p21 during the initial stages of myoblast differentiation. tated by their FLAG epitopes, and the extent of incorporation of Furthermore, at this stage in differentiation, when Mirk acts the label was determined by autoradiography after SDS-PAGE. as a survival factor for myoblasts, only Mirk, not p38 MAPK, Ectopic Mirk phosphorylated FLAG-p21 in vivo, whereas the phosphorylates p21. kinase-inactive YF-Mirk did not (Fig. 3C, 1st 2 lanes), and Phosphomimetic p21-S153D Is as Effective as Wild-type p21 served as the positive and negative controls, respectively. En- in Blocking Cell Cycle Progression—Ectopic expression of p21 dogenous Mirk in the differentiating C2C12 cells phosphoryl- has been shown by others (16) to decrease the percentage of ated wild-type p21 but not mutant p21-S153A (Fig. 3C, 3rd and myoblasts in S phase as assayed by BrdUrd incorporation 4th lanes), although similar amounts of p21 were immunopre- using fluorescence microscopy. Similarly, we found that tran- cipitated. In the cultures in which Mirk knockdown was accom- sient expression of GFP-p21 decreased the fraction of C2C12 plished (Fig. 3C, last 2 lanes), neither wild-type Mirk nor the myoblasts expressing GFP that showed BrdUrd incorporation p21-S153A mutant was phosphorylated much above back- from 30% in cells transfected with vector alone to 2.5% in cells ground levels. Therefore, endogenous Mirk phosphorylates p21 expressing GFP-p21 (Table I). Transient expression of the at Ser-153 in vivo in differentiating myoblasts. phosphomimetic GFP-p21-S153D caused a similar arrest of cell Mirk and p38 MAPK interact in vivo. Mirk is activated by growth, with only 1.9% of cells expressing this construct also the p38 MAPK kinase MKK3 (12), and p38 MAPK can seques- positive for BrdUrd. Thus, p21 phosphorylated by Mirk re- ter Mirk in vivo (13). Thus, we questioned whether phospho- mained capable of mediating growth arrest. These results were rylation of p21 by p38 MAPK occurred in differentiating myo- consistent with the known control of DNA replication by p21 blasts, as it does in colon carcinoma cells (23), and if so, and the retinoblastoma protein in terminally differentiated whether it was an essential prerequisite for phosphorylation of muscle cells (36). p21 by Mirk. In one set of cultures in the peptide mapping Phosphomimetic p21-S153D Translocates to the Cytoplasm— studies (Fig. 3B), 10 M SB203580 was added to inhibit p38 Serine 153 is within the nuclear localization signal of p21 and MAPK. During myoblast differentiation, p38 MAPK is acti- within its PCNA binding domain (reviewed in Ref. 37), so we vated and in turn activates the myogenic regulatory factors predicted that phosphorylation at this residue would alter both MEF2A, MEF2C, and MyoD (32–34). However, the in vivo the localization of p21 and its capacity to block DNA synthesis. peptide mapping studies (Fig. 3B) show that p38 MAPK does In the current study we have concentrated on the localization of not appear to phosphorylate p21 in differentiating myoblasts, p21. We transiently transfected GFP-p21-WT, the phosphomi- 25796 Mirk Blocks Apoptosis through Translocation of p21 TABLE II Localization of GFP-p21 constructs in C2C12 cells and NIH3T3 cells Cycling C2C12 myoblasts WT S153A S153D Nucleus cytoplasm 246 200 998 Nucleus only 1022 1082 278 % nucleus cytoplasm 19.4 15.6 78.3 (p 0.02) (p 0.0001) NIH3T3 fibroblasts WT S153A S153D Nucleus cytoplasm 164 141 420 Nucleus only 453 488 209 % nucleus cytoplasm 26.6 22.4 66.8 (p 0.0001) trials had demonstrated that GFP-p21 is always localized in the nucleus in this system, we used a one-tailed analysis to compare the proportion of transfected cells expressing the GFP constructs in both the cytoplasm and the nucleus to the pro- portion of cells expressing GFP-p21 exclusively in the nucleus. GFP-p21-WT was found in the cytoplasm in only 19.4% of myoblasts. Four times as many cells, 78.3%, expressed the Mirk-phosphomimetic GFP-p21-S153D construct in the cyto- plasm. This gave a highly significant (p 0.0001) difference in localization. In contrast, the nonphosphorylatable GFP-p21- S153A construct remained localized in the nucleus in myo- blasts, with only 15.6% of the cells expressing this construct in the cytoplasm, giving a statistically significant value of p 0.02 compared with wild-type p21. Similar results were ob- tained with NIH3T3 cells (Table II). Wild-type p21 was found in the cytoplasm of only 26.6% of transfected fibroblasts, whereas over twice as many transfected fibroblasts expressed the Mirk-phosphomimetic GFP-p21-S153D construct in the cy- toplasm. This was also a highly significant difference in local- ization (p 0.0001). The nonphosphorylatable GFP-p21-S153A construct remained localized in the nucleus in fibroblasts, with only 22.4% of the cells containing this construct in the cyto- plasm, a value that was not significantly different from cells FIG.4. Fluorescence microscopy demonstrated that the Mirk expressing wild-type p21. These data clearly demonstrate that phosphorylation site phosphomimetic mutant p21-S153D is mutation of p21 to mimic phosphorylation by Mirk caused a found in both the cytoplasm and the nucleus, whereas wild-type p21 and the nonphosphorylatable p21-S153A are localized ex- much larger proportion of the p21 to localize in the cytoplasm clusively in the nucleus in the majority of cells. GFP-p21-wild- in both myoblasts and fibroblasts. Mutation of an additional type, GFP-p21-S153A which is not phosphorylated by Mirk, and GFP- serine in the nuclear localization signal (Ser-160 to S160A or p21-S153D, the Mirk site-phosphomimetic construct, were transiently S160D) did not affect the cellular distribution of GFP-p21 (data expressed in C2C12 cells (A, 400 magnification) and in NIH3T3 cells (B, 1000 magnification). Nuclei were stained with DAPI, and identical not shown). fields were photographed for GFP and DAPI. Images were merged with Ectopic Mirk Translocates GFP-p21 to the Cytoplasm SPOT RT software. Scale bars 50 m. through Phosphorylation at Ser-153—NIH3T3 fibroblasts were transfected with ectopic wild-type Mirk or kinase-inactive YF- metic GFP-p21-S153D, and the nonphosphorylatable GFP-p21- Mirk and either wild-type GFP-p21 or GFP-p21-S153A. The S153A into cycling C2C12 myoblasts (Fig. 4A) and cycling cells expressing exogenous p21 and exogenous Mirk constructs NIH3T3 fibroblasts (Fig. 4B). NIH3T3 cells were used to de- were visualized by immunofluorescence. Cells expressing both termine the generality of these observations because Mirk is constructs were visualized by using a Green/Orange V2 filter widely distributed. Wild-type p21 and the nonphosphorylatable set, and the percentage of such cells in which p21 was pancel- GFP-p21-S153A construct were predominantly localized in the lular (versus restricted solely to the nucleus) was twice as large nucleus of both cell types. Nuclear localization was confirmed as the controls (Fig. 5). analysis showed this to be a highly by counterstaining the nuclei with DAPI. The GFP and the blue significant difference (p 0.0001). Expression of elevated lev- fluorescent DAPI were merged in the last lane of Fig. 4B and els of ectopic Mirk was sufficient to make the majority of showed overlap of each fluorochrome. In marked contrast to wild-type p21 pancellular, while having no effect on the p21 wild-type p21, the phosphomimetic GFP-p21-S153D construct mutant at the Mirk site (S153A). Moreover, the p21 transloca- was found in the cytoplasm as well as the nucleus in both tion was dependent on the kinase activity of Mirk. C2C12 myoblasts and NIH3T3 cells (Fig. 4). The GFP-p21- More Wild-type p21 Is Found in the Cytoplasm When Mirk Is S153D was found distributed throughout the entire cell, allow- Induced during Myoblast Differentiation—The localization ing visualization of cytoplasmic extensions. studies described above (Figs. 4 and 5) were performed in These observations were quantitated by counting 3824 cycling myoblasts. We next transfected the same three GFP- transfected myoblasts and 1875 transfected fibroblasts (Table p21 constructs into C2C12 cells, and we then stimulated them II) and subjecting the data to the test. Because preliminary to differentiate. Only the fused myoblasts were examined, and Mirk Blocks Apoptosis through Translocation of p21 25797 cells are shown in the top 2 rows). When myoblasts were cul- tured for 2 days in differentiation medium, however, p21 was found in the cytoplasm as well as the nucleus in fused myo- blasts (Fig. 7, bottom two rows). Note in particular in the Merge column of Fig. 7, the large myotubes that are stained with actin to delineate the entire cell body and with DAPI to delineate nuclei. 11 nuclei were found in the myotube in the 3rd row, whereas four nuclei were found in the myotube in the 4th row. Both of these myotubes show extensive localization of endoge- nous p21 in their cytoplasm (Fig. 7, p21 column, rows 3 and 4). These data clearly demonstrate that localization of endogenous p21 in the cytoplasm occurs as a natural part of myoblast differentiation. Thus the increase in concentration of the exog- enous FLAG-p21 within the cytoplasm of differentiating myo- FIG.5. Overexpression of Mirk causes a portion of the wild- tubes (Fig. 6) is reflected in a similar cytoplasmic enrichment of type p21 population to translocate to the cytoplasm in a Mirk the endogenous p21. kinase-dependent manner but cannot translocate p21 mutated Endogenous p21 Is Found within the Cytoplasm of Adult at the Mirk phosphorylation site. NIH3T3 fibroblasts were plated overnight in Labtek 2-well chamber slides (1.5 10 cells per well) and Human Muscle—p21 is known to play critical roles in early then transfected (2 g of plasmid DNA and 4 l of Lipofectamine 2000 muscle differentiation (18) and in the maintenance of adult per well) with GFP-p21(p21) or the nonphosphorylatable GFP-p21(p21- muscle tissue through the muscle progenitor (satellite) cell S153A), together with either wild-type (M) or kinase inactive (YF-M) population (19). However, there do not appear to be any exten- Mirk. Cells were transfected in DMEM containing 10% bovine calf serum. After 24 h of expression, cells were fixed with 4% paraformal- sive reports describing the presence or function of p21 in nor- dehyde, and the GFP-p21 protein was labeled by a 30-min incubation mal mature muscle. Therefore, we performed SAGE analysis with mouse anti-GFP monoclonal antibody (Santa Cruz Biotechnology using the NCBI/GEO data base, and we found that p21 is 8334; 1:500), followed by anti-mouse Alexa Fluor 488. Mirk was visu- alized by using rabbit polyclonal antibody to the Mirk C terminus at expressed to a higher extent in muscle biopsies from young 1:500, followed by anti-rabbit Alexa Fluor 594. An average of 1050 individuals, aged 21–31 years old, than in biopsies from most GFP-p21 expressing cells were observed for each transfectant. Cells older patients, aged 62–77 (GDS156), but that all skeletal were scored in 8 –10 random fields in each of three separate prepara- muscle biopsies contained p21 mRNA. We were unable to de- tions, and the number of cells labeled for both GFP-p21 and transfected tect p21 in formalin-fixed paraffin sections of human muscle Mirk was determined using a Green/Orange V2 filter set (Chroma) that allowed simultaneous visualization of both fluorophores. Combined even after antigen retrieval with citrate, EDTA, or proteolytic counts were analyzed by the test (Minitab) to determine the signif- digestion. This result is not unexpected given that p21 is nor- icance of differences between conditions. The total number of cells mally found in complexes, where the epitope may not be acces- scored per condition is labeled on each bar. sible to the antibody, particularly after formalin fixation. We therefore performed immunofluorescence on frozen sections of cells were stained for actin so the myotubes would be unam- adult human muscle. We were able to detect p21 in adult biguous. As predicted, in each of four experiments wild-type muscle using the C-19 antibody at a dilution of 1:200, followed GFP-p21 was also found in the cytoplasm of myotubes, whereas by goat anti-rabbit Alexa Fluor 594 at 1:500 (Fig. 8). The the nonphosphorylatable GFP-p21-S153A remained restricted distribution of p21 in the adult muscle fibers paralleled our to the nuclei of the multinucleated myotubes (Fig. 6). The observations of p21 localization in myotubes. p21 remained phosphomimetic GFP-p21-S153D served as the positive control concentrated in the nucleus but was distributed throughout the and partitioned into both cytoplasm and nucleus as expected. myofibrils. We were also able to see this pattern using the F-5 Thus p21 was shifted from the nucleus into the cytoplasm by anti-p21 antibody, thus demonstrating the specificity of our the presence of elevated levels of endogenous Mirk in differen- immunolabeling results (data not shown). Therefore, examina- tiating myotubes. tion of human skeletal muscle tissue confirmed that endoge- Changes in the Localization of Endogenous p21 during Dif- nous p21 partitioned into the cytoplasm as a natural part of ferentiation of C2C12 Myoblasts—The data thus far confirmed muscle differentiation. that a significant proportion of exogenous p21 is localized in the Phosphomimetic p21-S153D Is More Effective than Wild-type cytoplasm of differentiating myoblasts following phosphoryla- p21 in Blocking Caspase 3 Activation—Caspase 3 is the pri- tion by Mirk. We next investigated the localization of endoge- mary effector caspase in apoptosis. Caspase 3 is activated by nous p21 during differentiation of myoblasts. C2C12 myoblasts cleavage of its precursor, procaspase 3. C2C12 myoblasts were were cultured for 1 day in growth medium (GM), and then transiently transfected with either wild-type FLAG-p21, the switched to differentiation medium (DM). After 24 h in GM or phosphomimetic FLAG-p21-S153D, the nonphosphorylatable 48 h in DM, cells were fixed in paraformaldehyde, and endog- FLAG-p21-S153A, or vector alone. The constructs were allowed enous p21 was then visualized with the monoclonal antibody to express overnight; the cells were then cultured in differen- F-5. This antibody was raised against full-length p21, so anti- tiation medium for 16 h, and the amount of active caspase 3, body binding should not be affected by phosphorylation at released by cleavage, was determined by Western blotting. Ser-153 in the C terminus (Fig. 7). As an additional control, p21 Wild-type p21 caused a small 10% inhibition in the amount of was labeled in separate experiments using the polyclonal an- activated caspase 3, whereas the Mirk phosphomimetic p21- tibody C-19 (data not shown). The anti-p21 antibody was de- S153D construct blocked caspase 3 activation five times as tected with goat anti-mouse secondary antibody conjugated to much (Fig. 9). In contrast, little inhibition by the nonphospho- green fluorescent Alexa Fluor 488. Cells were also incubated rylatable construct was observed. In addition, the phosphomi- with Alexa Fluor 594 coupled to phalloidin to detect actin and DAPI for nuclear staining. All of the fields were merged to form metic p21 construct coimmunoprecipitated with ASK1 (data not shown), one upstream activator of this caspase cascade. the composite shown in the right-most column Fig. 7. Endogenous p21 was exclusively localized in the nucleus in ASK1 has been shown to be sequestered by wild-type p21 in proliferating myoblasts in growth medium. The signals for p21 other studies (22). Therefore, one mechanism by which Mirk and for DAPI completely coincided (Fig. 7, two fields of C2C12 blocks apoptosis in myoblasts is through phosphorylation of 25798 Mirk Blocks Apoptosis through Translocation of p21 FIG.6. When Mirk is up-regulated as a part of the myoblast differentia- tion program, both wild-type p21, as well as the Mirk-phosphorylation site phosphomimetic mutant p21-S153D, are translocated to the cytoplasm as shown by fluorescence microscopy. In contrast, the nonphosphorylatable p21-S153A is localized exclusively in the nuclei of the multinucleated myotubes. GFP-p21-wild-type, GFP-p21-S153A, and GFP-p21-S153D were transfected into C2C12 cells, which were then allowed to differentiate for 3 days. Similar data were seen in each of four experiments. FIG.7. Endogenous p21 is exclu- sively nuclear in cycling C2C12 cells in growth medium, but a portion of the endogenous p21 protein is trans- located to the cytoplasm when C2C12 cells are treated for 48 h with differ- entiation medium. Endogenous p21 was visualized with the F-5 monoclonal antibody and goat anti-mouse antibody conjugated to Alexa Fluor 488; the nuclei were stained with DAPI, and actin within the entire cell body was visualized with phalloidin conjugated to Alexa Fluor 594. Identical fields were photographed for each fluorochrome (1st three rows) and then merged with SPOT RT software. Identical results were obtained with the C-19 polyclonal antibody. Scale bar 50 m. FIG.8. p21 localizes in both the cy- toplasm and nucleus of adult skeletal muscle. Frozen sections of human mus- cle were analyzed for the localization of p21 by immunofluorescence using the polyclonal C-19 antibody (1:200) and goat anti-rabbit IgG Alexa Fluor 594 (1:500); nuclei were counterstained with DAPI. Scale bars 50 m. p21. A subpopulation of the p21 molecules is then retained in expressed for only about 1 day in growth medium (Fig. 10A) the cytoplasm where it blocks apoptosis through inhibition of and then were lost. We used this transient expression to deter- the activation of caspase 3. mine whether the phosphomimetic p21 construct could act as a Transient Expression of Phosphomimetic p21 Aids Myoblast survival factor in myoblasts at the time of plating at very low Survival—C2C12 cells were transiently transfected with either cell density for colony formation assays (Fig. 10B). The same wild-type FLAG-p21, the phosphomimetic FLAG-p21-S153D, transfection protocol was used, except that a vector control was or the nonphosphorylatable FLAG-p21-S153A, and the length included. Transient expression of the phosphomimetic p21- of time each construct was expressed was determined (Fig. S153D construct, and to a lesser extent the wild-type p21, 10A). After 24 h of transfection and expression, the cells were increased myoblast colony formation (Fig. 10B), a reflection of placed in growth medium for 0 –13 days. All of the constructs increased survival during the initial day on which cells were Mirk Blocks Apoptosis through Translocation of p21 25799 FIG.9. A p21 construct, phosphomimetic at the Mirk phospho- rylation site, is twice as effective as wild-type p21 in blocking the activation of caspase 3. C2C12 myoblasts were transfected with FLAG-p21 wild-type, FLAG-p21-S153A, FLAG-p21-S153D, or vector for 24 h, cultured in differentiation medium for 16 h, and then analyzed by SDS-PAGE and Western blotting for activated caspase 3, and nor- malized by Western blotting for tubulin and the expression of exoge- nous FLAG-p21 by Western blotting for FLAG. Mean S.D. shown of two experiments. plated. Transient expression of p21-S153D for only 1–2 days (Fig. 10A) thus inhibits caspase 3 activation during the initial plating period when cells are at very low density and suscep- tible to apoptosis. Loss of expression of the p21 construct after this period allows the cells to re-enter the cell cycle and grow to form a discernible colony. In contrast, the phosphorylation site mutant p21-S153A had no survival effect compared with the vector (Fig. 10B). Thus phosphorylation of p21 by Mirk enables p21 to oppose apoptosis and to enhance myoblast survival. FIG. 10. Transient expression of a p21 construct, phosphomi- DISCUSSION metic at the Mirk phosphorylation site, increases the viability In the current study we have demonstrated that the kinase of C2C12 cells. C2C12 myoblasts were transfected with FLAG-p21 Mirk mediates cell survival of myoblasts, during both growth wild-type, FLAG-p21-S153A, FLAG-p21-S153D, or vector for 4 h, al- lowed to express for 24 h (0 point) and then switched to serum-contain- and differentiation. These observations complement the known ing growth medium for 0 –13 days. A, each construct was expressed for survival function of Mirk in colon carcinoma cells (20). We also only about 1 day in growth medium following the transfection and show that at least some of the pro-survival functions of Mirk initial 24-h expression period, as determined by Western blotting for can be ascribed to its phosphorylation of the CDK inhibitor p21 the FLAG epitope. The zero time points for each construct were exam- ined in parallel to the zero time points for the other two constructs on and subsequent increased localization of p21 in the cytoplasm. each blot as internal controls. Ct, cross-reacting protein used as a Cytoplasmic p21 has a role in pro-survival signaling in differ- loading control. B, after 24 h, the cells were trypsinized and plated at entiating myoblasts (Fig. 9) and is maintained in the cytoplasm either 500 or 1000 cells in 100-mm tissue culture dishes, each in of mature myofibrils. The anti-apoptotic function of p21 in triplicate. Data shown are the mean of two such experiments, normal- ized to the number of colonies per 1000 cells plated. myogenesis is well documented. In C2 myoblasts, ectopic ex- pression of p21 has been shown to block apoptosis, whereas depletion of p21 by antisense oligonucleotides induced apopto- tion (42). The p21 gene is induced by the tumor suppressor p53 sis (15). A role for cytosolic p21 has also been demonstrated in (43) and by cellular senescence (44). p21 is a major mediator of neuronal differentiation. After functioning in the nucleus to the G growth arrest induced by activation of the tumor sup- block cell proliferation, cytoplasmic p21 promotes neuronal pressor p53 in response to DNA damage (reviewed in Ref. 45). differentiation where it regulates Rho-induced actin remodel- Other major functions for p21 are to promote assembly of cyclin ing leading to neurite outgrowth (38). Cytoplasmic p21 has also D-CDK4 complexes and to increase cyclin D1 accumulation by been shown recently (39) to enhance axonal regeneration and direct inhibition of GSK3-triggered nuclear export (46, 47). functional recovery after spinal cord injury. The cell cycle modulatory activity of p21 is tightly correlated Cell cycle progression is mediated by the sequential activa- with its nuclear localization. Movement of p21 from the nucleus tion of members of the CDK protein kinase family. Cell cycling to the cytoplasm blocks its cell cycle inhibitory activity and is blocked when cyclin-CDK complexes cannot form or when contributes to the cell growth induced by the oncogene Her-2/ the catalytic activity of these complexes is blocked through neu (21). Akt activated by Her-2/neu phosphorylates Thr-145 binding of a CDK inhibitor molecule. One of these inhibitors is and Ser-146 within the nuclear localization signal of p21 (21, Cip1/Waf1 p21 (40), which was originally identified as a PCNA- 30, 48). Phosphorylation of p21 by Akt has been shown to binding protein (41). p21 inhibits DNA replication by prevent- promote association with 14-3-3 proteins, which function as ing PCNA from contributing to DNA polymerase and func- nuclear export proteins (49). In the current study, we have 25800 Mirk Blocks Apoptosis through Translocation of p21 documented that the kinase Mirk, like Akt, also promotes localization of p21 in the cytoplasm of transformed cells might translocation of p21 to the cytoplasm by phosphorylation contribute not only to increased survival under environmental within the nuclear localization signal of p21. In the case of and/or pharmacological stress but may also contribute to cell Mirk, the phosphorylation site is serine 153, close to the Akt motility and thus enhance tumor cell invasion. sites of threonine 145 and serine 146. 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Chem. 279, 1885–1891 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology http://www.deepdyve.com/lp/american-society-for-biochemistry-and-molecular-biology/mirk-dyrk1b-mediates-survival-during-the-differentiation-of-PB6GN1gKQ2

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Publisher: American Society for Biochemistry and Molecular Biology
Copyright: Copyright © 2005 Elsevier Inc.
ISSN: 0021-9258
eISSN: 1083-351X
DOI: 10.1074/jbc.m413594200
Publisher site: See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 27, Issue of July 8, pp. 25788 –25801, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Mirk/Dyrk1B Mediates Survival during the Differentiation of C2C12 Myoblasts* Received for publication, December 2, 2004, and in revised form, March 29, 2005 Published, JBC Papers in Press, April 25, 2005, DOI 10.1074/jbc.M413594200 Stephen E. Mercer, Daina Z. Ewton, Xiaobing Deng, Seunghwan Lim, Thomas R. Mazur, and Eileen Friedman‡ From the Department of Pathology, Upstate Medical University, State University of New York, Syracuse, New York 13210 in controlling C2C12 myoblast differentiation following the The kinase Mirk/dyrk1B is essential for the differen- tiation of C2C12 myoblasts. Mirk reinforces the G /G commitment stage of myogenesis. Depletion of endogenous 0 1 arrest state in which differentiation occurs by directly Mirk by RNA interference blocked the transcription of myoge- Kip1 phosphorylating and stabilizing p27 and destabiliz- nin and the subsequent myoblast differentiation program (1). ing cyclin D1. We now demonstrate that Mirk is anti- Mirk controls the activation of the myogenin transcription fac- apoptotic in myoblasts. Knockdown of endogenous Mirk tor MEF2 by regulating nuclear accumulation of the MEF2 by RNA interference activated caspase 3 and decreased inhibitors, class II histone deacetylases (2). However, it has myoblast survival by 75%, whereas transient overex- been reported that embryonic knockout of Mirk did not block pression of Mirk increased cell survival. Mirk exerts its skeletal muscle development in the early embryo (3), suggest- anti-apoptotic effects during muscle differentiation at ing that Mirk may have a more significant function in muscle least in part through effects on the cell cycle inhibitor repair than in initial myogenesis. Cip1 and pro-survival molecule p21 . Overexpression and Mirk (Minibrain-related kinase; also known as Dyrk1B) is a RNA interference experiments demonstrated that Mirk member of an evolutionarily conserved family of proteins, the phosphorylates p21 within its nuclear localization do- Dyrk/Minibrain family of arginine-directed serine/threonine main at Ser-153 causing a portion of the typically nu- protein kinases (4 –7), that play roles in controlling the switch clear p21 to localize in the cytoplasm. Phosphomimetic from proliferation to differentiation in a wide variety of orga- GFP-p21-S153D was pancellular in both cycling C2C12 nisms. The roles of Dyrk/Minibrain/Mirk homologues in yeast myoblasts and NIH3T3 cells. Endogenous Mirk in myo- (Yak1) and slime mold (YakA) suggest that this group of ki- tubes and overexpressed Mirk in NIH3T3 cells were able nases helps to regulate the transition from growth to differen- to cause the pancellular localization of wild-type GFP- tiation in response to environmental stresses (8, 9). p21 but not the nonphosphorylatable mutant GFP-p21- Mirk is a unique, multifunctional kinase with primary S153A. Translocation to the cytoplasm enables p21 to block apoptosis through inhibitory interaction with activity in G phase and early differentiation. Specifically, pro-apoptotic molecules. Phosphomimetic p21-S153D Mirk elongates the G phase of the cell cycle in C2C12 myo- was more effective than wild-type p21 in blocking the blasts, Mv1Lu epithelial cells, and NIH3T3 fibroblasts by Kip1 activation of caspase 3. Transient expression of p21- stabilizing the CDK inhibitor p27 (10) and by destabiliz- S153D also increased myoblast viability in colony form- ing the G cyclin, cyclin D1 (11). Mirk has also been shown to ing assays, whereas the p21-S153A mutant had no effect. act as a transcriptional activator (2, 12, 13) and to inhibit cell This Mirk-dependent change in p21 intracellular local- motility (14). Because of these actions of Mirk on cell cycle ization is a natural part of myoblast differentiation. En- regulators active in G /G during myoblast differentiation, 0 1 dogenous p21 localized exclusively to the nuclei of pro- Cip1 we extended our study of Mirk substrates to p21 . The liferating myoblasts but was also found in the cytoplasm CDK inhibitor p21 is highly expressed in differentiating mus- of post-mitotic multinucleated myotubes and adult hu- cle in vivo (15) and has been implicated as playing a central man skeletal myofibers. role in mediating the pre-differentiation growth arrest in- duced by the major muscle regulatory factor MyoD (16). It is believed that this effect of p21 during myogenesis is because Although there is a large body of work describing the func- of inhibition of G cyclin-CDK complexes and subsequent tions of myogenic regulatory factors in skeletal muscle commit- blockade of retinoblastoma protein phosphorylation (16). ment and differentiation, much less is known regarding the Studies using mice deficient in members of the Cip/Kip CDK signaling pathways that control the specific molecular events inhibitor family have demonstrated that in the absence of p21, involved in terminal differentiation and maintenance of skele- mice can develop into normal adults (17). This appears to be tal muscle. We demonstrated recently (1) that a novel gene, due to redundant functions of p57, because mice deficient in which we cloned in 1997, the kinase Mirk, plays a critical role both p21 and p57 demonstrate severely arrested muscle devel- opment (18). Specifically, the double mutant mice displayed * This work was supported by United States Public Health Service Award RO1 CA67405 (to E. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 DM, differentiation medium; GM, growth medium; BrdUrd, bromode- U.S.C. Section 1734 solely to indicate this fact. oxyuridine; WT, wild type; FBS, fetal bovine serum; DMEM, Dulbecco’s ‡ To whom correspondence should be addressed: Pathology Dept., modified Eagle’s medium; DTT, dithiothreitol; PBS, phosphate-buffered Upstate Medical University, 750 East Adams St., Syracuse, NY 13210. saline; BSA, bovine serum albumin; GST, glutathione S-transferase; Tel.: 315-464-7138; Fax: 315-464-8419; E-mail: [email protected]. TUNEL, terminal dUTP nick-end labeling; DAPI, 4,6-diamidino-2- The abbreviations used are: Mirk, minibrain-related kinase; GFP, phenylindole hydrochloride; fmk, fluoromethyl ketone; CDK, cyclin-de- green fluorescent protein; CDK, cyclin-directed kinase; PCNA, prolifer- pendent kinase; hsp, heat shock protein; RNAi, RNA interference; ating cell nuclear antigen; MAPK, mitogen-activated protein kinase; siRNA, short interfering RNA. 25788 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Mirk Blocks Apoptosis through Translocation of p21 25789 g/liter glucose, containing 10% bovine calf serum. Cell cultures were increased proliferation and apoptosis of myoblasts and failed to typically maintained without the use of antibiotics. Cells were used for form myotubes. Significantly, although p21-deficient mice ap- experiments only from passages 3 to 10 from our frozen stocks. pear to develop normally, p21 is essential for normal regener- Transient Transfections—Two strategies were employed for tran- ation through the muscle progenitor (or satellite) cells that are sient transfections. In order to achieve high level short term expression responsible for repairing muscle following injury (19). It has (for GFP localization and in vivo labeling), we used a modification of the been suggested that muscle differentiation during embryogen- manufacturer’s standard protocol for Lipofectamine 2000 (Invitrogen). Specifically, we transfected cells with a 2:1 ratio (l/g) of Lipo- esis and during regeneration are fundamentally different, with fectamine 2000 to plasmid DNA immediately after plating freshly p57 playing a dominant role in embryogenesis and p21 being trypsinized cells. This increased transfection efficiency in the C2C12 the critical CDK inhibitor during regeneration (18). cell line from an average of 20 –30% to nearly 70 – 80%. Lipofectamine In the current study, we demonstrate that in the C2C12 2000 (4 –32 l, generally 16 l) and DNA (2–16 g, generally 8 g) were myoblast model of muscle regeneration, Mirk functions to pro- diluted in separate 500-l portions of Opti-MEM I (Invitrogen) or se- mote cell survival during the initial stages of differentiation, rum-free DMEM and combined after 5 min of incubation at room temperature. The DNA-Lipofectamine 2000 complexes were incubated similar to the known function of Mirk in mediating survival of for an additional 20 min at room temperature and then were added colon carcinoma cells in serum-free conditions (20). Undiffer- directly to 10 freshly plated cells in 3 ml of DMEM supplemented with entiated or partly differentiated cells are removed from cul- 10% FBS in 60-mm culture dishes. Media were typically replaced with tures of fusing myoblasts by apoptosis, causing a loss of 20 – DMEM containing 10% FBS 24 h after transfection. When lower levels 30% of C2C12 myoblasts during the first 48 h in differentiation of transfection were required, we transiently transfected cells using a medium (15). Mirk diminishes the extent of myoblast apoptosis 1:2:3 ratio of plasmid DNA/PLUS reagent/Lipofectamine in serum-free media. To lower the level of expression further for stability experi- during the differentiation process, at least in part by direct Cip1 ments, the p21 plasmids were diluted 1:5–1:10 with carrier plasmid modulation of p21 localization. Mirk phosphorylates p21 DNA. The media were replaced with 10% FBS/DMEM after4hof within its nuclear localization domain. This phosphorylation transfection in serum-free media. Antibiotics were not used at any time maintains a portion of p21 protein in the cytoplasm where p21 with cells used for transfection. has been reported to be unable to mediate cell cycle arrest (21) GST Fusion Proteins—p21 constructs were subcloned into the pGEX- and where p21 blocks caspase 3 activation (22). 4T1 vector (Amersham Biosciences) and expressed and purified as we have described in detail previously (23). Mirk and kinase-inactive Mirk EXPERIMENTAL PROCEDURES (YF) were subcloned into the pGEX-6P1 vector (Amersham Biosciences) Materials—Rabbit polyclonal antibodies were raised to unique se- and expressed in the Escherichia coli strain BL-21 (Amersham Bio- quences at the N terminus (amino acids 1–19) (20) and C terminus sciences). GST-Mirk proved to be a difficult protein to purify using (amino acids 595– 624) of Mirk and affinity-purified. The C-terminal standard techniques as it was rapidly incorporated into inclusion bodies antibody was raised to a slightly longer peptide than the C-terminal and tended to copurify with high quantities of the bacterial hsp70 antibody used in all our previous studies on Mirk (20), but it was only homologue, the dnaK gene product. This protein has been reported to homologous to Mirk/dyrk1B in BLAST analysis. This antibody detected play a role in degradation of abnormally folded proteins in E. coli (24). all three Mirk splice variants found in normal muscle as follows: 69, 70, The following protocol allowed the majority of the heat shock protein and 75 kDa, the 69- and 70-kDa variants found in C2C12 cells (1) and (hsp) to be eliminated from the final preparation. In addition, it appears the inducible 70-kDa Mirk form stably expressed in Mv1Lu cells (14), that the conditions also promoted hsp-dependent refolding of GST-Mirk but no other proteins at the dilutions used in the current study (West- (25, 26), resulting in a highly purified and active enzyme preparation. ern blotting data not shown). The C-terminal antibody was labeled with 300 ml of LB medium was seeded with 10% of an overnight culture of Alexa Fluor 594 using the Zenon rabbit IgG labeling kit (Molecular BL-21 carrying the desired plasmid and incubated at 37 °C for 1–2 h. Probes) for use in direct immunofluorescence experiments. Alexa Fluor Protein expression was induced with 0.1 mM isopropyl -D-thiogalacto- 488 and 594 (highly cross-adsorbed) secondary antibody conjugates and pyranoside for 6 h at 24 °C, and bacteria were collected by centrifuga- Alexa Fluor 594 phalloidin were purchased from Molecular Probes. tion and frozen at 70 °C. Pellets (averaging 2 g each) were resus- Antibodies to p21 (C-19 and F-5), GFP, myogenin, and tubulin were pended in 5 ml of ice-cold sonication buffer (140 mM NaCl, 2.7 mM KCl, from Santa Cruz Biotechnology. Rabbit monoclonal antibody to cleaved 10 mM NaH PO , 1.8 mM KH PO ,10mM MgCl ,5mM ATP, 5 mM DTT, 2 4 2 4 2 caspase 3 was from Cell Signaling. Antibody to the FLAG epitope was 20% glycerol, 2% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, from Sigma. Polyvinylidene difluoride transfer paper (Immobilon-P) 100 g/ml lysozyme, EDTA-free complete protease inhibitor set, pH 7.3; was purchased from Millipore. Lipofectamine, Lipofectamine 2000, and Roche Applied Science). The ATP and Mg are required for the activity PLUS reagent were from Invitrogen. All radioactive materials were of the heat shock protein (25, 26). The resuspended bacteria were purchased from PerkinElmer Life Sciences, and ECL reagents were incubated on ice for 15 min, and the bacteria were then lysed by from Amersham Biosciences. Tissue culture reagents were obtained sonication using a microtip (Misonix, setting 2.5) for 3 min with 5-s from Mediatech. SB203580 was purchased from Calbiochem. All other bursts separated by5sof rest. Lysates were clarified by centrifugation reagents were from Sigma. for 15 min at 15,000 g to remove insoluble material. 13-ml portions of Plasmids—pCDNA3.1-HisA-Mirk and the kinase-inactive forms the cleared lysate were added to 1 ml (bed volume) of glutathione- pCDNA3.1-HisA-Mirk (KR) and pCDNA3.1-HisA-Mirk (YF) had been Sepharose 4B (Amersham Biosciences) in a 15-ml conical tube, and an Cip1 generated previously. pMT-5/p21 was the kind gift of Dr. M.-C. additional 750 l of 100 mM ATP, 150 lof1 M MgCl , and a Complete Cip1 Hung. Wild-type p21 was subcloned into pGEX-4T1 (Amersham Protease Inhibitor Mini-Tab (Roche Applied Science) was then added to Biosciences), and site-directed mutagenesis was performed using the each sample. The beads were then incubated at 4 °C overnight with GeneEditor in vitro site-directed mutagenesis system (Promega). Mu- end-on rotation. Beads were washed four times for 10 min with 10 ml of tants were subsequently subcloned into pCMV-tag2B (Stratagene) to standard wash buffer (290 mM NaCl, 2.7 mM KCl, 10 mM NaH PO , 1.8 2 4 yield FLAG-p21 and pEGFP-C2 (Clontech) to yield GFP-p21 for mam- mM KH PO , 10% glycerol, 0.1% Triton X-100, pH 7.3) to remove the 2 4 malian expression. All mutant p21 constructs were sequenced to con- majority of contaminants from the beads. The beads were then washed firm the mutated sequence. five times for 1 h with 10-ml portions of Mg/ATP wash buffer (290 mM RNA Interference—A sequence within the murine Mirk coding do- NaCl, 2.7 mM KCl, 10 mM NaH PO , 1.8 mM KH PO ,5mM MgCl ,2 2 4 2 4 2 main si1, GACCTACAAGCACATCATT, was used to knock down en- mM ATP, 2 mM DTT, 10% glycerol, 0.1% Triton X-100, pH 7.3) to remove dogenous Mirk, whereas another sequence mutant at two positions to the majority of the heat shock protein contaminant. The beads were murine Mirk si2, GCTCTCTGTGGACCTCA, served as the mutant con- then washed three times for 5 min with standard wash buffer. All trol (1, 10). Each was inserted into the pSilencer plasmid (Ambion). washes and incubations were performed at 4 °C with end-on rotation. Cell Culture—C2C12 mouse myoblasts and NIH3T3 cells were ob- The PreScission enzyme, which is active at 4 °C, was used to cleave tained from the ATCC and cultured according to their recommenda- Mirk from GST. The efficiency of PreScission protease cleavage was tions. C2C12 cells were maintained in growth medium (GM; Dulbecco’s enhanced significantly by first eluting the GST fusion proteins from the modified Eagle’s medium, 4 mML-glutamine, 4.5 g/liter glucose, con- beads by the addition of 1 ml of glutathione elution buffer (75 mM taining 20% fetal bovine serum) and induced to undergo differentiation HEPES, pH 7.4, 150 mM NaCl, 20 mM reduced glutathione, 5 mM DTT, by switching to differentiation medium (DM; Dulbecco’s modified Ea- 0.1% Triton X-100). Elution was typically carried out overnight at 4 °C gle’s medium containing 2% horse serum). NIH3T3 cells were main- with end-on rotation. The eluate was concentrated; the glutathione was tained in Dulbecco’s modified Eagle’s medium, 4 mML-glutamine, 4.5 dialyzed out, and the elution buffer was exchanged for PreScission 25790 Mirk Blocks Apoptosis through Translocation of p21 cleavage buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, Triton in PBS (PBST) for 5 min, and blocked with 3% BSA/PBS for 30 0.01% Triton X-100, pH 7.0) by three concentrations with an Amicon min. Slides were incubated for 20 min with 2 units of Alexa Fluor 594 Ultra-4 30,000 molecular weight cut-off centrifugal filter device (Milli- phalloidin (Molecular Probes) diluted in 3% BSA/PBS, washed twice, pore). Protein concentration was measured using the Bradford protein and then incubated for 5 min with a 2 ng/ml solution of 4,6-diamidino- assay, and 20 units of PreScission enzyme were added per mg of pro- 2-phenylindole hydrochloride (DAPI) in PBST. Following three 5-min tein. Proteolytic cleavage was carried out overnight at 4 °C with end-on washes with PBST, slides were rinsed with distilled water, blotted dry, rotation. The cleaved protein solution was then incubated two times for and mounted with Biomedia Gel/Mount. Preparations were analyzed 4 h at 4 °C with equal volumes (bed volume) of glutathione-Sepharose and photographed as described below. 4B in order to clear the sample of uncleaved protein, GST, and the Statistical Analysis of Localization—Cell counts were obtained at PreScission enzyme (also a GST fusion protein). The resulting sample 400 using a Nikon Eclipse E50i fluorescent microscope. For C2C12 was concentrated, and the cleavage buffer was replaced with PBS with cells, at least 300 cells were observed in 8 –10 random fields in each of 10% glycerol and protease inhibitors using centrifugal filtration. The four separate preparations, and the number of cells with localization of product retained high activity for up to 1 year when stored at 20 °C. green fluorescence in the nucleus only and localization of green fluo- In Vitro Kinase Assays—2–5 g of GST-p21 fusion proteins bound to rescence in the nucleus and cytoplasm was determined. Two separate glutathione-Sepharose beads were washed twice with kinase assay preparations of NIH3T3 fibroblasts were examined in the same way. buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM MgCl , 0.5 mM Combined counts were analyzed by the test (Minitab) to determine dithiothreitol) and then incubated with 100 ng of recombinant Mirk for the significance of differences between the WT and mutant constructs. 20 min at 30 °C in 30 l of kinase assay buffer containing 10 M Immunofluorescence of Cultured Cells—C2C12 myoblasts were 32 5 unlabeled ATP plus 5 Ci of [- P]ATP. Reaction products were ana- plated in Lab-Tek 2-well chamber slides (2 10 cells per well), lyzed by SDS-PAGE and autoradiography. cultured for 1 day in growth media, and then switched to differenti- In Vivo Labeling and Immunoprecipitations—2 10 C2C12 cells ation media. After 24 h of expression in GM or 48 h in DM, cells were were plated in 60-mm dishes and transfected immediately with 12 gof washed twice with PBS, fixed in 4% paraformaldehyde for 5 min, FLAG-p21 constructs and 4 g of HisA-Mirk using 32 l of Lipo- washed twice for 5 min with PBS, permeabilized with 0.1% Triton fectamine 2000 in DMEM containing 10% FBS. Following 24 h of X-100/PBS (wash buffer; used in all subsequent washes), and blocked expression, cells were switched to differentiation media for 19 h, then to with 10% normal goat serum/PBS for 30 min. Dual staining for phosphate-free media for 1 h, and then incubated with 400 Ci of endogenous p21 and Mirk was accomplished by simultaneously incu- [ P]orthophosphate for4hin2mlof reduced phosphate medium (75% bating slides for 30 min with 1:500 dilutions of anti-p21 mouse phosphate-free DMEM, 25% DMEM). Cells were washed twice and then monoclonal antibody (F-5, Santa Cruz Biotechnology) and anti-C- lysed in 0.5 ml of buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM terminal Mirk antibody conjugated to Alexa Fluor 594. As a control NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, Roche Complete for nonspecific antibody interactions, we also performed identical Protease Inhibitor Mixture, and Sigma Phosphatase Inhibitor Mixture experiments with the C-19 polyclonal antibody (Santa Cruz Biotech- I and II (lysis buffer). Lysates were precleared by rocking incubation at nology) and our anti-N-terminal Mirk antibody. Additional controls 4 °C with 50 l of protein A-agarose (Santa Cruz Biotechnology) for 1 h included use of direct immunofluorescence (using the Zenon labeling and then pelleted in a microcentrifuge at 10,000 g for 20 min to system) and substitution of the primary antibody with nonspecific remove insoluble material. FLAG constructs were isolated using 15 l mouse or rabbit IgG as appropriate. All labeling incubations were of pre-washed EZview Anti-FLAG M2 Affinity Gel (Sigma), which was diluted in 10% normal goat serum/PBS. After three 5-min washes, incubated with the cell lysate at 4 °C for 2 h and then washed five times cells were incubated with a 1:1000 dilution of goat anti-mouse anti- with lysis buffer before analysis. Because both endogenous and FLAG- body conjugated to Alexa Fluor 488 (Molecular Probes). Slides were p21 ran extremely close to the IgG light chain on SDS-PAGE, we used washed two times for 5 min. During single-labeling experiments for the TrueBlot anti-mouse IgG secondary detection system (eBioscience; detection of p21, cells were incubated for 20 min with 2 units of Alexa 1:1000) for Western analysis of immunoprecipitated p21. Fluor 594 phalloidin (Molecular Probes), diluted in 3% BSA/PBS, and Two-dimensional Phosphopeptide Analysis—Chymotrypsin or tryp- washed twice. When using direct labeling, cells were fixed a second sin cleavage and two-dimensional analysis of p21 phosphopeptides were time (4% paraformaldehyde for 5 min) to stabilize the Zenon label carried out as described (23). Following an in vitro kinase assay or after the secondary antibody washes. Nuclei were counterstained immunoprecipitation of phospho-labeled proteins, the substrate was with a 5-min incubation bya2ng/mlsolutionof DAPI. Following resolved by SDS-PAGE and transferred to a nitrocellulose membrane, three 5-min washes, slides were rinsed with distilled water, blotted and the protein band was identified by autoradiography and excised dry, and mounted with Biomedia Gel/Mount. Images were obtained from the membrane. Proteins were digested with sequencing grade as described below. chymotrypsin or trypsin (20 gin200 l of freshly prepared 50 mM Immunofluorescence of Human Muscle Frozen Sections—Anony- NH HCO overnight at 37 °C), and the polypeptides were spotted onto mous samples of flash-frozen human muscle tissue on slides were 4 3 20 20-cm thin layer cellulose plates and separated in the first dimen- obtained from the Department of Pathology, State University of New sion by electrophoresis for 45 min at 1000 V on an HTLE-7002 electro- York, Upstate Medical University, in accordance with institutional phoresis system (C.B.S. Scientific) using buffer containing 2.2% formic review procedures for clinical specimen use. Slides were thawed at acid and 7.8% acetic acid, pH 1.9. Peptides were separated in the second room temperature for 15 min, hydrated with PBS for 15 min, and dimension by chromatography for9hin phosphochromatography buffer then fixed in 4% paraformaldehyde for 1 min at room temperature or (38% 1-butanol, 25% pyridine, and 7.5% acetic acid). Dried plates were ice-cold acetone for 10 min at 0 °C. Slides were rinsed three times for exposed to Eastman Kodak Biomax MS film for 2– 8 days. 5 min with PBS. The sections were then demarcated with a PAP pen Immunodetection—Following treatment as indicated and washing (Zymed Laboratories Inc.), permeabilized for 20 min with 0.2% Triton with phosphate-buffered saline, cells were lysed as described previously X-100 in PBS (wash buffer; used in all subsequent steps), and blocked (23). Depending upon the experiment, 20 –50 g of the cell lysate was with 10% normal goat serum, 0.2% Triton, PBS for 30 min. Mirk was resolved by SDS-PAGE and blotted onto polyvinylidene difluoride mem- visualized with polyclonal antibody to either the N or C terminus of branes. The blots were blocked in Tris-buffered saline containing 0.1% Mirk (both at 1:500 dilution for 1 h), whereas p21 was visualized with Tween 20 (TBST) and 5% nonfat dry milk for 1 h and incubated for 1–2 either the polyclonal C-19 antibody (1:250 for 1 h) or the monoclonal h at room temperature with the primary antibody (1:1000 dilution of F-5 antibody (1:50 for 2 h). Nonspecific IgG isolated from the same Cip1 antibodies to p21 , tubulin, and MyoD). Proteins were subsequently species as the primary antibody diluted to an equivalent mass/con- detected by enhanced chemiluminescence. Immunoblots were scanned centration was used as a negative control. All labeling incubations using a Lacie Silverscanner DTP, and densitometry was performed were diluted in 10% normal goat serum, 0.2% Triton, PBS. After three using the IP Lab Gel program (Scanalytics). 5-min washes, cells were incubated for 30 min with a 1:1000 dilution Localization of GFP-p21 Mutant Constructs—C2C12 myoblasts were of goat anti-IgG antibody Alexa Fluor conjugate (Molecular Probes). plated in Lab-Tek 2-well chamber slides (3.5–5.0 10 cells per well) Sections were washed twice for 5 min. In some experiments, sections and then immediately transfected with GFP-p21-WT, nonphosphory- were incubated for 20 min with 2 units of Alexa Fluor 594 phalloidin latable S153A, or the phosphomimetic S153D (3 g of plasmid DNA and (Molecular Probes), diluted in 3% BSA/PBS, and washed twice. Nuclei 6 l of Lipofectamine 2000 per well). NIH3T3 cells were plated at 1.5 were counterstained by a 5-min incubation with a 2 ng/ml solution of 10 cells per well, incubated overnight before transfection, and then DAPI. Following three 5-min washes, slides were rinsed with distilled transfected with a mixture of 3 g of plasmid DNA, 5 l of PLUS water, blotted dry, and mounted with Biomedia Gel/Mount. Images reagent, and 9 l of Lipofectamine per well. After 24 h of expression, were obtained as described below. cells were washed three times with PBS, fixed in 4% paraformaldehyde Imaging—Monochrome fluorescence images were obtained at 400 for 5 min, washed twice for 5 min with PBS, permeabilized with 0.1% or 1000 (as indicated) using a Diagnostic Instruments SPOT RT Mirk Blocks Apoptosis through Translocation of p21 25791 camera mounted on a Nikon Eclipse E50i fluorescent microscope. SPOT the cultures were undergoing apoptosis. In contrast, the RT software version 4.0.9 was used to pseudocolor the images, adjust amount of YF-Mirk decreased far more, about l0-fold. To de- the RGB histogram, and merge the images. Image manipulation con- termine what percentage of Mirk left after 48 h was endoge- sisted of resetting the zero point of the RGB histogram of the green and nous, another set of transfections was performed. Cell lysates red fluorescent channels to stretch the darker areas of the image yield- were analyzed for expression of ectopic YF-Mirk and for endog- ing a uniform black background consistent with the image viewed through the microscope. Final figures were arranged using Adobe Pho- enous Mirk from the cultures transfected with the vector alone toshop version 7.0. (Fig. 1B, bottom). These blots were exposed for a much longer BrdUrd Incorporation Assay—C2C12 myoblasts were plated over- time to visualize endogenous Mirk, which accounted for ⁄3 of night in Labtek 2-well chamber slides (10 cells per well) and then the Mirk detected in cultures transfected with YF-Mirk at the transfected (2 g of plasmid DNA, 4 l of PLUS, and 4 l of Lipo- 48-h point. Only about 8% of the YF-Mirk was left after 48 h, fectamine per well) with GFP vector, GFP-p21-WT, or the phosphomi- metic GFP-p21-S153D. Cells were transfected in serum-free media for whereas about 50% of wild-type Mirk was retained. Half-life 4 h, and an equal volume of 20% FBS/DMEM was then added. BrdUrd measurements showed that this effect was not because of dif- incorporation was measured using the cell proliferation assay kit (Am- ferences in protein stability (data not shown). Thus both bio- ersham Biosciences). After 24 h of expression, cells were incubated for chemical analysis and immunofluorescence studies demon- 1 h with BrdUrd labeling reagent diluted 1:500 in 10% FBS/DMEM. strated that about 4 –5 times as many C2C12 myoblasts Cells were rinsed twice with PBS and fixed for 30 min at room temper- survived apoptosis in cultures undergoing differentiation if ature with acid alcohol (90% absolute ethanol, 5% glacial acetic acid, 5% water). Cells were then rinsed three times with PBS, blocked for 30 min they expressed ectopic wild-type Mirk than if they expressed in 10% normal goat serum/PBST, and then incubated for1hat room ectopic kinase-inactive YF-Mirk. temperature with a DNase I solution containing a 1:100 dilution of In a recent report (1), we showed that depletion of endoge- anti-BrdUrd antibody (Amersham Biosciences). The denatured GFP nous Mirk by RNA interference in C2C12 myoblasts undergo- protein was labeled by a 30-min incubation with anti-GFP antibody ing differentiation blocked the induction of myogenin and con- (Santa Cruz Biotechnology 8334; 1:500). BrdUrd was visualized using anti-mouse Alexa Fluor 594; GFP was visualized with anti-rabbit Alexa tractile proteins and subsequent myoblast fusion. In these Fluor 488. At least 300 GFP-expressing cells were observed in 8 –10 experiments, we also observed a loss of those cells in which random fields in each of three separate preparations, and the number of Mirk had been depleted, visualized by expression of cotrans- cells labeled for both GFP and BrdUrd was determined using a Green/ fected DsRed by fluorescence microscopy. To test directly Orange V2 filter set (Chroma Technology) that allowed simultaneous whether Mirk played some role in myoblast survival, we de- visualization of both fluorophores. As an additional control, the propor- pleted Mirk in C2C12 cells by RNA interference using RNAi to tion of cells incorporating BrdUrd was also assessed in a set of non- transfected slides stained with DAPI. Combined counts were analyzed Mirk and a mutant RNAi as the control, and we measured cell by the test (Minitab) to determine the significance of differences viability by colony formation after Mirk depletion (Fig. 1, C and between the GFP vector and GFP-p21 constructs. D). C2C12 cells were cotransfected with the neomycin resist- ance gene and with the pSilencer vector encoding either RNAi RESULTS to Mirk, a mutant RNAi, or vector alone, and the extent of Mirk Mirk Promotes Myoblast Survival—Undifferentiated or depletion was analyzed by Western blotting (Fig. 1C). The partly differentiated cells are removed from cultures of fusing Mirk-depleted cells and controls were plated at single cell den- myoblasts by apoptosis. Programmed cell death of 20 –30% of sity and selected in growth medium containing G418 for 3 C2C12 myoblasts is seen during the first 48 h after myoblasts weeks to determine the number of cells capable of proliferation are transferred from growth medium to differentiation medium to form a colony. Colony formation was reduced 75% in the (15). We had observed in earlier studies that the serine/threo- C2C12 cells experiencing Mirk knockdown at the time of plat- nine kinase Mirk afforded colon carcinoma cells increased sur- ing compared with cells transfected with the mutant RNAi or vival capabilites, whereas mutant forms of kinase-inactive the vector control (Fig. 1D). Parallel studies with NIH3T3 cells Mirk did not (20). Mirk is highly expressed in skeletal muscle showed that the cells in colonies that did arise after Mirk cells and in myoblasts, so we speculated that one function of knockdown expressed Mirk (data not shown). Thus these colo- Mirk was to enhance the survival of differentiating myoblasts. nies presumably had arisen either from cells that had not been Mirk is enriched in NIH3T3 cells in G /G and down-regulated 0 1 transfected or from cells that had lost the Mirk RNAi plasmid. when cells are treated with mitogens (10). Likewise, Mirk was These data indicate that knockdown of Mirk levels at the time found to be enriched in C2C12 myoblasts undergoing mitosis C2C12 myoblasts were seeded as single cells had reduced their and in early G (Fig. 1A). Levels of Mirk rapidly increase in viability and thus their ability to give rise to colonies, and C2C12 cells arresting in G /G when placed in DM (1, 11). We 0 1 indicate that Mirk functions as a survival factor. hypothesized that if Mirk mediated cell survival, C2C12 cells We next assayed whether depletion of Mirk would render expressing ectopic wild-type Mirk would survive in greater cells more susceptible to apoptosis in short term experiments numbers during myoblast differentiation than cells expressing without chemical selection of transfected cells (Fig. 1E). We kinase-inactive YF-Mirk. C2C12 cells were transfected with depleted Mirk in C2C12 cells by RNA interference, with a either wild-type Mirk, YF mutant Mirk, or vector alone. The mutant RNAi and the empty vector as controls, and we used constructs were allowed to express for 18 h, and the cells were cotransfected DsRed to mark the transfectants. After 24 h to then transferred to differentiation medium for 0 – 48 h. Expres- induce Mirk knockdown, we transferred the cells to differenti- sion of Mirk or YF-Mirk was determined by immunofluores- ation medium for 1 day, and we identified the apoptotic cells cence analysis. Each construct was expressed in roughly the within the DsRed population by incubation with a fluorescent same percentage of C2C12 cells, 30 –34%, after culture for 18 h conjugate of the permeable caspase inhibitor VAD-fmk. 48% of in growth medium. When cells were cultured in DM for 48 h, cells with Mirk knockdown were undergoing apoptosis com- four times as many cells in the differentiating cultures were pared with 22.9 and 20.1%, respectively, of cells treated with found expressing ectopic wild-type Mirk as expressing kinase- mutant RNAi or vector, a highly significant difference (Fig. inactive YF-Mirk. These data supported the hypothesis that Mirk provided some survival advantage to differentiating myo- 1E). A similar knockdown experiment was performed in a time course format by measuring the abundance of Mirk, and the blasts. In parallel transfections, cell lysates were analyzed for expression of ectopic Mirk and YF-Mirk normalized to -tubu- apoptosis effector activated caspase 3 by Western blotting (Fig. lin by Western blotting (Fig. 1B, top). The amount of wild-type 1F). After 1 day, Mirk levels were depleted to about 50% of Mirk declined about 2-fold by 48 h, during which time cells in control values, and caspase 3 was activated to 160% of control 25792 Mirk Blocks Apoptosis through Translocation of p21 FIG.1. Mirk mediates myoblast survival. A, Mirk is expressed in cycling C2C12 myoblasts. Fluorescence microscopy demonstrates that Mirk expression is greatest in C2C12 cells in mitosis and in cells in early G . Affinity-purified anti-peptide antibody to the Mirk C terminus was conjugated to Alexa Fluor 594 and used to detect endogenous Mirk in C2C12 cells in growth medium. Nuclei were stained with DAPI, and identical fields were merged with SPOT RT software. Similar results were obtained with antibody directed to the unique N terminus of Mirk. B, top, ectopic Mirk maintains the survival of differentiating C2C12 cells. Cells were transiently transfected with wild-type Mirk or kinase-inactive mutant YF-Mirk, allowed to express for 18 h, and then transferred to differentiation medium for up to 48 h. Lysates were analyzed for expression of Mirk Mirk Blocks Apoptosis through Translocation of p21 25793 values. After 2 days, there was less Mirk knockdown and less activation of caspase 3. After 3 days, there was no Mirk knock- down and no activation of caspase 3 (Fig. 1F). Thus, depletion of Mirk protein correlated with increased abundance of acti- vated caspase 3 in multiple experiments and led to loss of cells by apoptosis. In another experiment, we measured the effect of the caspase inhibitor benzyloxycarbonyl-VAD-fmk on differen- tiation-induced apoptosis, as assayed by the TUNEL reaction FIG.2. Mirk phosphorylates transfected FLAG-p21 in vivo. Top (Fig. 1G). The caspase inhibitor blocked apoptosis to a similar panel, autoradiogram after SDS-PAGE of in vivo phosphorylation of extent, 40% in control cultures, 50% in mutant RNAi-treated wild-type FLAG-p21 cotransfected into NIH3T3 cells with either wild- cultures, and 33% in cultures where Mirk knockdown occurred. type or kinase-inactive Mirk and allowed to coexpress for 24 h. After 4 h of labeling, FLAG-p21 was immunoprecipitated (IP) with anti-FLAG These studies employing either Mirk knockdown or Mirk over- M2 antibody, and the immunoprecipitates were resolved on SDS-PAGE expression demonstrate that Mirk has anti-apoptotic survival with FLAG-p21 migrating at the expected position. FLAG-p21 was functions in C2C12 myoblasts. phosphorylated only in the cotransfections with wild-type Mirk. The Mirk Phosphorylates p21 in Vivo—The CDK inhibitor nonspecific doublet seen at the top of each lane serves as the loading Cip1 control. Bottom panel, Anti-FLAG Western blot (WB) showed that equal p21 has been implicated in the control of apoptosis during amounts of FLAG-p21 were immunoprecipitated. The upper band rep- myoblast fusion (15), possibly through stabilization by ERKs resents light chain, and the lower band represents FLAG-p21. There Kip1 (27). Mirk phosphorylates another CDK inhibitor p27 in appears to be a slight decrease in mobility in the p21 phosphorylated by vivo (10), so we hypothesized that Mirk might also phosphoryl- Mirk/dyrk1B. Cip1 ate the closely related p21 . A wild-type p21 construct with an N-terminal FLAG epitope tag was cotransfected into ilar data were obtained when Mirk was coexpressed with p21 in NIH3T3 fibroblasts with either wild-type or kinase-inactive C2C12 myoblasts (data not shown). The many functions of p21 Mirk and allowed to coexpress for 24 h. After4hof labeling rely on its association with a variety of proteins, including with [ P]orthophosphate, FLAG-p21 was immunoprecipitated cyclins, CDKs, PCNA, and others, which associate with p21 with anti-FLAG M2 antibody, and the immunoprecipitates within specific domains. It was essential to determine the phos- were resolved by SDS-PAGE and analyzed by autoradiography phorylation site of Mirk in p21 in order to be able to postulate (Fig. 2). FLAG-p21 migrated at the expected molecular weight the biological effect of this phosphorylation. For initial studies, but was phosphorylated only in the cotransfections with wild- we measured the capacity of Mirk to phosphorylate in vitro a type Mirk. FLAG-p21 was not labeled when coexpressed with series of GST-p21 constructs that were mutated to the non- kinase-inactive KR-Mirk or vector (Fig. 2, upper panel). West- phosphorylatable alanine residue at known phosphorylation ern blotting for the FLAG epitope (Fig. 2, lower panel) demon- sites: Ser-130 (the p38 MAPK site (23)), Thr-57 (the GSK3 site strated that equal amounts of FLAG-p21 were immunoprecipi- (28)), and Ser-146 (a protein kinase C site (29) and one of two tated. The lower band of the doublet was FLAG-p21, and the Akt sites (30)). Mirk phosphorylated each of these constructs to upper band was light chain. The FLAG-p21 protein exhibited a similar degree (data not shown), indicating that none of these retarded mobility only when coexpressed with wild-type Mirk sites was the Mirk phosphorylation site. (Fig. 2, center lane), consistent with its phosphorylation. Sim- We then compared human and mouse p21 sequences for and -tubulin by Western blotting. Bottom, to determine whether the amount of Mirk remaining in cells expressing ectopic YF-Mirk after 48 h was either ectopic or endogenous Mirk, cells were transiently transfected with kinase-inactive mutant YF-Mirk, or vector, allowed to express for 18 h, and then transferred to differentiation medium for up to 48 h. Lysates were analyzed for expression of Mirk and -tubulin by Western blotting. The YF-Mirk and vector blots were exposed for the same amount of time to allow direct comparison. One of duplicate experiments with similar results is shown. C, depletion of Mirk in cycling myoblasts by RNA interference reduced C2C12 cell viability in colony formation assays. Cells were plated at 5 10 per 60-mm dish, three dishes per point for 24 h, and then were transfected for4hin serum-free medium with 5 g of pSilencer DNA encoding either RNAi to Mirk (RNAi), a mutant RNAi (Mt), or vector (Vc) together with 0.5 g of neomycin resistance plasmid, using 10 l each of Lipofectamine and PLUS reagent. Fetal bovine serum was then added to 10% to maintain cell viability during expression. Cells were incubated for 20 h before trypsinization and then lysed for Western blotting (WB). Ct, control, cross-reacting band showing similar loading. D, same experiment as in C, but transfected cells were reseeded at the same cell density and selected in 400 g/ml G418 for 3 weeks. Mean colony number S.E. is shown. E, Mirk knockdown increases apoptosis measured by fluorescence microscopy using an activated caspase 3 inhibitor. C2C12 myoblasts were plated overnight in LabTek 2-well chamber slides (2 10 cells per well) and then cotransfected with 0.5 g of pDsRed and 1.5 g of pSilencer vector (Vec), mutant si or RNAi to Mirk (4 l of PLUS and 4 l of Lipofectamine per well). Cells were transfected in serum-free media for 4 h, and an equal volume of 20% FBS/DMEM was then added. Following 24 h of expression, cells were incubated with differentiation media for 24 h. Activated caspase was then labeled by a 30-min incubation with 10 M CaspACE FITC-VAD-fmk in situ marker (Promega) diluted in DM. This is a fluorescent conjugate of the permeable caspase inhibitor VAD-fmk that was used as an in situ marker for apoptosis. Slides were washed and mounted with Biomedia GelMount. At least 200 DsRed expressing cells were observed in each of four separate preparations, and the number of cells labeled for both DsRed and the FITC-VAD-fmk marker was determined using a Green/Orange V2 filter set (Chroma) that allowed simultaneous visualization of both fluorophores. Combined counts were analyzed by the test to determine the significance of differences between the RNAi constructs. Efficiency of cotransfection was determined to be greater than 85% in parallel experiments using a combination of GFP and DsRed. The number of cells scored per assay conditions is shown. F, knockdown of endogenous Mirk by RNA interference inhibits the activation of caspase 3 in differentiating C2C12 cells. C2C12 cells (5 10 per 60-mm dish) were transfected with pSilencer plasmid encoding RNAi to Mirk (RNAi) or mutant RNAi (Mt) for 24 h and then switched to differentiation medium for 1–3 days, as noted. The relative abundance of Mirk/tubulin and of activated caspase 3/tubulin in lysates was determined by Western blotting on the upper and lower sections of same blot that was cut in half and then reunited for the exposure, with tubulin used as the loading control. The relative abundance of either protein in cells treated with RNAi to Mirk was then normalized to their relative abundance in cells treated with mutant RNAi. Mean S.E. (if greater than 5%) is shown from data from three experiments. G, apoptosis induced by differentiation medium or Mirk knockdown blocked to a similar extent by a caspase inhibitor. C2C12 myoblasts were plated, transfected, and cultured as in E, except they were cotransfected with 0.5 g of pEGFP. A parallel set of cultures was incubated with DM containing 20 M of the cell-permeable caspase inhibitor benzyloxycarbonyl-VAD-fmk (Promega). After 24 h in DM, DNA breaks in apoptotic cells were labeled with tetramethylrhodamine-dUTP by terminal deoxynucleotidyltransferase-mediated in situ end labeling (TUNEL) using the in situ cell death detection kit (Roche Applied Science). At least 300 GFP-expressing cells were observed in each of four separate preparations, so that an average of 1250 cells were scored per point. The number of GFP-expressing cells labeled with the TUNEL marker was determined using a Green/Orange V2 filter set (Chroma) that allowed simultaneous visualization of both fluorophores. Combined counts were analyzed by the test. Efficiency of cotransfection was determined to be greater than 85% in parallel experiments using a combination of GFP and DsRed. 25794 Mirk Blocks Apoptosis through Translocation of p21 conserved threonine or serine residues within three or four residues of an arginine, the usual site of phosphorylation by Mirk, and we mutated each of these six amino acids to the nonphosphorylatable alanine residue as follows: S2A, S15A, S27A, T97A, T145A, S153A. Mutation of only one site, serine 153 to alanine, caused a reduction in in vitro phosphorylation by Mirk (data not shown). We confirmed that Mirk phospho- rylated p21 at Ser-153 in vitro by performing two-dimensional phosphopeptide mapping following digestion of the phosphoryl- ated p21 protein by trypsin. Three phosphopeptides were de- rived from wild-type p21 after in vitro phosphorylation by Mirk, but only one phosphopeptide was seen after phosphoryl- ation of the mutant p21-S153A construct (Fig. 3A). The slower migrating peptide on chromatography (Fig. 3A, long arrow) was probably a partial digestion product. The tryptic peptide containing serine 153 consists of amino acids 143–154 and is flanked by two KRR sequences, so trypsin could cleave at multiple points within both of these regions. Alternative cleav- age may also have been promoted by steric hindrance resulting from phosphorylation of Ser-153. Supporting this interpreta- tion was the observation that the relative abundance of the two peptides varied in different experiments. However, the same general pattern was seen in three separate experiments, dem- onstrating that Mirk phosphorylates p21 at serine 153. In Vivo Phosphorylation of FLAG-p21 by Cotransfected Mirk Occurs at Ser-153 in Differentiating Myoblasts—In vivo phos- phorylation is known to be more specific than in vitro phospho- The peptide identified by the longer arrow may be a partial digestion product containing Ser-153, due to steric interference of the enzyme by the phosphate group. Similar data were seen in two additional peptide maps. B, Mirk phosphorylates p21 at Ser-153 in vivo as shown by peptide mapping with chymotrypsin. Wild-type FLAG-p21, mutant p21-S153A, and mutant p21-S130A were each cotransfected with wild- type Mirk into C2C12 cells. Constructs were allowed to express over- night, and cells were then placed in myoblast differentiation medium for24h,thelast4hof which cells were labeled with [ P]orthophos- phate. In one dish transfected with wild-type p21, 10 M SB203580 was added at the same time as the label. FLAG-p21 was immunoprecipi- tated with anti-FLAG M2 antibody; the immunoprecipitates were di- gested with chymotrypsin and then subjected to two-dimensional pep- tide mapping. The p21-S153A mutant incorporated about 40% as much label as wild-type p21 when normalized to the total amount of p21 immunoprecipitated. Chymotrypsin was used because it yields a sim- pler map with a broader distribution of peptides. The peptide indicated by the arrow is lost in the p21-S153A mutant and is the presumed 152–159-amino acid peptide. C, depletion of endogenous Mirk by RNA interference blocks the phosphorylation of transfected wild-type p21 but not p21-S153A mutant at the Mirk phosphorylation site. Cells were plated at 5 10 per 60-mm dish, cultured for 16 h, and then were transfected for4hin serum-free medium with 2.5 g of either FLAG- p21 or FLAG-p21-S153A, and 2.5 g of pSilencer DNA encoding either RNAi to Mirk (RNAi) or vector. As controls, FLAG-p21 was cotrans- fected with either wild-type Mirk (M) or kinase-inactive YF-Mirk (YF). Fetal bovine serum was then added to 10% to maintain cell viability during overnight expression, and cells were switched to Opti-MEM differentiation medium for 24 h, with 325 Ci of [ P]orthophosphate added to each dish for the last 4 h following1hin phosphate-free medium. Anti-FLAG epitope immunoprecipitates were separated by SDS-PAGE, and the FLAG-p21 bands were detected by autoradiogra- phy (top lanes) and then by Western blotting for FLAG. The amounts of p21 and Mirk in the lysates were determined by Western blotting (lower 2 bands). D, SB203580 inhibits myoblast differentiation as measured by the level of expression of the myogenic regulatory factor myogenin that controls the differentiation program. C2C12 cells were placed in differ- entiation medium 10 M SB203580 for 1 and 2 days, and the abun- dance of myogenin was determined by Western blotting. Ct, cross- reacting protein used as a loading control. E, relative stability of FIG.3. Mirk phosphorylates p21 at Ser-153 in vitro and in mutant p21 constructs. C2C12 cells were transfected with either mu- differentiating myoblasts. A, Mirk phosphorylates p21 at Ser-153 in tant FLAG-p21-S153D, FLAG-p21-S153A, or wild-type FLAG-p21, and vitro as shown by peptide mapping with trypsin. Wild-type (Wt) and constructs were allowed to express for 24 h. Cycloheximide at 20 g/ml mutant p21-S153A constructs were phosphorylated in vitro by purified was added to each culture, and FLAG-p21 and tubulin abundances recombinant Mirk, digested with trypsin, and then subjected to two- were determined at the indicated times by Western blotting and nor- dimensional peptide mapping. The peptide containing Ser-153 and con- malized to the 0 time values. Mean S.E. is shown from two separate sisting of amino acids 143–154 is shown by a short arrow in both panels. experiments. Mirk Blocks Apoptosis through Translocation of p21 25795 TABLE I rylation, especially on a substrate such as p21, which exists in BrdUrd incorporation in C2C12 cells expressing GFP-p21 constructs vivo in several different complexes, and is only found as a free None Vector p21(WT) p21(S153D) molecule immediately after synthesis (31). In fact, in our pre- vious study, p38 MAPK phosphorylated p21 strongly at Thr-57 GFP 910 973 922 935 and weakly at Ser-130 in vitro, but in colon carcinoma cells p38 GFP BrdUrd 272 292 23 18 % S phase 29.9 30.0 2.5 1.9 MAPK only phosphorylated p21 at Ser-130 (23). Mirk was (p 0.0001) (p 0.0001) cotransfected into proliferating myoblasts with either wild-type FLAG-p21, the Mirk site mutant FLAG-p21-S153A, or the p38 site mutant FLAG-p21-S130A. The cells were then switched to at least within the time frame of these experiments. Mutation differentiation media for 24 h and labeled with [ P]orthophos- of p21 at the p38 MAPK site of Ser-130 and treatment of cells phate for the last 4 h. The immunoprecipitated p21 constructs with the p38 inhibitor SB203580 did not eliminate any of the were digested with chymotrypsin before phosphopeptide map- three phosphorylated peptides. In addition, the Ser-153-con- ping. Trypsin digestion yields 17 fragments from p21, whereas taining peptide was phosphorylated in the FLAG-p21-S130A chymotrypsin yields 11, and only 6 of these peptide fragments mutant construct to a similar extent as in wild-type p21, dem- contain either serine or threonine residues, resulting in a much onstrating that phosphorylation of p21 at Ser-130 is not an simpler pattern. Additionally, the position of the peptide con- essential prerequisite for phosphorylation by Mirk (Fig. 3B). taining the p38 MAPK phosphorylation site on p21 is readily The SB203580 preparation used in these experiments was ac- identifiable in the chymotrypsin map (23). Mirk was found to tive because exposure of C2C12 cells in DM for 1 or 2 days to phosphorylate p21 at Ser-153 in differentiating myoblasts, in this drug prevented induction of myogenin, an essential myo- accordance with Mirk’s in vitro phosphorylation of p21. Muta- genic regulatory factor, whose transcription is mediated by the tion of p21 to S153A at the Mirk phosphorylation site blocked p38 MAPK substrate MEF2 (Fig. 3D). Treatment of differenti- phosphorylation in vivo. FLAG-p21 was phosphorylated on ating C2 myoblasts with SB203580 at 5 M for 48 h has been three phosphopeptides in vivo, whereas FLAG-p21-S153A was reported to block the kinase activity of p38 on the exogenous phosphorylated on only two phosphopeptides (Fig. 3B). The substrate myelin basic protein but not to block the phosphoryl- peptide containing Ser-153 is indicated by an arrow in each ation of p38 (35). Other investigators as well as ourselves (data panel of Fig. 3B. This peptide consists of amino acids 152–159 not shown) have also noted this discrepancy. However, our and contains only one serine and no threonines. Mutation of studies do not entirely depend on the use of the inhibitor. There this peptide at serine 153 to alanine blocked its in vivo phos- was no loss in phosphorylation of the p38 site mutant, p21- phorylation. Therefore, p21 is phosphorylated at Ser-153 in S130A, during myoblast differentiation because the peptide differentiating myoblasts by Mirk. containing this mutant sequence was as equally phosphoryl- Knockdown of endogenous Mirk by RNAi blocked phospho- ated as the wild-type peptide (Fig. 3B). Therefore, we could rylation of wild-type FLAG-p21, but not FLAG-p21-S153A, mu- detect no phosphorylation of p21 by p38 MAPK during this tant at the Mirk phosphorylation site (Fig. 3C), confirming that initial period of myogenic differentiation. Mirk phosphorylates p21 at Ser-153 in differentiating myo- The phosphorylation of p21 by p38 MAPK at Ser-130 in- blasts in vivo. C2C12 cells were transfected with either wild- creased the stability of p21 in colon carcinoma cells (23). How- type or mutant p21 together with the pSilencer plasmid for ever, phosphorylation of p21 by Mirk did not stabilize p21 in Mirk RNAi or the vector alone. Parallel cultures were trans- C2C12 cells. In fact, p21-S153D was slightly less stable than fected with wild-type p21 and either wild-type Mirk or kinase- wild-type p21, as demonstrated by cycloheximide arrest exper- inactive YF-Mirk. Cells in DM were metabolically labeled with iments (Fig. 3E). Therefore, neither Mirk nor p38 MAPK sta- [ P]orthophosphate. The p21 constructs were immunoprecipi- bilizes p21 during the initial stages of myoblast differentiation. tated by their FLAG epitopes, and the extent of incorporation of Furthermore, at this stage in differentiation, when Mirk acts the label was determined by autoradiography after SDS-PAGE. as a survival factor for myoblasts, only Mirk, not p38 MAPK, Ectopic Mirk phosphorylated FLAG-p21 in vivo, whereas the phosphorylates p21. kinase-inactive YF-Mirk did not (Fig. 3C, 1st 2 lanes), and Phosphomimetic p21-S153D Is as Effective as Wild-type p21 served as the positive and negative controls, respectively. En- in Blocking Cell Cycle Progression—Ectopic expression of p21 dogenous Mirk in the differentiating C2C12 cells phosphoryl- has been shown by others (16) to decrease the percentage of ated wild-type p21 but not mutant p21-S153A (Fig. 3C, 3rd and myoblasts in S phase as assayed by BrdUrd incorporation 4th lanes), although similar amounts of p21 were immunopre- using fluorescence microscopy. Similarly, we found that tran- cipitated. In the cultures in which Mirk knockdown was accom- sient expression of GFP-p21 decreased the fraction of C2C12 plished (Fig. 3C, last 2 lanes), neither wild-type Mirk nor the myoblasts expressing GFP that showed BrdUrd incorporation p21-S153A mutant was phosphorylated much above back- from 30% in cells transfected with vector alone to 2.5% in cells ground levels. Therefore, endogenous Mirk phosphorylates p21 expressing GFP-p21 (Table I). Transient expression of the at Ser-153 in vivo in differentiating myoblasts. phosphomimetic GFP-p21-S153D caused a similar arrest of cell Mirk and p38 MAPK interact in vivo. Mirk is activated by growth, with only 1.9% of cells expressing this construct also the p38 MAPK kinase MKK3 (12), and p38 MAPK can seques- positive for BrdUrd. Thus, p21 phosphorylated by Mirk re- ter Mirk in vivo (13). Thus, we questioned whether phospho- mained capable of mediating growth arrest. These results were rylation of p21 by p38 MAPK occurred in differentiating myo- consistent with the known control of DNA replication by p21 blasts, as it does in colon carcinoma cells (23), and if so, and the retinoblastoma protein in terminally differentiated whether it was an essential prerequisite for phosphorylation of muscle cells (36). p21 by Mirk. In one set of cultures in the peptide mapping Phosphomimetic p21-S153D Translocates to the Cytoplasm— studies (Fig. 3B), 10 M SB203580 was added to inhibit p38 Serine 153 is within the nuclear localization signal of p21 and MAPK. During myoblast differentiation, p38 MAPK is acti- within its PCNA binding domain (reviewed in Ref. 37), so we vated and in turn activates the myogenic regulatory factors predicted that phosphorylation at this residue would alter both MEF2A, MEF2C, and MyoD (32–34). However, the in vivo the localization of p21 and its capacity to block DNA synthesis. peptide mapping studies (Fig. 3B) show that p38 MAPK does In the current study we have concentrated on the localization of not appear to phosphorylate p21 in differentiating myoblasts, p21. We transiently transfected GFP-p21-WT, the phosphomi- 25796 Mirk Blocks Apoptosis through Translocation of p21 TABLE II Localization of GFP-p21 constructs in C2C12 cells and NIH3T3 cells Cycling C2C12 myoblasts WT S153A S153D Nucleus cytoplasm 246 200 998 Nucleus only 1022 1082 278 % nucleus cytoplasm 19.4 15.6 78.3 (p 0.02) (p 0.0001) NIH3T3 fibroblasts WT S153A S153D Nucleus cytoplasm 164 141 420 Nucleus only 453 488 209 % nucleus cytoplasm 26.6 22.4 66.8 (p 0.0001) trials had demonstrated that GFP-p21 is always localized in the nucleus in this system, we used a one-tailed analysis to compare the proportion of transfected cells expressing the GFP constructs in both the cytoplasm and the nucleus to the pro- portion of cells expressing GFP-p21 exclusively in the nucleus. GFP-p21-WT was found in the cytoplasm in only 19.4% of myoblasts. Four times as many cells, 78.3%, expressed the Mirk-phosphomimetic GFP-p21-S153D construct in the cyto- plasm. This gave a highly significant (p 0.0001) difference in localization. In contrast, the nonphosphorylatable GFP-p21- S153A construct remained localized in the nucleus in myo- blasts, with only 15.6% of the cells expressing this construct in the cytoplasm, giving a statistically significant value of p 0.02 compared with wild-type p21. Similar results were ob- tained with NIH3T3 cells (Table II). Wild-type p21 was found in the cytoplasm of only 26.6% of transfected fibroblasts, whereas over twice as many transfected fibroblasts expressed the Mirk-phosphomimetic GFP-p21-S153D construct in the cy- toplasm. This was also a highly significant difference in local- ization (p 0.0001). The nonphosphorylatable GFP-p21-S153A construct remained localized in the nucleus in fibroblasts, with only 22.4% of the cells containing this construct in the cyto- plasm, a value that was not significantly different from cells FIG.4. Fluorescence microscopy demonstrated that the Mirk expressing wild-type p21. These data clearly demonstrate that phosphorylation site phosphomimetic mutant p21-S153D is mutation of p21 to mimic phosphorylation by Mirk caused a found in both the cytoplasm and the nucleus, whereas wild-type p21 and the nonphosphorylatable p21-S153A are localized ex- much larger proportion of the p21 to localize in the cytoplasm clusively in the nucleus in the majority of cells. GFP-p21-wild- in both myoblasts and fibroblasts. Mutation of an additional type, GFP-p21-S153A which is not phosphorylated by Mirk, and GFP- serine in the nuclear localization signal (Ser-160 to S160A or p21-S153D, the Mirk site-phosphomimetic construct, were transiently S160D) did not affect the cellular distribution of GFP-p21 (data expressed in C2C12 cells (A, 400 magnification) and in NIH3T3 cells (B, 1000 magnification). Nuclei were stained with DAPI, and identical not shown). fields were photographed for GFP and DAPI. Images were merged with Ectopic Mirk Translocates GFP-p21 to the Cytoplasm SPOT RT software. Scale bars 50 m. through Phosphorylation at Ser-153—NIH3T3 fibroblasts were transfected with ectopic wild-type Mirk or kinase-inactive YF- metic GFP-p21-S153D, and the nonphosphorylatable GFP-p21- Mirk and either wild-type GFP-p21 or GFP-p21-S153A. The S153A into cycling C2C12 myoblasts (Fig. 4A) and cycling cells expressing exogenous p21 and exogenous Mirk constructs NIH3T3 fibroblasts (Fig. 4B). NIH3T3 cells were used to de- were visualized by immunofluorescence. Cells expressing both termine the generality of these observations because Mirk is constructs were visualized by using a Green/Orange V2 filter widely distributed. Wild-type p21 and the nonphosphorylatable set, and the percentage of such cells in which p21 was pancel- GFP-p21-S153A construct were predominantly localized in the lular (versus restricted solely to the nucleus) was twice as large nucleus of both cell types. Nuclear localization was confirmed as the controls (Fig. 5). analysis showed this to be a highly by counterstaining the nuclei with DAPI. The GFP and the blue significant difference (p 0.0001). Expression of elevated lev- fluorescent DAPI were merged in the last lane of Fig. 4B and els of ectopic Mirk was sufficient to make the majority of showed overlap of each fluorochrome. In marked contrast to wild-type p21 pancellular, while having no effect on the p21 wild-type p21, the phosphomimetic GFP-p21-S153D construct mutant at the Mirk site (S153A). Moreover, the p21 transloca- was found in the cytoplasm as well as the nucleus in both tion was dependent on the kinase activity of Mirk. C2C12 myoblasts and NIH3T3 cells (Fig. 4). The GFP-p21- More Wild-type p21 Is Found in the Cytoplasm When Mirk Is S153D was found distributed throughout the entire cell, allow- Induced during Myoblast Differentiation—The localization ing visualization of cytoplasmic extensions. studies described above (Figs. 4 and 5) were performed in These observations were quantitated by counting 3824 cycling myoblasts. We next transfected the same three GFP- transfected myoblasts and 1875 transfected fibroblasts (Table p21 constructs into C2C12 cells, and we then stimulated them II) and subjecting the data to the test. Because preliminary to differentiate. Only the fused myoblasts were examined, and Mirk Blocks Apoptosis through Translocation of p21 25797 cells are shown in the top 2 rows). When myoblasts were cul- tured for 2 days in differentiation medium, however, p21 was found in the cytoplasm as well as the nucleus in fused myo- blasts (Fig. 7, bottom two rows). Note in particular in the Merge column of Fig. 7, the large myotubes that are stained with actin to delineate the entire cell body and with DAPI to delineate nuclei. 11 nuclei were found in the myotube in the 3rd row, whereas four nuclei were found in the myotube in the 4th row. Both of these myotubes show extensive localization of endoge- nous p21 in their cytoplasm (Fig. 7, p21 column, rows 3 and 4). These data clearly demonstrate that localization of endogenous p21 in the cytoplasm occurs as a natural part of myoblast differentiation. Thus the increase in concentration of the exog- enous FLAG-p21 within the cytoplasm of differentiating myo- FIG.5. Overexpression of Mirk causes a portion of the wild- tubes (Fig. 6) is reflected in a similar cytoplasmic enrichment of type p21 population to translocate to the cytoplasm in a Mirk the endogenous p21. kinase-dependent manner but cannot translocate p21 mutated Endogenous p21 Is Found within the Cytoplasm of Adult at the Mirk phosphorylation site. NIH3T3 fibroblasts were plated overnight in Labtek 2-well chamber slides (1.5 10 cells per well) and Human Muscle—p21 is known to play critical roles in early then transfected (2 g of plasmid DNA and 4 l of Lipofectamine 2000 muscle differentiation (18) and in the maintenance of adult per well) with GFP-p21(p21) or the nonphosphorylatable GFP-p21(p21- muscle tissue through the muscle progenitor (satellite) cell S153A), together with either wild-type (M) or kinase inactive (YF-M) population (19). However, there do not appear to be any exten- Mirk. Cells were transfected in DMEM containing 10% bovine calf serum. After 24 h of expression, cells were fixed with 4% paraformal- sive reports describing the presence or function of p21 in nor- dehyde, and the GFP-p21 protein was labeled by a 30-min incubation mal mature muscle. Therefore, we performed SAGE analysis with mouse anti-GFP monoclonal antibody (Santa Cruz Biotechnology using the NCBI/GEO data base, and we found that p21 is 8334; 1:500), followed by anti-mouse Alexa Fluor 488. Mirk was visu- alized by using rabbit polyclonal antibody to the Mirk C terminus at expressed to a higher extent in muscle biopsies from young 1:500, followed by anti-rabbit Alexa Fluor 594. An average of 1050 individuals, aged 21–31 years old, than in biopsies from most GFP-p21 expressing cells were observed for each transfectant. Cells older patients, aged 62–77 (GDS156), but that all skeletal were scored in 8 –10 random fields in each of three separate prepara- muscle biopsies contained p21 mRNA. We were unable to de- tions, and the number of cells labeled for both GFP-p21 and transfected tect p21 in formalin-fixed paraffin sections of human muscle Mirk was determined using a Green/Orange V2 filter set (Chroma) that allowed simultaneous visualization of both fluorophores. Combined even after antigen retrieval with citrate, EDTA, or proteolytic counts were analyzed by the test (Minitab) to determine the signif- digestion. This result is not unexpected given that p21 is nor- icance of differences between conditions. The total number of cells mally found in complexes, where the epitope may not be acces- scored per condition is labeled on each bar. sible to the antibody, particularly after formalin fixation. We therefore performed immunofluorescence on frozen sections of cells were stained for actin so the myotubes would be unam- adult human muscle. We were able to detect p21 in adult biguous. As predicted, in each of four experiments wild-type muscle using the C-19 antibody at a dilution of 1:200, followed GFP-p21 was also found in the cytoplasm of myotubes, whereas by goat anti-rabbit Alexa Fluor 594 at 1:500 (Fig. 8). The the nonphosphorylatable GFP-p21-S153A remained restricted distribution of p21 in the adult muscle fibers paralleled our to the nuclei of the multinucleated myotubes (Fig. 6). The observations of p21 localization in myotubes. p21 remained phosphomimetic GFP-p21-S153D served as the positive control concentrated in the nucleus but was distributed throughout the and partitioned into both cytoplasm and nucleus as expected. myofibrils. We were also able to see this pattern using the F-5 Thus p21 was shifted from the nucleus into the cytoplasm by anti-p21 antibody, thus demonstrating the specificity of our the presence of elevated levels of endogenous Mirk in differen- immunolabeling results (data not shown). Therefore, examina- tiating myotubes. tion of human skeletal muscle tissue confirmed that endoge- Changes in the Localization of Endogenous p21 during Dif- nous p21 partitioned into the cytoplasm as a natural part of ferentiation of C2C12 Myoblasts—The data thus far confirmed muscle differentiation. that a significant proportion of exogenous p21 is localized in the Phosphomimetic p21-S153D Is More Effective than Wild-type cytoplasm of differentiating myoblasts following phosphoryla- p21 in Blocking Caspase 3 Activation—Caspase 3 is the pri- tion by Mirk. We next investigated the localization of endoge- mary effector caspase in apoptosis. Caspase 3 is activated by nous p21 during differentiation of myoblasts. C2C12 myoblasts cleavage of its precursor, procaspase 3. C2C12 myoblasts were were cultured for 1 day in growth medium (GM), and then transiently transfected with either wild-type FLAG-p21, the switched to differentiation medium (DM). After 24 h in GM or phosphomimetic FLAG-p21-S153D, the nonphosphorylatable 48 h in DM, cells were fixed in paraformaldehyde, and endog- FLAG-p21-S153A, or vector alone. The constructs were allowed enous p21 was then visualized with the monoclonal antibody to express overnight; the cells were then cultured in differen- F-5. This antibody was raised against full-length p21, so anti- tiation medium for 16 h, and the amount of active caspase 3, body binding should not be affected by phosphorylation at released by cleavage, was determined by Western blotting. Ser-153 in the C terminus (Fig. 7). As an additional control, p21 Wild-type p21 caused a small 10% inhibition in the amount of was labeled in separate experiments using the polyclonal an- activated caspase 3, whereas the Mirk phosphomimetic p21- tibody C-19 (data not shown). The anti-p21 antibody was de- S153D construct blocked caspase 3 activation five times as tected with goat anti-mouse secondary antibody conjugated to much (Fig. 9). In contrast, little inhibition by the nonphospho- green fluorescent Alexa Fluor 488. Cells were also incubated rylatable construct was observed. In addition, the phosphomi- with Alexa Fluor 594 coupled to phalloidin to detect actin and DAPI for nuclear staining. All of the fields were merged to form metic p21 construct coimmunoprecipitated with ASK1 (data not shown), one upstream activator of this caspase cascade. the composite shown in the right-most column Fig. 7. Endogenous p21 was exclusively localized in the nucleus in ASK1 has been shown to be sequestered by wild-type p21 in proliferating myoblasts in growth medium. The signals for p21 other studies (22). Therefore, one mechanism by which Mirk and for DAPI completely coincided (Fig. 7, two fields of C2C12 blocks apoptosis in myoblasts is through phosphorylation of 25798 Mirk Blocks Apoptosis through Translocation of p21 FIG.6. When Mirk is up-regulated as a part of the myoblast differentia- tion program, both wild-type p21, as well as the Mirk-phosphorylation site phosphomimetic mutant p21-S153D, are translocated to the cytoplasm as shown by fluorescence microscopy. In contrast, the nonphosphorylatable p21-S153A is localized exclusively in the nuclei of the multinucleated myotubes. GFP-p21-wild-type, GFP-p21-S153A, and GFP-p21-S153D were transfected into C2C12 cells, which were then allowed to differentiate for 3 days. Similar data were seen in each of four experiments. FIG.7. Endogenous p21 is exclu- sively nuclear in cycling C2C12 cells in growth medium, but a portion of the endogenous p21 protein is trans- located to the cytoplasm when C2C12 cells are treated for 48 h with differ- entiation medium. Endogenous p21 was visualized with the F-5 monoclonal antibody and goat anti-mouse antibody conjugated to Alexa Fluor 488; the nuclei were stained with DAPI, and actin within the entire cell body was visualized with phalloidin conjugated to Alexa Fluor 594. Identical fields were photographed for each fluorochrome (1st three rows) and then merged with SPOT RT software. Identical results were obtained with the C-19 polyclonal antibody. Scale bar 50 m. FIG.8. p21 localizes in both the cy- toplasm and nucleus of adult skeletal muscle. Frozen sections of human mus- cle were analyzed for the localization of p21 by immunofluorescence using the polyclonal C-19 antibody (1:200) and goat anti-rabbit IgG Alexa Fluor 594 (1:500); nuclei were counterstained with DAPI. Scale bars 50 m. p21. A subpopulation of the p21 molecules is then retained in expressed for only about 1 day in growth medium (Fig. 10A) the cytoplasm where it blocks apoptosis through inhibition of and then were lost. We used this transient expression to deter- the activation of caspase 3. mine whether the phosphomimetic p21 construct could act as a Transient Expression of Phosphomimetic p21 Aids Myoblast survival factor in myoblasts at the time of plating at very low Survival—C2C12 cells were transiently transfected with either cell density for colony formation assays (Fig. 10B). The same wild-type FLAG-p21, the phosphomimetic FLAG-p21-S153D, transfection protocol was used, except that a vector control was or the nonphosphorylatable FLAG-p21-S153A, and the length included. Transient expression of the phosphomimetic p21- of time each construct was expressed was determined (Fig. S153D construct, and to a lesser extent the wild-type p21, 10A). After 24 h of transfection and expression, the cells were increased myoblast colony formation (Fig. 10B), a reflection of placed in growth medium for 0 –13 days. All of the constructs increased survival during the initial day on which cells were Mirk Blocks Apoptosis through Translocation of p21 25799 FIG.9. A p21 construct, phosphomimetic at the Mirk phospho- rylation site, is twice as effective as wild-type p21 in blocking the activation of caspase 3. C2C12 myoblasts were transfected with FLAG-p21 wild-type, FLAG-p21-S153A, FLAG-p21-S153D, or vector for 24 h, cultured in differentiation medium for 16 h, and then analyzed by SDS-PAGE and Western blotting for activated caspase 3, and nor- malized by Western blotting for tubulin and the expression of exoge- nous FLAG-p21 by Western blotting for FLAG. Mean S.D. shown of two experiments. plated. Transient expression of p21-S153D for only 1–2 days (Fig. 10A) thus inhibits caspase 3 activation during the initial plating period when cells are at very low density and suscep- tible to apoptosis. Loss of expression of the p21 construct after this period allows the cells to re-enter the cell cycle and grow to form a discernible colony. In contrast, the phosphorylation site mutant p21-S153A had no survival effect compared with the vector (Fig. 10B). Thus phosphorylation of p21 by Mirk enables p21 to oppose apoptosis and to enhance myoblast survival. FIG. 10. Transient expression of a p21 construct, phosphomi- DISCUSSION metic at the Mirk phosphorylation site, increases the viability In the current study we have demonstrated that the kinase of C2C12 cells. C2C12 myoblasts were transfected with FLAG-p21 Mirk mediates cell survival of myoblasts, during both growth wild-type, FLAG-p21-S153A, FLAG-p21-S153D, or vector for 4 h, al- lowed to express for 24 h (0 point) and then switched to serum-contain- and differentiation. These observations complement the known ing growth medium for 0 –13 days. A, each construct was expressed for survival function of Mirk in colon carcinoma cells (20). We also only about 1 day in growth medium following the transfection and show that at least some of the pro-survival functions of Mirk initial 24-h expression period, as determined by Western blotting for can be ascribed to its phosphorylation of the CDK inhibitor p21 the FLAG epitope. The zero time points for each construct were exam- ined in parallel to the zero time points for the other two constructs on and subsequent increased localization of p21 in the cytoplasm. each blot as internal controls. Ct, cross-reacting protein used as a Cytoplasmic p21 has a role in pro-survival signaling in differ- loading control. B, after 24 h, the cells were trypsinized and plated at entiating myoblasts (Fig. 9) and is maintained in the cytoplasm either 500 or 1000 cells in 100-mm tissue culture dishes, each in of mature myofibrils. The anti-apoptotic function of p21 in triplicate. Data shown are the mean of two such experiments, normal- ized to the number of colonies per 1000 cells plated. myogenesis is well documented. In C2 myoblasts, ectopic ex- pression of p21 has been shown to block apoptosis, whereas depletion of p21 by antisense oligonucleotides induced apopto- tion (42). The p21 gene is induced by the tumor suppressor p53 sis (15). A role for cytosolic p21 has also been demonstrated in (43) and by cellular senescence (44). p21 is a major mediator of neuronal differentiation. After functioning in the nucleus to the G growth arrest induced by activation of the tumor sup- block cell proliferation, cytoplasmic p21 promotes neuronal pressor p53 in response to DNA damage (reviewed in Ref. 45). differentiation where it regulates Rho-induced actin remodel- Other major functions for p21 are to promote assembly of cyclin ing leading to neurite outgrowth (38). Cytoplasmic p21 has also D-CDK4 complexes and to increase cyclin D1 accumulation by been shown recently (39) to enhance axonal regeneration and direct inhibition of GSK3-triggered nuclear export (46, 47). functional recovery after spinal cord injury. The cell cycle modulatory activity of p21 is tightly correlated Cell cycle progression is mediated by the sequential activa- with its nuclear localization. Movement of p21 from the nucleus tion of members of the CDK protein kinase family. Cell cycling to the cytoplasm blocks its cell cycle inhibitory activity and is blocked when cyclin-CDK complexes cannot form or when contributes to the cell growth induced by the oncogene Her-2/ the catalytic activity of these complexes is blocked through neu (21). Akt activated by Her-2/neu phosphorylates Thr-145 binding of a CDK inhibitor molecule. One of these inhibitors is and Ser-146 within the nuclear localization signal of p21 (21, Cip1/Waf1 p21 (40), which was originally identified as a PCNA- 30, 48). Phosphorylation of p21 by Akt has been shown to binding protein (41). p21 inhibits DNA replication by prevent- promote association with 14-3-3 proteins, which function as ing PCNA from contributing to DNA polymerase and func- nuclear export proteins (49). In the current study, we have 25800 Mirk Blocks Apoptosis through Translocation of p21 documented that the kinase Mirk, like Akt, also promotes localization of p21 in the cytoplasm of transformed cells might translocation of p21 to the cytoplasm by phosphorylation contribute not only to increased survival under environmental within the nuclear localization signal of p21. In the case of and/or pharmacological stress but may also contribute to cell Mirk, the phosphorylation site is serine 153, close to the Akt motility and thus enhance tumor cell invasion. sites of threonine 145 and serine 146. A phosphomimetic mu- Acknowledgments—We thank Dr. Anna-Luise Katzenstein for pro- tant, p21-S153D, was found in the cytoplasm in 78% of C2C12 viding us with specimens of normal muscle tissue, Dr. Howard Chang myoblasts and 67% of NIH3T3 fibroblasts, whereas wild-type for assistance with frozen sections, and Julie Lippa for assistance with paraffin section immunohistochemistry. p21 and the nonphosphorylatable mutant p21-S153A were found in the cytoplasm in only 20 –25% of transfected myo- REFERENCES blasts and fibroblasts. The phosphomimetic mutant, p21- 1. Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) S153D, was still as capable of blocking myoblast cell cycling as J. Biol. Chem. 278, 41347– 41354 2. Deng, X., Ewton, D., Mercer, S., and Friedman, E. (2005) J. Biol. Chem. 280, ectopic wild-type p21 (Table I). Because the p21-S153D con- 4894 – 4905 struct was overexpressed, we speculate that enough p21-S153D 3. 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Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

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Mirk/Dyrk1B Mediates Survival during the Differentiation of C2C12Myoblasts *

Mirk/Dyrk1B Mediates Survival during the Differentiation of C2C12Myoblasts *

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Mirk/Dyrk1B Mediates Survival during the Differentiation of C2C12Myoblasts *

Mirk/Dyrk1B Mediates Survival during the Differentiation of C2C12Myoblasts *

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