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Ionizing Radiation Activates Nuclear Protein Phosphatase-1 by ATM-dependent Dephosphorylation

Ionizing Radiation Activates Nuclear Protein Phosphatase-1 by ATM-dependent Dephosphorylation THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 44, Issue of November 1, pp. 41756 –41761, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Ionizing Radiation Activates Nuclear Protein Phosphatase-1 by ATM-dependent Dephosphorylation* Received for publication, July 25, 2002, and in revised form, August 25, 2002 Published, JBC Papers in Press, August 28, 2002, DOI 10.1074/jbc.M207519200 Chang Y. Guo‡, David L. Brautigan§, and James M. Larner‡ From the ‡Department of Radiation Oncology, University of Virginia Health System, Charlottesville, Virginia 22908 and §Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908 lian PP1, prevents normal mitotic progression and normal po- Ionizing radiation (IR) is known to activate multiple signaling pathways, resulting in diverse stress re- lar growth. Expression of mammalian PP1 fully complements sponses including apoptosis, cell cycle arrest, and gene the bimG11 phenotype. Additionally, microinjection of neutral- induction. IR-activated cell cycle checkpoints are regu- izing PP1 antibody into mammalian cells in early mitosis lated by Ser/Thr phosphorylation, so we tested to see if causes metaphase arrest (9, 10). protein phosphatases were targets of an IR-activated Although PP1 is required for cell cycle progression, few sub- damage-sensing pathway. Jurkat cells were subjected to strates of PP1 have been identified, and therefore, its role in IR or sham radiation followed by brief P metabolic these processes remains poorly defined. Several lines of evi- labeling. Nuclear extracts were subjected to microcystin dence suggest that in the absence of DNA damage, Rb is reg- affinity chromatography to recover phosphatases, and ulated by PP1. Mitotic extracts dephosphorylate Rb in the the proteins were analyzed by two-dimensional gel elec- absence but not the presence of inhibitor 2, a protein specific for trophoresis. Protein sequencing revealed that the mi- PP1 (11). Microinjection of PP1 into G cells results in accu- crocystin-bound proteins with the greatest reduction in mulation of dephosphorylated Rb and inhibition of S phase P intensity following IR were the  and  isoforms of progression (12). Other potential PP1 substrates that regulate protein phosphatase 1 (PP1). Both of these PP1 isoforms mitotic progression include Cdk1 and lamin B (13), the dephos- contain an Arg-Pro-Ile/Val-Thr-Pro-Pro-Arg sequence phorylation of which may be necessary for the formation of the near the C terminus, a known site of phosphorylation by nuclear envelope. Histones H1 and H3 may also be dephospho- Cdc/Cdk kinases, and phosphorylation attenuates phos- rylated by PP1 in mitosis (14 –16). phatase activity. In wild-type Jurkat cells or ataxia tel- PP1 plays an important role in cell cycle progression and is a angiectasia (AT) cells that are stably transfected with potentially important downstream effector of IR-stimulated full-length ATM kinase, IR resulted in net dephospho- damage-sensing pathways resulting in checkpoint activation. rylation of this site in PP1 and produced activation of PP1. However, in AT cells that are deficient in ATM, IR Here we show that IR causes dephosphorylation of a Thr site in failed to induce dephosphorylation or activation of PP1. PP1 catalytic subunit (PP1c) resulting in activation of PP1. The IR-induced PP1 activation in the nucleus may be a crit- IR-induced dephosphorylation of this site is shown to be de- ical component in an ATM-mediated pathway control- pendent on ATM (mutated in ataxia telangiectasia), the gene ling checkpoint activation. product that is deficient in the human autosomal recessive disease. MATERIALS AND METHODS Ionizing radiation (IR) is known to activate multiple signal- ing pathways resulting in diverse stress responses including Cell Culture—Jurkat cells (a human T cell lymphoma cell line) were grown in RPMI 1640 medium (Life Technologies, Inc.) with penicillin apoptosis, cell cycle arrest, and gene induction (1, 2). Many of and streptomycin and 10% fetal bovine serum. FT/pEBS7 and FT/ these responses are controlled by phosphorylation and dephos- pEBS7-YZ5 cells were both derived from the AT22IJE-T line (17), an phorylation of Ser and Thr residues in proteins. PP1 is a major immortalized fibroblast line containing a homozygous frameshift mu- protein Ser/Thr phosphatase conserved among eukaryotic spe- tation at codon 762 of the ATM gene. AT22IJE-T cells were transfected cies that regulates a variety of key steps in metabolism, repli- with the mammalian expression vector pEBS7 (18) containing either cation, transcription, and the cell cycle (3– 6). PP1 is required the hygromycin resistance marker to yield FT/pEBS7 cells or with full-length ATM open reading frame to yield FT/pEBS7-YZ5 cells. FT/ for completion of mitosis in many eukaryotic organisms. For pEBS7 and FT/pEBS7-YZ5 were generously provided by Y. Shiloh (Tel example, in Aspergillus nidulans and in Schizosaccharomyces Aviv University) and grown in Dulbecco’s modified Eagle’s medium pombe proteins with 80% identity to mammalian PP1 are re- with 15% fetal bovine serum and 100 g/ml hygromycin B. All cells quired for separation of daughter nuclei, completion of an- were in an exponential growth phase at the time of radiation. aphase, and chromosome segregation (7, 8). The Aspergillus Radiation Treatment—Cell cultures were irradiated with a Varian mutant bimG11, which encodes a protein similar to mamma- linear accelerator at a dose rate of 9 Gy per min. During irradiation, the cultures were maintained in a container designed to mimic the condi- tions of the cell culture incubator (5% CO and 95% air at 37 °C). * This study was supported by National Institutes of Health Grants Preparation of Nuclear Extracts—Cells were collected by centrifuga- CA 72622 (to J. M. L.) and GM 56362 and CA 40042 (to D. L. B.). The tion in an ice-cold preparation buffer consisting of 20 mM HEPES pH costs of publication of this article were defrayed in part by the payment 7.4, 110 mM potassium acetate, 2 mM magnesium acetate, and 5 mM of page charges. This article must therefore be hereby marked “adver- EGTA. The cell pellet was resuspended in the same buffer containing 1 tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate TM g/ml aprotinin, 1 mM Pefabloc , 0.2 mM PMSF, and 1 mM dithiothre- this fact. itol at 5  10 cells/ml. Digitonin was added to a final concentration of To whom correspondence should be addressed: University of 50 g/ml to permeabilize the plasma membrane and release the cytosol. Virginia Health System, Box 800383, Charlottesville, VA 22908-0383. The cell suspension was placed on ice for 5 min and then diluted 10-fold Tel.: 434-924-5191; Fax: 434-982-3262; E-mail: [email protected]. The abbreviations used are: IR, ionizing radiation; PP1, protein in complete preparation buffer. Following centrifugation, the pellet phosphatase 1; Gy, gray; PMSF, phenylmethylsulfonyl fluoride. containing intact nuclei was extracted with either nuclear lysis buffer 41756 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. IR Activates PP1 in an ATM-dependent Manner 41757 (1% Nonidet P-40, 150 mM NaCl, 10 mM sodium phosphate, pH 7.2, 2 mM EDTA, 50 mM sodium fluoride, 0.2 mM sodium vanadate, 1 mM PMSF, and 1 g/ml aprotinin) or for the PP1 activity assay lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris-Cl, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0.2 mM sodium vanadate, and 0.2 mM PMSF). Western Analyses—Samples (80 –100 g of protein) were run on 12% SDS-polyacrylamide gels and transferred (with Tris/glycine/methanol buffer, 100 V for 1 h) to nitrocellulose. Reactive proteins were detected with horseradish peroxidase-conjugated antibodies and detected by chemiluminescence. The following commercial antibodies were used: anti-phospho-PP1 (Thr-320) antibody (Cell Signaling Technology), anti-PP1c (Santa Cruz Biotechnology, Inc.), and anti-Cdk2 (Upstate Biotechnology). Anti-PP1 and anti-PP1 were raised and purified against synthetic peptides corresponding to the C-terminal regions of the  and  isoforms, respectively. Microcystin Affinity Purification—80 l of a 50% slurry of microcys- tin-agarose (Upstate Biotechnology Inc.) was added to nuclear extract containing 4.0 mg of total protein and incubated for3hat4 °C, after FIG.1. IR dephosphorylates PP1  and . Asynchronously grow- which the beads were washed three times with ice-cold nuclear lysis ing Jurkat cells were subjected to either 0 or 10 Gy. Post-IR, the cells buffer. The protein was then dissociated from the beads with SDS were labeled with [ P]orthophosphate (0.6 mCi/ml) for 45 min after boiling sample buffer and assayed by two-dimensional gel analysis. which they were washed with cold phosphate-buffered saline. Extracts Histone H1 Kinase Assays—Cells were collected, washed with cold were prepared and equal amounts of protein subjected to microcystin phosphate-buffered saline, and resuspended in a lysis buffer. The sus- affinity chromatography as previously described (38). The pellets were pensions were kept on ice for 30 min, and the lysate was collected by heated with sample buffer and were subjected to two-dimensional gel centrifugation at l5,000  g for 20 min at 4 °C. Protein concentrations electrophoresis. The gels were then silver-stained and exposed to films were determined using a Bradford assay. Extracts were diluted to 1 to make autoradiographs. The four proteins circled were sequenced by mg/ml with lysis buffer. Immunoprecipitation with anti-Cdk2 was per- tandem mass spectroscopy. formed by incubating 0.5 mg of extract and 4 g of antibody for1hat 4 °C. The immune complexes were then incubated at 4 °C with 20 lof RESULTS a 50% suspension of protein A-agarose and washed three times first with lysis buffer and then with kinase buffer (l0 mM Tris, pH 7.4, 150 IR Induces in Vivo Dephosphorylation of PP1—We tested if mM NaCl, l0 mM MgCl , and 0.5 mM dithiothreitol) at 4 °C. The pellets 2 protein Ser/Thr phosphatases were targets of an IR-activated were incubated for 15 min at 37 °C with 40 l of the kinase assay buffer damage-sensing pathway. Jurkat cells were labeled with inor- containing 25 M ATP, 2.5 Ci of [ P]ATP, and histone H1 at 1.0 32 ganic P for 45 min after irradiation or sham irradiation. mg/ml. 8 lof6 electrophoresis sample buffer was added to 40 lof Nuclear extracts were prepared and subjected to microcystin the supernatant and boiled for 5 min and run on a 12.5% SDS-poly- affinity chromatography, followed by two-dimensional gel anal- acrylamide gel. The gel was fixed and stained with 0.25% Coomassie ysis (Fig. 1). Microcystin binds with nanomolar affinity to the Blue (in 45% methanol, 10% acetic acid), destained (40% methanol, 10% acetic acid), dried, and exposed to x-ray film. For quantification, the catalytic cleft of protein phosphatases (20) and can be used to P incorporation was determined histone H1 bands were excised and rapidly and quantitatively recover the various forms of PP1 by liquid scintillation counting. The amount of Cdk2 protein was deter- and PP2A from extracts (21) together with their multiple reg- mined by Western analysis. ulatory subunits (22). On the two-dimensional gel, more than Phosphorylation Assay—The purified catalytic subunit of rabbit skel- 50 distinct silver-stained proteins were visualized in the elu- etal muscle PP1c/ (19) (1.6 g) was incubated for 30 min at 30 °C with tion from the microcystin beads, and about a dozen of these varying concentrations of purified Cdk2/cyclin A kinase in a total vol- 32 32 ume of 40 l of kinase buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 were P-labeled. The levels of P incorporated into one group mM MgCl , 0.5 mM dithiothreitol, and 0.1 mM ATP). An aliquot of the of proteins retained by the microcystin matrix dramatically reaction mixture was added to 6 sample buffer and heated at 100 °C decreased following IR, and we centered our attention on these for 4 min and resolved by 12% SDS-PAGE. Phosphorylated PP1c and proteins (Fig. 1). The major form, both by silver staining and PP1c were analyzed by Western analysis. P labeling, was the protein in spot 3. However, all of these To assay the ability of endogenous Cdk2 to phosphorylate the Thr- spots were dephosphorylated in response to IR, based on P 320 site in PP1c, Jurkat cells were subjected to 10 Gy, and Cdk2 was labeling. Tandem mass spectrometric sequencing revealed immunoprecipitated from 0.5 mg of cellular extract at various times post-IR with 4 g of Cdk2 antibody. The immunoprecipitates were these proteins were the  and  isoforms of PP1 (Table I). The washed three times with ice-cold lysis buffer and three times with differences in migration in the first dimension (isoelectric ice-cold kinase buffer without ATP and then incubated with purified focusing) for these PP1 isoforms are probably because of for- catalytic subunit of rabbit skeletal muscle PP1c (1.6 g) at 30 °C for 45 mation of intramolecular disulfides and/or differences in oxida- min in a total volume of 40 l of kinase buffer. Phosphorylated and total tion state of the multiple Cys residues in the catalytic subunits. PP1c from the supernatant and Cdk2 in the pellet were analyzed by Regardless, the results show IR caused loss of P indicating immunoblot analysis. that PP1 is phosphorylated in living cells and IR causes net PP1 Activity Assay—PP1 was immunoprecipitated with 5 g of anti- PP1c monoclonal antibody from either an aliquot of the purified PP1c- dephosphorylation. Cdk2 reaction mixture (0.5 g of PP1c diluted in 1 ml of PP1 activity Effects of IR on Nuclear PP1 in Jurkat and AT Cells—PP1 lysis buffer (see “Preparation of Nuclear Extracts”)) or from nuclear and  isoforms contain a C-terminal RP(I/V)TPPR motif known lysis solution (1.0 mg of nuclear protein) prepared from Jurkat, FT/ to be a preferred site for Cdk/Cdc phosphorylation (23). Phos- pEBS7, and FT/pEBS7-YZ5. The immune complexes were then incu- phorylation of the threonine in this motif occurs in yeast as well bated with 30 l of a 50% suspension of protein A-agarose beads washed as mammalian PP1 (24) and attenuates PP1 activity. We and three times with ice-cold lysis buffer followed by ice-cold Ser/Thr assay buffer (50 mM Tris-HCl, pH 7.0, 0.1 mM CaCl ). PP1 activity was others (25, 26) have shown that IR inhibits Cdk2/cyclin A and assayed using a Ser/Thr phosphatase assay kit (Upstate Biotechnology Cdk2/cyclin E activity, and both of these kinases phosphorylate Inc.). The PP1 immune complex beads in 50 l of Ser/Thr assay buffer PP1 at Thr-320. We reasoned that DNA damage might acti- were incubated with the phosphopeptide KRpTIRR at 30 °C for 30 min. vate PP1 through reduced phosphorylation of this Thr site. The beads were pelleted, and 25 l of supernatant was analyzed for free Jurkat cells were irradiated and nuclear extracts were assayed phosphate in the malachite green assay by dilution with 100 lof for phosphatase activity at various times post-IR (Fig. 2a). developing solution (malachite green). After incubation for 15 min, the There was more than a doubling in PP1 activity over 90 min. In release of phosphate was quantified by measuring the absorbance at 620 nm in a microtiter plate reader. parallel nuclear extracts were subjected to immunoblot analy- 41758 IR Activates PP1 in an ATM-dependent Manner TABLE I Tandem mass spectrometry identification of dephosphorylated proteins Spot Identified as Peptide mass (Da) pI Sequence Accession NCBI no. 1 PP1C/ 37.5 5.94 (16–26) LLEVQGSRPGK 4506003 (27–36) NVQLTENEIRG (306–317) YGQFSGLNPGGR PP1C/ 37.2 5.84 (26–35) IVQMTEAEVR 4506005 (132–140) IYGFYDECK (168–186) IFCCHGGLSPDLQSMEQIRRI (304–319) YQYGGLNSGRPVTPPR 2 PP1C/ 37.5 5.94 (61–74) ICGDIHGQYYDLLR 4506003 (235–246) FLHKHDLDLICR (306–323) YGQFSGLNPGGRPITPPR PP1C/ 37.2 5.84 (111–121) IKYPENFFLLR 4506005 (150–167) TFTDCFNCLPIAAIVDEK (246–259) AHQVVEDGYEFFAK 3 PP1C/ 37.5 5.94 (75–96) LFEYGGFPPESNYLFLGDYVDR 4506003 PP1C/ 37.2 5.84 (60–73) ICGDIHGQYTDLLR 4506005 (111–121) IKYPENFFLLR (168–186) IFCCHGGLSPDLQSMEQIR (246–260) AHQVVEDGYEFFAKR 4 PP1C/ 37.5 5.94 (16–26) LLEVQGSRPGK 4506003 (27–36) NVQLTENEIR (222–234) GVSFTFGAEVVAK (306–323) YGQFSGLNPGGRPITPPR PP1C/ 37.2 5.84 (98–110) QSLETICLLLAYK 4506005 (132–141) IYGFYDECKR (168–186) IFCCHGGLSPDLQSMEQIR (246–260) AHQVVEDGYEFFAKR FIG.2. a, IR activates nuclear PP1. Jurkat cells (circles) were sub- jected to 10 Gy, and nuclear extracts were prepared at various times post-IR. PP1c was then immunoprecipitated, and its activity was meas- FIG.3. Thr-320 phosphorylation influences PP1 activity. Puri- ured using the phosphopeptide substrate KRpTIRR as described under fied catalytic subunit of rabbit skeletal muscle PP1 was incubated for 30 “Materials and Methods.” Quantitation (squares) of the optical density min at 30 °C with the indicated concentrations of purified Cdk2 kinase of the immunoblot shown in b was measured by Image Quant 5.0 as described under “Materials and Methods.” a, an aliquot of the reac- (Molecular Dynamics, Sunnyvale, CA). b, IR induces dephosphorylation tion mixture was subjected to immunoblot analysis with phospho- on Thr-320 of nuclear PP1 catalytic subunit. Asynchronously growing PP1c/ Thr-320 antibody and anti-PP1c. Quantitation (squares)ofthe Jurkat cells received either 0 or 10 Gy irradiation, and nuclear extracts optical density of the immunoblot is shown in b. b, PP1c was immuno- were prepared at the indicated times post-IR and subjected to immu- precipitated from the remaining reaction mixture and its activity was noblot analysis with phospho-PP1c (Thr-320) and anti-PP1c. measured as described for Fig. 2a. sis with a phospho-PP1 (Thr-320) antibody. As shown in Fig. crease in PP1 activity. PP1 levels themselves, which serve as a 2, a and b, 10 Gy resulted in a time-dependent dephosphoryl- loading control, were unchanged, arguing that there was in- ation of Thr-320 of PP1, which parallels the IR-induced in- creased specific activity, not synthesis or import of PP1. IR Activates PP1 in an ATM-dependent Manner 41759 FIG.4. IR decreases the ability of endogenous Cdk2 to phos- phorylate the Thr-320 site in PP1c. Jurkat cells were subjected to 10 Gy, and Cdk2 was immunoprecipitated from nuclear extracts at various times post-IR. The pellet was incubated with purified PP1c at 30 °C for 45 min as described under “Materials and Methods.” The supernatant was subjected to immunoblot analysis with phospho-PP1c/ (Thr-320) antibody and anti-PP1c. To verify that Thr-320 phosphorylation regulates PP1 activ- ity under our assay conditions, purified skeletal muscle PP1 protein was incubated with purified Cdk2 kinase and subjected to immunoprecipitation with anti-PP1c. PP1 activity in the immunoprecipitates was assayed with a phosphopeptide as substrate. As expected, increasing doses of Cdk2 resulted in increased phosphorylation of the Thr-320 site (Fig. 3a). Phos- phorylation of this site in PP1 corresponded to decreased PP1 activity (Fig. 3b). Thus, in vitro and in living cells the level of Thr-320 phosphorylation inversely correlated with the specific activity of PP1. To establish that IR inhibited the activity of the endogenous Cdk2 kinase that was phosphorylating PP1, Cdk2 was immuno- precipitated at various intervals after 10 Gy. As shown in Fig. 4, the activity of endogenous Cdk2 toward PP1 at the Thr-320 site decreased in a time-dependent manner following IR. IR Both Decreases Cdk2 Kinase and Increases PP1 Phospha- tase Activity in an ATM-dependent Manner—We previously found that IR-induced dephosphorylation of histone H1 is ATM- dependent (14). This is because of reduced Cdk2 kinase and increased nuclear phosphatase activity. IR (10 Gy) inhibits Cdk2 activity in an ATM-dependent manner as shown in Fig. 5a. Cdk2 from AT (PEBS) cells lacking active ATM kinase was inhibited only 40% whereas Cdk2 from reconstituted AT cells (YZ-5) that express ATM was inhibited by 84%. The radiation- induced dephosphorylation of PP1 Thr-320 was ATM-depend- ent. We compared AT cells (PEBS) and AT cells transfected with full-length ATM (YZ-5) that were irradiated with a dose of 10 Gy. Nuclear extracts were subjected to immunoblot analysis that showed IR failed to dephosphorylate PP1 Thr-320 in AT cells (Fig. 5b). However, in AT cells transfected with full- length ATM, IR-induced dephosphorylation of Thr-320 occurred in a time-dependent manner starting 15 min post- irradiation. This genetic system was used to demonstrate FIG.5. IR-induced nuclear PP1c dephosphorylation and acti- that IR-induced dephosphorylation of the Thr-320 site of PP1 vation are ATM-dependent. a, in vitro H1 kinase activity was deter- is dependent on ATM. mined on immunoprecipitates with antibodies to Cdk2 at various times The ATM kinase is also necessary for the IR-induced in- following 10 Gy. All experiments were performed 3 times with similar crease in PP1 activity. Nuclear extracts were prepared from AT results, and a representative experiment is shown. Equal loading of H1 and Cdk2 is shown as controls. Percentage inhibition of kinase activity cells deficient in ATM and AT cells expressing ectopic full- was determined by excising H1 bands from the gel and measuring P length ATM. Samples were subjected to immunoprecipitation incorporation by liquid scintillation counting. b, AT fibroblasts trans- with anti-PP1 and assayed for PP1 activity. As shown in Fig. fected with either empty vector FT/pEBS7 (PEBS) or recombinant wild 5c, the IR-induced time-dependent 2-fold increase in PP1 ac- type ATM FT/pEBS7-YZ5 (YZ-5) were irradiated with 10 Gy, and nu- clear extracts were subjected to immunoblot analysis with either a tivity was only observed in cells expressing ATM. Interestingly, phospho-PP1c (Thr-320) antibody or anti-PP1c. c, AT fibroblasts PEBS the time course of IR activation of PP1 parallels that of and YZ-5 were irradiated with 10 Gy, and PP1c was immunoprecipi- IR-induced H1 dephosphorylation, which we have previously tated from nuclear extracts and assayed for PP1 activity as described reported (14). for Fig. 2a. Three genes code for four distinct isoforms of PP1 in mam- mals called , 1, 2, and  (3). Using isoform-specific antibod- sponding sequence motif in both these PP1 isoforms. Consist- ies to immunoprecipitate PP1c  and  from cell lysates, we ent with the metabolic P-labeling results (Fig. 1), both the tested if the Thr-320 site was dephosphorylated in response to and  PP1 isoforms were dephosphorylated at the Thr-320 site IR in Jurkat, AT, and reconstituted AT cells. The numbering of in response to IR (Fig. 6a). Furthermore, the dephosphoryl- the Thr-320 is not identical in  and  but lies in the corre- ation of both the  and  isoforms was ATM-dependent, as 41760 IR Activates PP1 in an ATM-dependent Manner tase (Cdc25) would be regulating another phosphatase (PP1) via a phosphatase-kinase-phosphatase cascade (Cdc25-Cdk2- PP1). Our data do not establish that Cdk2 is the in vivo kinase of PP1. There may be other kinases that phosphorylate the Thr-320 site in PP1, but they would also need to be regulated in response to IR by an ATM-dependent pathway. Alternatively, ATM could directly (or indirectly) phosphorylate a nuclear PP1 subunit that restrains PP1 in its phosphorylated, low activity form. Phosphorylation would release inhibition and allow autodephos- phorylation of Thr-320 and activation of PP1 toward other sub- strates. NIPP is one example of a nuclear PP1 regulatory subunit whose activity is regulated by phosphorylation (35, 36). What are the downstream nuclear substrates of PP1 that may be critical in the damage response? Unlike protein serine/ threonine kinases, PP1 catalytic subunit does not manifest a FIG.6. IR-induced dephosphorylation of PP1  and PP1  is ATM-dependent. Jurkat (a) and AT fibroblasts PEBS and YZ-5 (b) high degree of sequence specificity and dephosphorylates mul- were irradiated with 10 Gy. Nuclear extracts were prepared and sub- tiple substrates. The substrate specificity of PP1 is thought to jected to immunoprecipitation with the indicated isoform-specific anti- be modulated through the formation of heterodimeric com- PP1c antibodies. Immunoblot analysis was performed as described for plexes with regulatory subunits. Regulatory proteins that tar- Fig. 2. IP, immunoprecipitate. get PP1 to its substrates in response to DNA damage are not known, and these may bind both  and  isoforms or there may shown in Fig. 6b. Decreased phosphospecific staining of PP1 be individual regulatory subunits for these isoforms. Specific and  was only seen in irradiated cells that were expressing PP1 isoforms have been shown to have distinct functions and ATM (YZ-5 clone). Cells that did not express ATM (PEBS) did locations. For example, the PP1 isoform has been shown to not show diminished phospho-Thr-320 staining in response regulate the G /S transition by dephosphorylating Rb (12), and to IR. 1 the PP1 isoform is chromatin-associated. Because histone H1 DISCUSSION and histone H3 have been implicated as PP1 substrates (14, 16, IR is known to activate the G , S, and G checkpoints (2, 27) 37), it is likely that the PP1 isoform may regulate IR-induced 1 2 in an ATM-dependent manner. The ATM gene is mutated in H1 and H3 dephosphorylation. the autosomal recessive disease ataxia telangiectasia, charac- In summary, the PP1  and  isoforms are dynamically terized by neuronal degeneration resulting in ataxia, oculocu- phosphorylated in Jurkat cells and in response to IR become taneous telangiectasia, immune dysfunction, and cancer pre- activated by dephosphorylation of Thr-320 to function as a disposition (28). ATM is thought to be an upstream sensor of downstream effector of an ATM-dependent damage-sensing DNA damage as well as oxidative stress. ATM transmits the pathway. Regardless of the mechanism by which ATM acti- damage signal downstream through its C-terminal phospho- vates PP1, defining the regulatory subunits that target PP1 to inositol 3-kinase domain. ATM has been shown to directly its respective substrates may reveal novel targets for chemo phosphorylate several proteins and to enhance the phosphoryl- and radiation sensitizers. Drugs that function at the level of ation of other proteins by activating downstream protein ki- these targets may have low toxicity and, therefore, be more nases. These targets include the nuclear tyrosine kinase c-Abl, efficacious than less specific inhibitors. Chk2, nibrin, p53, and BRCA1, all of which have been impli- cated in DNA damage responses (29 –33). REFERENCES Interestingly, ATM has also been shown to control the dam- 1. Liu, V. F., Boubnov, N. V., and Weaver, D. T. (1995) Stem Cells 13, Suppl. 1, 117–128 age-induced dephosphorylation of Ser-376 in p53 (34) and to 2. Larner, J. M., Lee, H., and Hamlin, J. L. (1997) Cancer Surv. 29, 25– 45 regulate H1 dephosphorylation following IR. Thus, ATM acti- 3. Cohen, P. T. (2002) J. Cell Sci. 115, 241–256 4. Clarke, P. R., Hoffmann, I., Draetta, G., and Karsenti, E. (1993) Mol. Biol. Cell vates the phosphatase(s) responsible for dephosphorylating H1 4, 397– 411 and p53. We have recently demonstrated that IR causes disso- 5. van Zyl, W., Huang, W., Sneddon, A. A., Stark, M., Camier, S., Werner, M., ciation of the B55 regulatory subunit, which has been impli- Marck, C., Sentenac, A., and Broach, J. R. (1992) Mol. Cell. Biol. 12, 4946 – 4959 cated in mitotic progression, from heterotrimeric nuclear PP2A 6. Virshup, D. M., Russo, A. A., and Kelly, T. J. (1992) Mol. Cell. Biol. 12, in an ATM-dependent manner (38). Although the functional 4883– 4895 7. Ohkura, H., Kinoshita, N., Miyatani, S., Toda, T., and Yanagida, M. (1989) Cell significance of IR-induced dissociation of B55 from PP2A het- 57, 997–1007 erotrimer is unknown, it is likely that IR-induced subunit 8. Hughes, M., Arundhati, A., Lunness, P., Shaw, P. J., and Doonan, J. H. (1996) exchange allows PP2A to execute diverse functions in the dam- EMBO J. 15, 4574 – 4583 9. Doonan, J. H., MacKintosh, C., Osmani, S., Cohen, P., Bai, G., Lee, E. Y., and age response. 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Ionizing Radiation Activates Nuclear Protein Phosphatase-1 by ATM-dependent Dephosphorylation

Guo, Chang Y.; Brautigan, David L.; Larner, James M.
Journal of Biological Chemistry – Nov 1, 2002
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 44, Issue of November 1, pp. 41756 –41761, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Ionizing Radiation Activates Nuclear Protein Phosphatase-1 by ATM-dependent Dephosphorylation* Received for publication, July 25, 2002, and in revised form, August 25, 2002 Published, JBC Papers in Press, August 28, 2002, DOI 10.1074/jbc.M207519200 Chang Y. Guo‡, David L. Brautigan§, and James M. Larner‡ From the ‡Department of Radiation Oncology, University of Virginia Health System, Charlottesville, Virginia 22908 and §Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908 lian PP1, prevents normal mitotic progression and normal po- Ionizing radiation (IR) is known to activate multiple signaling pathways, resulting in diverse stress re- lar growth. Expression of mammalian PP1 fully complements sponses including apoptosis, cell cycle arrest, and gene the bimG11 phenotype. Additionally, microinjection of neutral- induction. IR-activated cell cycle checkpoints are regu- izing PP1 antibody into mammalian cells in early mitosis lated by Ser/Thr phosphorylation, so we tested to see if causes metaphase arrest (9, 10). protein phosphatases were targets of an IR-activated Although PP1 is required for cell cycle progression, few sub- damage-sensing pathway. Jurkat cells were subjected to strates of PP1 have been identified, and therefore, its role in IR or sham radiation followed by brief P metabolic these processes remains poorly defined. Several lines of evi- labeling. Nuclear extracts were subjected to microcystin dence suggest that in the absence of DNA damage, Rb is reg- affinity chromatography to recover phosphatases, and ulated by PP1. Mitotic extracts dephosphorylate Rb in the the proteins were analyzed by two-dimensional gel elec- absence but not the presence of inhibitor 2, a protein specific for trophoresis. Protein sequencing revealed that the mi- PP1 (11). Microinjection of PP1 into G cells results in accu- crocystin-bound proteins with the greatest reduction in mulation of dephosphorylated Rb and inhibition of S phase P intensity following IR were the  and  isoforms of progression (12). Other potential PP1 substrates that regulate protein phosphatase 1 (PP1). Both of these PP1 isoforms mitotic progression include Cdk1 and lamin B (13), the dephos- contain an Arg-Pro-Ile/Val-Thr-Pro-Pro-Arg sequence phorylation of which may be necessary for the formation of the near the C terminus, a known site of phosphorylation by nuclear envelope. Histones H1 and H3 may also be dephospho- Cdc/Cdk kinases, and phosphorylation attenuates phos- rylated by PP1 in mitosis (14 –16). phatase activity. In wild-type Jurkat cells or ataxia tel- PP1 plays an important role in cell cycle progression and is a angiectasia (AT) cells that are stably transfected with potentially important downstream effector of IR-stimulated full-length ATM kinase, IR resulted in net dephospho- damage-sensing pathways resulting in checkpoint activation. rylation of this site in PP1 and produced activation of PP1. However, in AT cells that are deficient in ATM, IR Here we show that IR causes dephosphorylation of a Thr site in failed to induce dephosphorylation or activation of PP1. PP1 catalytic subunit (PP1c) resulting in activation of PP1. The IR-induced PP1 activation in the nucleus may be a crit- IR-induced dephosphorylation of this site is shown to be de- ical component in an ATM-mediated pathway control- pendent on ATM (mutated in ataxia telangiectasia), the gene ling checkpoint activation. product that is deficient in the human autosomal recessive disease. MATERIALS AND METHODS Ionizing radiation (IR) is known to activate multiple signal- ing pathways resulting in diverse stress responses including Cell Culture—Jurkat cells (a human T cell lymphoma cell line) were grown in RPMI 1640 medium (Life Technologies, Inc.) with penicillin apoptosis, cell cycle arrest, and gene induction (1, 2). Many of and streptomycin and 10% fetal bovine serum. FT/pEBS7 and FT/ these responses are controlled by phosphorylation and dephos- pEBS7-YZ5 cells were both derived from the AT22IJE-T line (17), an phorylation of Ser and Thr residues in proteins. PP1 is a major immortalized fibroblast line containing a homozygous frameshift mu- protein Ser/Thr phosphatase conserved among eukaryotic spe- tation at codon 762 of the ATM gene. AT22IJE-T cells were transfected cies that regulates a variety of key steps in metabolism, repli- with the mammalian expression vector pEBS7 (18) containing either cation, transcription, and the cell cycle (3– 6). PP1 is required the hygromycin resistance marker to yield FT/pEBS7 cells or with full-length ATM open reading frame to yield FT/pEBS7-YZ5 cells. FT/ for completion of mitosis in many eukaryotic organisms. For pEBS7 and FT/pEBS7-YZ5 were generously provided by Y. Shiloh (Tel example, in Aspergillus nidulans and in Schizosaccharomyces Aviv University) and grown in Dulbecco’s modified Eagle’s medium pombe proteins with 80% identity to mammalian PP1 are re- with 15% fetal bovine serum and 100 g/ml hygromycin B. All cells quired for separation of daughter nuclei, completion of an- were in an exponential growth phase at the time of radiation. aphase, and chromosome segregation (7, 8). The Aspergillus Radiation Treatment—Cell cultures were irradiated with a Varian mutant bimG11, which encodes a protein similar to mamma- linear accelerator at a dose rate of 9 Gy per min. During irradiation, the cultures were maintained in a container designed to mimic the condi- tions of the cell culture incubator (5% CO and 95% air at 37 °C). * This study was supported by National Institutes of Health Grants Preparation of Nuclear Extracts—Cells were collected by centrifuga- CA 72622 (to J. M. L.) and GM 56362 and CA 40042 (to D. L. B.). The tion in an ice-cold preparation buffer consisting of 20 mM HEPES pH costs of publication of this article were defrayed in part by the payment 7.4, 110 mM potassium acetate, 2 mM magnesium acetate, and 5 mM of page charges. This article must therefore be hereby marked “adver- EGTA. The cell pellet was resuspended in the same buffer containing 1 tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate TM g/ml aprotinin, 1 mM Pefabloc , 0.2 mM PMSF, and 1 mM dithiothre- this fact. itol at 5  10 cells/ml. Digitonin was added to a final concentration of To whom correspondence should be addressed: University of 50 g/ml to permeabilize the plasma membrane and release the cytosol. Virginia Health System, Box 800383, Charlottesville, VA 22908-0383. The cell suspension was placed on ice for 5 min and then diluted 10-fold Tel.: 434-924-5191; Fax: 434-982-3262; E-mail: [email protected]. The abbreviations used are: IR, ionizing radiation; PP1, protein in complete preparation buffer. Following centrifugation, the pellet phosphatase 1; Gy, gray; PMSF, phenylmethylsulfonyl fluoride. containing intact nuclei was extracted with either nuclear lysis buffer 41756 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. IR Activates PP1 in an ATM-dependent Manner 41757 (1% Nonidet P-40, 150 mM NaCl, 10 mM sodium phosphate, pH 7.2, 2 mM EDTA, 50 mM sodium fluoride, 0.2 mM sodium vanadate, 1 mM PMSF, and 1 g/ml aprotinin) or for the PP1 activity assay lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris-Cl, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0.2 mM sodium vanadate, and 0.2 mM PMSF). Western Analyses—Samples (80 –100 g of protein) were run on 12% SDS-polyacrylamide gels and transferred (with Tris/glycine/methanol buffer, 100 V for 1 h) to nitrocellulose. Reactive proteins were detected with horseradish peroxidase-conjugated antibodies and detected by chemiluminescence. The following commercial antibodies were used: anti-phospho-PP1 (Thr-320) antibody (Cell Signaling Technology), anti-PP1c (Santa Cruz Biotechnology, Inc.), and anti-Cdk2 (Upstate Biotechnology). Anti-PP1 and anti-PP1 were raised and purified against synthetic peptides corresponding to the C-terminal regions of the  and  isoforms, respectively. Microcystin Affinity Purification—80 l of a 50% slurry of microcys- tin-agarose (Upstate Biotechnology Inc.) was added to nuclear extract containing 4.0 mg of total protein and incubated for3hat4 °C, after FIG.1. IR dephosphorylates PP1  and . Asynchronously grow- which the beads were washed three times with ice-cold nuclear lysis ing Jurkat cells were subjected to either 0 or 10 Gy. Post-IR, the cells buffer. The protein was then dissociated from the beads with SDS were labeled with [ P]orthophosphate (0.6 mCi/ml) for 45 min after boiling sample buffer and assayed by two-dimensional gel analysis. which they were washed with cold phosphate-buffered saline. Extracts Histone H1 Kinase Assays—Cells were collected, washed with cold were prepared and equal amounts of protein subjected to microcystin phosphate-buffered saline, and resuspended in a lysis buffer. The sus- affinity chromatography as previously described (38). The pellets were pensions were kept on ice for 30 min, and the lysate was collected by heated with sample buffer and were subjected to two-dimensional gel centrifugation at l5,000  g for 20 min at 4 °C. Protein concentrations electrophoresis. The gels were then silver-stained and exposed to films were determined using a Bradford assay. Extracts were diluted to 1 to make autoradiographs. The four proteins circled were sequenced by mg/ml with lysis buffer. Immunoprecipitation with anti-Cdk2 was per- tandem mass spectroscopy. formed by incubating 0.5 mg of extract and 4 g of antibody for1hat 4 °C. The immune complexes were then incubated at 4 °C with 20 lof RESULTS a 50% suspension of protein A-agarose and washed three times first with lysis buffer and then with kinase buffer (l0 mM Tris, pH 7.4, 150 IR Induces in Vivo Dephosphorylation of PP1—We tested if mM NaCl, l0 mM MgCl , and 0.5 mM dithiothreitol) at 4 °C. The pellets 2 protein Ser/Thr phosphatases were targets of an IR-activated were incubated for 15 min at 37 °C with 40 l of the kinase assay buffer damage-sensing pathway. Jurkat cells were labeled with inor- containing 25 M ATP, 2.5 Ci of [ P]ATP, and histone H1 at 1.0 32 ganic P for 45 min after irradiation or sham irradiation. mg/ml. 8 lof6 electrophoresis sample buffer was added to 40 lof Nuclear extracts were prepared and subjected to microcystin the supernatant and boiled for 5 min and run on a 12.5% SDS-poly- affinity chromatography, followed by two-dimensional gel anal- acrylamide gel. The gel was fixed and stained with 0.25% Coomassie ysis (Fig. 1). Microcystin binds with nanomolar affinity to the Blue (in 45% methanol, 10% acetic acid), destained (40% methanol, 10% acetic acid), dried, and exposed to x-ray film. For quantification, the catalytic cleft of protein phosphatases (20) and can be used to P incorporation was determined histone H1 bands were excised and rapidly and quantitatively recover the various forms of PP1 by liquid scintillation counting. The amount of Cdk2 protein was deter- and PP2A from extracts (21) together with their multiple reg- mined by Western analysis. ulatory subunits (22). On the two-dimensional gel, more than Phosphorylation Assay—The purified catalytic subunit of rabbit skel- 50 distinct silver-stained proteins were visualized in the elu- etal muscle PP1c/ (19) (1.6 g) was incubated for 30 min at 30 °C with tion from the microcystin beads, and about a dozen of these varying concentrations of purified Cdk2/cyclin A kinase in a total vol- 32 32 ume of 40 l of kinase buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 were P-labeled. The levels of P incorporated into one group mM MgCl , 0.5 mM dithiothreitol, and 0.1 mM ATP). An aliquot of the of proteins retained by the microcystin matrix dramatically reaction mixture was added to 6 sample buffer and heated at 100 °C decreased following IR, and we centered our attention on these for 4 min and resolved by 12% SDS-PAGE. Phosphorylated PP1c and proteins (Fig. 1). The major form, both by silver staining and PP1c were analyzed by Western analysis. P labeling, was the protein in spot 3. However, all of these To assay the ability of endogenous Cdk2 to phosphorylate the Thr- spots were dephosphorylated in response to IR, based on P 320 site in PP1c, Jurkat cells were subjected to 10 Gy, and Cdk2 was labeling. Tandem mass spectrometric sequencing revealed immunoprecipitated from 0.5 mg of cellular extract at various times post-IR with 4 g of Cdk2 antibody. The immunoprecipitates were these proteins were the  and  isoforms of PP1 (Table I). The washed three times with ice-cold lysis buffer and three times with differences in migration in the first dimension (isoelectric ice-cold kinase buffer without ATP and then incubated with purified focusing) for these PP1 isoforms are probably because of for- catalytic subunit of rabbit skeletal muscle PP1c (1.6 g) at 30 °C for 45 mation of intramolecular disulfides and/or differences in oxida- min in a total volume of 40 l of kinase buffer. Phosphorylated and total tion state of the multiple Cys residues in the catalytic subunits. PP1c from the supernatant and Cdk2 in the pellet were analyzed by Regardless, the results show IR caused loss of P indicating immunoblot analysis. that PP1 is phosphorylated in living cells and IR causes net PP1 Activity Assay—PP1 was immunoprecipitated with 5 g of anti- PP1c monoclonal antibody from either an aliquot of the purified PP1c- dephosphorylation. Cdk2 reaction mixture (0.5 g of PP1c diluted in 1 ml of PP1 activity Effects of IR on Nuclear PP1 in Jurkat and AT Cells—PP1 lysis buffer (see “Preparation of Nuclear Extracts”)) or from nuclear and  isoforms contain a C-terminal RP(I/V)TPPR motif known lysis solution (1.0 mg of nuclear protein) prepared from Jurkat, FT/ to be a preferred site for Cdk/Cdc phosphorylation (23). Phos- pEBS7, and FT/pEBS7-YZ5. The immune complexes were then incu- phorylation of the threonine in this motif occurs in yeast as well bated with 30 l of a 50% suspension of protein A-agarose beads washed as mammalian PP1 (24) and attenuates PP1 activity. We and three times with ice-cold lysis buffer followed by ice-cold Ser/Thr assay buffer (50 mM Tris-HCl, pH 7.0, 0.1 mM CaCl ). PP1 activity was others (25, 26) have shown that IR inhibits Cdk2/cyclin A and assayed using a Ser/Thr phosphatase assay kit (Upstate Biotechnology Cdk2/cyclin E activity, and both of these kinases phosphorylate Inc.). The PP1 immune complex beads in 50 l of Ser/Thr assay buffer PP1 at Thr-320. We reasoned that DNA damage might acti- were incubated with the phosphopeptide KRpTIRR at 30 °C for 30 min. vate PP1 through reduced phosphorylation of this Thr site. The beads were pelleted, and 25 l of supernatant was analyzed for free Jurkat cells were irradiated and nuclear extracts were assayed phosphate in the malachite green assay by dilution with 100 lof for phosphatase activity at various times post-IR (Fig. 2a). developing solution (malachite green). After incubation for 15 min, the There was more than a doubling in PP1 activity over 90 min. In release of phosphate was quantified by measuring the absorbance at 620 nm in a microtiter plate reader. parallel nuclear extracts were subjected to immunoblot analy- 41758 IR Activates PP1 in an ATM-dependent Manner TABLE I Tandem mass spectrometry identification of dephosphorylated proteins Spot Identified as Peptide mass (Da) pI Sequence Accession NCBI no. 1 PP1C/ 37.5 5.94 (16–26) LLEVQGSRPGK 4506003 (27–36) NVQLTENEIRG (306–317) YGQFSGLNPGGR PP1C/ 37.2 5.84 (26–35) IVQMTEAEVR 4506005 (132–140) IYGFYDECK (168–186) IFCCHGGLSPDLQSMEQIRRI (304–319) YQYGGLNSGRPVTPPR 2 PP1C/ 37.5 5.94 (61–74) ICGDIHGQYYDLLR 4506003 (235–246) FLHKHDLDLICR (306–323) YGQFSGLNPGGRPITPPR PP1C/ 37.2 5.84 (111–121) IKYPENFFLLR 4506005 (150–167) TFTDCFNCLPIAAIVDEK (246–259) AHQVVEDGYEFFAK 3 PP1C/ 37.5 5.94 (75–96) LFEYGGFPPESNYLFLGDYVDR 4506003 PP1C/ 37.2 5.84 (60–73) ICGDIHGQYTDLLR 4506005 (111–121) IKYPENFFLLR (168–186) IFCCHGGLSPDLQSMEQIR (246–260) AHQVVEDGYEFFAKR 4 PP1C/ 37.5 5.94 (16–26) LLEVQGSRPGK 4506003 (27–36) NVQLTENEIR (222–234) GVSFTFGAEVVAK (306–323) YGQFSGLNPGGRPITPPR PP1C/ 37.2 5.84 (98–110) QSLETICLLLAYK 4506005 (132–141) IYGFYDECKR (168–186) IFCCHGGLSPDLQSMEQIR (246–260) AHQVVEDGYEFFAKR FIG.2. a, IR activates nuclear PP1. Jurkat cells (circles) were sub- jected to 10 Gy, and nuclear extracts were prepared at various times post-IR. PP1c was then immunoprecipitated, and its activity was meas- FIG.3. Thr-320 phosphorylation influences PP1 activity. Puri- ured using the phosphopeptide substrate KRpTIRR as described under fied catalytic subunit of rabbit skeletal muscle PP1 was incubated for 30 “Materials and Methods.” Quantitation (squares) of the optical density min at 30 °C with the indicated concentrations of purified Cdk2 kinase of the immunoblot shown in b was measured by Image Quant 5.0 as described under “Materials and Methods.” a, an aliquot of the reac- (Molecular Dynamics, Sunnyvale, CA). b, IR induces dephosphorylation tion mixture was subjected to immunoblot analysis with phospho- on Thr-320 of nuclear PP1 catalytic subunit. Asynchronously growing PP1c/ Thr-320 antibody and anti-PP1c. Quantitation (squares)ofthe Jurkat cells received either 0 or 10 Gy irradiation, and nuclear extracts optical density of the immunoblot is shown in b. b, PP1c was immuno- were prepared at the indicated times post-IR and subjected to immu- precipitated from the remaining reaction mixture and its activity was noblot analysis with phospho-PP1c (Thr-320) and anti-PP1c. measured as described for Fig. 2a. sis with a phospho-PP1 (Thr-320) antibody. As shown in Fig. crease in PP1 activity. PP1 levels themselves, which serve as a 2, a and b, 10 Gy resulted in a time-dependent dephosphoryl- loading control, were unchanged, arguing that there was in- ation of Thr-320 of PP1, which parallels the IR-induced in- creased specific activity, not synthesis or import of PP1. IR Activates PP1 in an ATM-dependent Manner 41759 FIG.4. IR decreases the ability of endogenous Cdk2 to phos- phorylate the Thr-320 site in PP1c. Jurkat cells were subjected to 10 Gy, and Cdk2 was immunoprecipitated from nuclear extracts at various times post-IR. The pellet was incubated with purified PP1c at 30 °C for 45 min as described under “Materials and Methods.” The supernatant was subjected to immunoblot analysis with phospho-PP1c/ (Thr-320) antibody and anti-PP1c. To verify that Thr-320 phosphorylation regulates PP1 activ- ity under our assay conditions, purified skeletal muscle PP1 protein was incubated with purified Cdk2 kinase and subjected to immunoprecipitation with anti-PP1c. PP1 activity in the immunoprecipitates was assayed with a phosphopeptide as substrate. As expected, increasing doses of Cdk2 resulted in increased phosphorylation of the Thr-320 site (Fig. 3a). Phos- phorylation of this site in PP1 corresponded to decreased PP1 activity (Fig. 3b). Thus, in vitro and in living cells the level of Thr-320 phosphorylation inversely correlated with the specific activity of PP1. To establish that IR inhibited the activity of the endogenous Cdk2 kinase that was phosphorylating PP1, Cdk2 was immuno- precipitated at various intervals after 10 Gy. As shown in Fig. 4, the activity of endogenous Cdk2 toward PP1 at the Thr-320 site decreased in a time-dependent manner following IR. IR Both Decreases Cdk2 Kinase and Increases PP1 Phospha- tase Activity in an ATM-dependent Manner—We previously found that IR-induced dephosphorylation of histone H1 is ATM- dependent (14). This is because of reduced Cdk2 kinase and increased nuclear phosphatase activity. IR (10 Gy) inhibits Cdk2 activity in an ATM-dependent manner as shown in Fig. 5a. Cdk2 from AT (PEBS) cells lacking active ATM kinase was inhibited only 40% whereas Cdk2 from reconstituted AT cells (YZ-5) that express ATM was inhibited by 84%. The radiation- induced dephosphorylation of PP1 Thr-320 was ATM-depend- ent. We compared AT cells (PEBS) and AT cells transfected with full-length ATM (YZ-5) that were irradiated with a dose of 10 Gy. Nuclear extracts were subjected to immunoblot analysis that showed IR failed to dephosphorylate PP1 Thr-320 in AT cells (Fig. 5b). However, in AT cells transfected with full- length ATM, IR-induced dephosphorylation of Thr-320 occurred in a time-dependent manner starting 15 min post- irradiation. This genetic system was used to demonstrate FIG.5. IR-induced nuclear PP1c dephosphorylation and acti- that IR-induced dephosphorylation of the Thr-320 site of PP1 vation are ATM-dependent. a, in vitro H1 kinase activity was deter- is dependent on ATM. mined on immunoprecipitates with antibodies to Cdk2 at various times The ATM kinase is also necessary for the IR-induced in- following 10 Gy. All experiments were performed 3 times with similar crease in PP1 activity. Nuclear extracts were prepared from AT results, and a representative experiment is shown. Equal loading of H1 and Cdk2 is shown as controls. Percentage inhibition of kinase activity cells deficient in ATM and AT cells expressing ectopic full- was determined by excising H1 bands from the gel and measuring P length ATM. Samples were subjected to immunoprecipitation incorporation by liquid scintillation counting. b, AT fibroblasts trans- with anti-PP1 and assayed for PP1 activity. As shown in Fig. fected with either empty vector FT/pEBS7 (PEBS) or recombinant wild 5c, the IR-induced time-dependent 2-fold increase in PP1 ac- type ATM FT/pEBS7-YZ5 (YZ-5) were irradiated with 10 Gy, and nu- clear extracts were subjected to immunoblot analysis with either a tivity was only observed in cells expressing ATM. Interestingly, phospho-PP1c (Thr-320) antibody or anti-PP1c. c, AT fibroblasts PEBS the time course of IR activation of PP1 parallels that of and YZ-5 were irradiated with 10 Gy, and PP1c was immunoprecipi- IR-induced H1 dephosphorylation, which we have previously tated from nuclear extracts and assayed for PP1 activity as described reported (14). for Fig. 2a. Three genes code for four distinct isoforms of PP1 in mam- mals called , 1, 2, and  (3). Using isoform-specific antibod- sponding sequence motif in both these PP1 isoforms. Consist- ies to immunoprecipitate PP1c  and  from cell lysates, we ent with the metabolic P-labeling results (Fig. 1), both the tested if the Thr-320 site was dephosphorylated in response to and  PP1 isoforms were dephosphorylated at the Thr-320 site IR in Jurkat, AT, and reconstituted AT cells. The numbering of in response to IR (Fig. 6a). Furthermore, the dephosphoryl- the Thr-320 is not identical in  and  but lies in the corre- ation of both the  and  isoforms was ATM-dependent, as 41760 IR Activates PP1 in an ATM-dependent Manner tase (Cdc25) would be regulating another phosphatase (PP1) via a phosphatase-kinase-phosphatase cascade (Cdc25-Cdk2- PP1). Our data do not establish that Cdk2 is the in vivo kinase of PP1. There may be other kinases that phosphorylate the Thr-320 site in PP1, but they would also need to be regulated in response to IR by an ATM-dependent pathway. Alternatively, ATM could directly (or indirectly) phosphorylate a nuclear PP1 subunit that restrains PP1 in its phosphorylated, low activity form. Phosphorylation would release inhibition and allow autodephos- phorylation of Thr-320 and activation of PP1 toward other sub- strates. NIPP is one example of a nuclear PP1 regulatory subunit whose activity is regulated by phosphorylation (35, 36). What are the downstream nuclear substrates of PP1 that may be critical in the damage response? Unlike protein serine/ threonine kinases, PP1 catalytic subunit does not manifest a FIG.6. IR-induced dephosphorylation of PP1  and PP1  is ATM-dependent. Jurkat (a) and AT fibroblasts PEBS and YZ-5 (b) high degree of sequence specificity and dephosphorylates mul- were irradiated with 10 Gy. Nuclear extracts were prepared and sub- tiple substrates. The substrate specificity of PP1 is thought to jected to immunoprecipitation with the indicated isoform-specific anti- be modulated through the formation of heterodimeric com- PP1c antibodies. Immunoblot analysis was performed as described for plexes with regulatory subunits. Regulatory proteins that tar- Fig. 2. IP, immunoprecipitate. get PP1 to its substrates in response to DNA damage are not known, and these may bind both  and  isoforms or there may shown in Fig. 6b. Decreased phosphospecific staining of PP1 be individual regulatory subunits for these isoforms. Specific and  was only seen in irradiated cells that were expressing PP1 isoforms have been shown to have distinct functions and ATM (YZ-5 clone). Cells that did not express ATM (PEBS) did locations. For example, the PP1 isoform has been shown to not show diminished phospho-Thr-320 staining in response regulate the G /S transition by dephosphorylating Rb (12), and to IR. 1 the PP1 isoform is chromatin-associated. Because histone H1 DISCUSSION and histone H3 have been implicated as PP1 substrates (14, 16, IR is known to activate the G , S, and G checkpoints (2, 27) 37), it is likely that the PP1 isoform may regulate IR-induced 1 2 in an ATM-dependent manner. The ATM gene is mutated in H1 and H3 dephosphorylation. the autosomal recessive disease ataxia telangiectasia, charac- In summary, the PP1  and  isoforms are dynamically terized by neuronal degeneration resulting in ataxia, oculocu- phosphorylated in Jurkat cells and in response to IR become taneous telangiectasia, immune dysfunction, and cancer pre- activated by dephosphorylation of Thr-320 to function as a disposition (28). ATM is thought to be an upstream sensor of downstream effector of an ATM-dependent damage-sensing DNA damage as well as oxidative stress. ATM transmits the pathway. Regardless of the mechanism by which ATM acti- damage signal downstream through its C-terminal phospho- vates PP1, defining the regulatory subunits that target PP1 to inositol 3-kinase domain. ATM has been shown to directly its respective substrates may reveal novel targets for chemo phosphorylate several proteins and to enhance the phosphoryl- and radiation sensitizers. 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Published: Nov 1, 2002

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