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Actopaxin Interacts with TESK1 to Regulate Cell Spreading on Fibronectin *

Actopaxin Interacts with TESK1 to Regulate Cell Spreading on Fibronectin * THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 22, Issue of June 3, pp. 21680–21688, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Actopaxin Interacts with TESK1 to Regulate Cell Spreading on Fibronectin* Received for publication, January 20, 2005, and in revised form, March 24, 2005 Published, JBC Papers in Press, April 6, 2005, DOI 10.1074/jbc.M500752200 David P. LaLonde, Michael C. Brown, Brian P. Bouverat‡, and Christopher E. Turner§ From the Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York 13210 proteins, including the LIM kinases (LIMK) (1). Closely re- The focal adhesion protein actopaxin contributes to integrin-actin associations and is involved in cell adhe- lated to the LIM family of kinases are TESK1 and TESK2 sion, spreading, and motility. Herein, we identify and (testicular protein kinases), which were originally identified characterize an association between actopaxin and the in testicular cells but have since been found to be ubiqui- serine/threonine kinase testicular protein kinase 1 tously expressed (2–5). Unlike LIMK, regulation of TESK1 (TESK1), a ubiquitously expressed protein previously activity by Rho GTPases has not been confirmed, although reported to regulate cellular spreading and focal adhe- recent studies in Drosophila implicate a role in the Rac sion formation via phosphorylation of cofilin. The inter- pathway associated with both eye development and spermat- action between actopaxin and TESK1 is direct and the ogenesis (6). However, this kinase has been shown to be binding sites were mapped to the carboxyl terminus of activated upon matrix adhesion and is regulated by binding both proteins. The association between actopaxin and of 14-3-3 and Sprouty4 (7, 8). TESK1 is negatively regulated by adhesion to fibronec- TESK1, as is the case with LIMK, regulates integrin-depend- tin, and a phosphomimetic actopaxin mutant that pro- motes cell spreading also exhibits impaired binding to ent focal adhesion assembly and actin organization through TESK1. Binding of actopaxin to TESK1 inhibits TESK1 phosphorylation of the amino terminus of the F-actin-severing kinase activity in vitro. Expression of the carboxyl ter- protein cofilin (3). Phosphorylation on serine 3, which is re- minus of actopaxin has previously been reported to re- versed by the serine phosphatases slingshot and chronophin, tard cell spreading. This effect was reversed following has been shown to decrease cofilin activity by interfering with overexpression of TESK1 and was found to be depend- its ability to bind F-actin (9–11). Cofilin is a critical regulator ent on an inability of actopaxin carboxyl terminus ex- of both growth factor and matrix-dependent actin reorganiza- pressing cells to promote cofilin phosphorylation upon tion, affecting lamellipodia formation, cell spreading, motility, matrix adhesion and caused by retention of TESK1 by and polarity (12–16). For instance, cofilin’s F-actin severing this actopaxin mutant. Thus, the association between activity potentiates Arp2/3-mediated actin assembly that is actopaxin and TESK1, which is likely regulated by phos- phorylation of actopaxin, regulates TESK1 activity and required for epidermal growth factor-induced lamellipodia for- subsequent cellular spreading on fibronectin. mation (17). The focal adhesion protein actopaxin is the -isoform of the parvin family and binds actin through a pair of calponin homol- Integrin-mediated adhesion to the extracellular matrix ogy (CH) domains (18–20). It interacts with the integrin-linked (ECM) leads to extensive actin reorganization that is regu- kinase (ILK) and the focal adhesion scaffolding proteins paxillin lated predominantly by the Rho family of GTPases, Cdc42, Rac, and Hic-5 (18, 21, 22). Affixin, the -isoform of the parvin family, and Rho, to stimulate formation of the actin-dependent struc- has been found to bind the putative Rac/Cdc42 guanine nucleo- tures filopodia, lamellipodia, and stress fibers, respectively (1). tide exchange factor PIX suggesting the possibility of this inter- Activated Rho family members interact with numerous effec- action for actopaxin as well (23). The associations between acto- tors, including the p21-associated kinase (PAK), the Wiskott- paxin, ILK, and paxillin constitute an evolutionarily conserved Aldrich Syndrome protein (WASP), and the Rho-associated integrin-actin linkage important in muscle cytoarchitecture and kinase (ROCK). PAK and ROCK share some common target contribute to the regulation of cellular spreading and adhesion in mesenchymal cells (18, 22, 24–26). Recent evidence suggests that * This work was supported by National Institutes of Health Grant Erk-dependent phosphorylation of the actopaxin amino terminus RO1 HL070244 (to C. E. T.). The costs of publication of this article were regulates cellular spreading and motility via modulation of Rho defrayed in part by the payment of page charges. This article must family signaling (27). therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. In this study we performed a yeast two-hybrid screen of a ‡ Current address: Dept. of Pharmacology and Physiology, University human placenta library using actopaxin as bait and identified of Rochester Medical Center, Rochester, NY 14642. TESK1 as a direct binding partner. We have established that § To whom correspondence should be addressed: Dept. of Cell and Developmental Biology, State University of New York Upstate Medical an association between actopaxin and TESK1 negatively regu- University, 750 East Adams St., Syracuse, NY 13210. Tel.: 315-464- lates TESK1 kinase activity and thus phosphorylation of cofi- 8598; Fax: 315-464-8535; E-mail: [email protected]. lin. Furthermore, this association is negatively modulated by The abbreviations used are: ECM, extracellular matrix; aa, amino adhesion to fibronectin, most likely through phosphorylation of acids; CH, calponin homology; Erk, extracellular signal-regulated ki- nase; FAK, focal adhesion kinase; ILK, integrin-linked kinase; LIMK, the amino terminus of actopaxin. Consequently, the association LIM kinase; MBP, myelin basic protein; TESK1 and TESK2, testicular between TESK1 and actopaxin provides a mechanism for the protein kinases 1 and 2; XAC, Xenopus actin depolymerizing factor/ regulation of cell spreading and potentially cell migration via cofilin; PAK, p21-associated kinase; ROCK, Rho-associated kinase; GFP, green fluorescent protein. modulation of cofilin activity. 21680 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Actopaxin Regulation of TESK1 Function 21681 EXPERIMENTAL PROCEDURES Myc-TESK1 was then resuspended in reaction buffer and incubated at room temperature for 30 min in the presence of 5 Ci of [- P]ATP, 10 Antibodies and Materials—Human plasma fibronectin was purchased g of focal adhesion kinase (MBP), and various GST-actopaxin con- from Sigma or BD Biosciences. Monoclonal antibody to the Xpress tag was structs as indicated. The reaction was terminated by boiling in sample purchased from Invitrogen. -Actinin and MOPC monoclonal antibodies buffer and the samples resolved on a 15% SDS-PAGE gel, followed were obtained from Sigma. Focal adhesion kinase (FAK) and ILK mono- by Coomassie Blue staining and autoradiography. Phosphorylation clonal antibodies were purchased from BD Transduction Laboratories. levels were quantitated with a Storm PhosphorImager (Amersham The 9E10 anti-Myc monclonal antibody developed by J. M. Bishop was Biosciences). obtained from the Developmental Studies Hybridoma Bank developed Respreadings and Immunofluorescence—These assays were per- under the auspices of the NICHD and maintained by The University of formed as described previously (27, 31). Area values obtained were Iowa, Department of Biological Sciences, Iowa City, IA. Omni-probe (di- compared with GFP-expressing control cells in the same experiment rected against region of Xpress epitope tag) and GFP polyclonal antibodies and further normalized against attached, unspread cells to quantitate were obtained from Santa Cruz Biotechnology. Polyclonal antibody to comparative spreading capacities. For phosphocofilin staining, cover- cofilin phosphorylated upon serine 3 was provided by Dr. James Bamburg slips were fixed in cytoskeletal stabilization buffer (3.7% formaldehyde, (Colorado State University) (28). 0.32 M sucrose, 10 mM MES, pH 6.1, 3 mM MgCl , 138 mM KCl, 2 mM Yeast Two-hybrid Assay—The MoBiTec Grow’n’Glow Two-Hybrid EGTA) for 15 min, then processed as described (27, 31). System (MoBiTec, Goettingen, Germany) based on the Brent LexA Interaction Trap System was used to identify proteins that interact RESULTS with actopaxin. Full-length rat actopaxin and the “nonspecific” protein bait, mouse p53 (aa 72–390), were cloned into pEG202 (His selectable Actopaxin Interacts with TESK1 in Vitro—To identify acto- marker) as a COOH-terminal fusion to LexA. To moderate the inherent paxin-binding proteins, a yeast two-hybrid screen was per- transactivation potential of full-length actopaxin, we utilized the low formed with full-length rat actopaxin fused to LexA as a bait. sensitivity Saccharomyces cerevisiae strain EGY188 that contains two Ten positive clones were isolated from the screening of 1  10 copies of the LexA operator upstream of the LEU2 reporter integrated recombinant colonies. To confirm the observed interactions, in the genome. A GFP reporter plasmid (pGNG1) with a URA3 select- selected clones were subjected to an additional round of screen- able marker was used in place of a -galactosidase reporter, allowing for simple screening by UV transillumination. A human placenta cDNA ing using either p53 as a nonspecific protein bait or actopaxin. library in pJG4-5 (TRP1 selectable marker), fused to the activation Two of the plasmids that were positive for actopaxin binding domain B42, was employed in the screen. Plasmids were sequentially and at the same time negative for p53 association encoded transformed by lithium acetate, into EGY188. Positive pJG4-5 library/ sequence representing the serine/threonine kinase testicular prey plasmids were isolated, and a directed interaction of the positive, protein kinase 1 (TESK1) (data not shown). isolated library/prey with nonspecific protein bait, p53, was performed. One plasmid insert contained DNA encoding aa 529–626, A directed interaction with actopaxin plasmid was also performed to confirm the association. True positive clones were sequenced at the comprising the carboxyl terminus of TESK1, as well as 80 BioResource Center of Cornell University (Ithaca, NY). nucleotides from the 3-untranslated region. The second plas- Plasmids—To generate full-length human TESK1, PCR primer pairs mid contained aa 544–626 and 74 nucleotides from the 3- were synthesized representing the 5 and 3 termini of the published untranslated region. It is notable that the principal difference TESK1 coding sequence incorporating EcoRI and XbaI restriction sites. between TESK1 and its family member TESK2 is the presence A human heart cDNA library (Clontech) was used for PCR amplifica- of this proline-rich carboxyl terminus extension within tion. The product obtained was identical to that reported previously, except that nucleotides 31–111, representing amino acids 11–37, were TESK1 (5). absent. Notably this stretch of coding sequence represents human exon An association between actopaxin and TESK1 was first con- 2 suggesting the existence of a new TESK1 splice isoform. Full-length firmed using GST binding assays. These studies were re- rat TESK1 and TESK2 Myc-tagged constructs were generously pro- stricted to the analysis of exogenous TESK1 due to the lack of vided by Dr. Kensaku Mizuno (Tohoku University, Sendai, Japan) (2, an available antibody to the endogenous form. Myc-tagged 4). A pGEX-4T TESK1 carboxyl-terminal (aa 529–626) fusion protein TESK1 and TESK2 were expressed in HeLa cells and actopaxin was generated for use in precipitation assays. A construct encoding binding tested using GST-actopaxin fusion proteins. TESK1 TESK1 1–528 with an amino-terminal GFP tag was created via PCR amplification. Actopaxin constructs were as described previously (18, was found to specifically bind full-length GST-actopaxin (Fig. 22, 27, 29). GFP-XAC (Xenopus actin depolymerizing factor/cofilin), 1A). In contrast, GST-actopaxin did not precipitate TESK2, hereafter referred to as GFP-cofilin, constructs were provided by Dr. consistent with the absence of the carboxyl-terminal extension James Bamburg (Colorado State University) (30). of TESK1 in this isoform (Fig. 1A). The binding of ILK served Cell Culture and Transfections—HeLa cells were maintained in as a positive control. Additional GST pull-down assays estab- Dulbecco’s modified Eagle’s medium (Mediatech) supplemented with lished the carboxyl-terminal actopaxin amino acids 223–372 as 10% fetal bovine serum (Atlanta Biologicals). Transfections were performed with FuGENE 6 (Roche Applied Science) according to the binding region for TESK1 (Fig. 1, B and C). This region of manufacturer’s protocols. actopaxin consists of a portion of the intra-CH linker domain Binding Assays—GST binding assays were performed essentially as and the second CH domain. It also contains the binding sites described previously (18). For in vivo binding experiments, cells were for paxillin and ILK (18, 22). lysed in co-immunoprecipitation buffer (50 mM Tris-HCl, pH 7.6, 0.5% The yeast two-hybrid screen identified the carboxyl-terminal Nonidet P-40, 100 mM NaCl, 10% glycerol, 1 mM MgCl ,10 g/ml 98 residues of TESK1 as the site of interaction with actopaxin. leupeptin, 1 mM NaVO ,and1mM NaF). Lysates were centrifuged to remove cellular debris, and then Xpress-actopaxin was precipitated A construct was created consisting of this portion of TESK1 using Omni-probe antibody (anti-epitope tag of Xpress-actopaxin) and (529–626) fused to the carboxyl terminus of GFP. Pull-down protein A/G beads (Santa Cruz Biotechnology). Immunoprecipitated assays confirmed the binding of GFP-TESK1 529–626 to GST- proteins were subsequently solubilized in sample buffer and analyzed actopaxin (Fig. 2A). Furthermore, a GST fusion construct of by Western blotting. this region of TESK1 was created to verify binding with acto- In Vitro Kinase Assays—Kinase assays were performed essentially as paxin. GST binding assays performed using HeLa cell lysates described previously (3). HeLa cells expressing Myc-TESK1 were lysed in kinase immunoprecipitation buffer (50 mM Tris-HCl, pH 7.6, 150 mM demonstrated binding of endogenous actopaxin to GST-TESK1 NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl flu- 529–626, while the GST-paxillin LD4 motif served as a posi- oride, 10 g/ml leupeptin), incubated on ice, and centrifuged to remove tive control for actopaxin binding, as reported previously (Fig. cellular debris. The resulting supernatant was incubated at 4 °C for 2 h 2B) (18). It has been suggested that ILK and actopaxin are with protein A/G beads and either control antibody (MOPC) or 9E10 obligate binding partners (32). However, ILK was not present anti-Myc antibody. Immune complexes were then washed three times in in the GST-TESK1 precipitate, indicating that TESK1 can bind wash buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% Nonidet P-40) a pool of actopaxin that is not concurrently bound to ILK (Fig. and twice in reaction buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM NaF, 1 mM NaVO ,5mM MnCl ,5mM MgCl ). Immunoprecipitated 2B). Conversely, the presence of ILK and actopaxin in the LD4 4 2 2 21682 Actopaxin Regulation of TESK1 Function FIG.2. The carboxyl terminus of TESK1 binds a pool of acto- paxin that is not associated with ILK. A, HeLa cells were trans- fected with GFP-TESK1 amino acids 529–626 and then lysed and incubated with either GST or GST-actopaxin fusion proteins. GFP- TESK1 529–626 was precipitated by GST-actopaxin but not by the GST control. GST-actopaxin also bound paxillin, as described previously, but not -actinin. B, HeLa lysates were incubated with GST, GST-TESK1 529–626, or GST-paxillin LD4. Bound proteins were analyzed by SDS- PAGE and blotted with antibodies to ILK, actopaxin, and -actinin. Actopaxin was precipitated by both GST-paxillin LD4 and GST-TESK1 529–626. However, ILK was only co-precipitated in the GST-paxillin FIG.1. TESK1 binds the carboxyl terminus of actopaxin in LD4 lane. These results indicate that TESK1 binds to a pool of acto- vitro. A, HeLa cells were transfected with either Myc-TESK1 or Myc- paxin, which does not concurrently bind ILK. TESK2 then lysed in GST binding buffer and subjected to pull-down assays with both GST and GST full-length actopaxin. Samples were analyzed by Western blotting with antibodies to -actinin, ILK, and lamellipodia extension associated with cell migration. To de- Myc (9E10). GST-actopaxin precipitated TESK1 but not TESK2, while termine whether the interaction between TESK1 and acto- -actinin served as a negative control. ILK served as a positive control paxin might be modulated during cell spreading, we performed for GST-actopaxin binding. B, to delineate the region of actopaxin co-immunoprecipitations of co-transfected proteins in HeLa involved in TESK1 binding, GST fusion protein pull-down binding assays were performed as above, with the addition of amino-terminal cells that were growing asynchronously in culture versus cells (amino acids 1–222) and carboxyl-terminal (amino acids 223–372) GST- that had been actively spreading upon 10 g/ml fibronectin for actopaxin fusion proteins. TESK1 was precipitated with full-length and 90 min. Actopaxin precipitated Myc-TESK1 less efficiently in carboxyl-terminal actopaxin, while -actinin served as a negative con- spreading cells than unstimulated cells (Fig. 4A). trol. C, Coomassie Blue staining of GST fusion proteins. Lane 1, GST; lane 2, GST-actopaxin 1–372; lane 3, GST-actopaxin 1–222; and lane 4, Adhesion-dependent phosphorylation of actopaxin affects GST-actopaxin 223–372. cell spreading and migration. This post-translational modifica- tion of actopaxin may exert its effects by altering its association pull-down shows that existing actopaxin/ILK associations were with various binding partners. To test whether this is the case not disrupted in these lysates as paxillin LD4 only binds ILK for TESK1, HeLa cells were co-transfected with Myc-TESK1 indirectly through actopaxin (22, 33). and either wild type or phosphomimetic (S4/8D) Xpress-acto- Actopaxin and TESK1 Interact in Vivo—The association be- paxin constructs (27). Immunoprecipitations using an antibody tween TESK1 and actopaxin was demonstrated in vivo using to the actopaxin epitope-tag showed that the phosphomimetic co-immunoprecipitation experiments. Epitope-tagged versions of S4/8D Xpress-actopaxin failed to precipitate Myc-TESK1, these proteins were co-expressed in HeLa cells and immunopre- while the wild-type Xpress-actopaxin displayed robust binding cipitated using a polyclonal antibody (Omni-probe) to the epitope- (Fig. 4B). The specificity of this result was confirmed by the tag of actopaxin. Myc-TESK1, but not FAK, was co-immunopre- observation that ILK was precipitated by both Xpress-acto- cipitated with full-length Xpress-actopaxin (Fig. 3A). Consistent paxin constructs (Fig. 4B). with the GST pull-down assays, TESK1 co-immunoprecipitated As the sites of actopaxin phosphorylation are on the amino with the carboxyl terminus of actopaxin (aa 223–372) (Fig. 3B). terminus of the protein, while the carboxyl terminus mediates FAK served as a negative control, while ILK binding to the TESK1 binding, the actopaxin phosphorylation-dependent ab- actopaxin carboxyl terminus was used as a positive control (Fig. rogation of TESK1 binding may involve allosteric regulation 3B). These data demonstrate that actopaxin and TESK1 are between the amino and carboxyl terminus of actopaxin. Thus, associated in asynchronously growing cells. we next co-transfected Myc-TESK1 with either full-length (aa The Association between TESK1 and Actopaxin Is Negatively 1–372) or carboxyl-terminal (aa 223–372) Xpress-actopaxin Regulated during Adhesion to Fibronectin—Cell attachment constructs. Notably, the carboxyl terminus of actopaxin con- and spreading on the ECM leads to the activation of integrins tains the TESK1 binding site but lacks the phosphorylation and, in turn, Rho GTPases and is a widely accepted model for sites (Fig. 1B). Both populations of cells were placed in suspen- Actopaxin Regulation of TESK1 Function 21683 sion proteins as compared with GST alone (Fig. 5A,B). Both of these actopaxin fusion proteins bind TESK1 (Fig. 1B). Con- versely, a GST fusion protein consisting of the amino terminus (aa 1–222) of actopaxin, which does not bind TESK1, failed to inhibit TESK1 kinase activity (Fig. 1B and Fig. 5, A and B). Therefore, Myc-TESK1 kinase activity is reduced by interac- tion with actopaxin. Interestingly, the difference in activity of TESK1 in the presence versus the absence of actopaxin that we observed is equivalent to the relative changes in TESK1 activ- ity that occurred upon adhesion to fibronectin (3), with a near doubling of kinase activity. A low level of phosphorylation of the actopaxin amino terminus (aa 1–222) and full-length GST fusion proteins was also observed in these assays, suggesting TESK1 kinase activity may, in turn, regulate actopaxin func- tion in vivo via direct phosphorylation (Fig. 5C). Expression of the Carboxyl Terminus of Actopaxin Inhibits Adhesion-dependent Cofilin Phosphorylation—Since the car- boxyl terminus of actopaxin retains binding to TESK1 during adhesion and inhibits its kinase activity in vitro, this mutant can be used as a tool to evaluate the effects on cell function of alteration of the physiologic association between TESK1 and actopaxin. Specifically, we evaluated how introduction of this mutant influenced phosphorylation of the TESK1 substrate cofilin during cell spreading on fibronectin. HeLa cells were co-transfected with GFP-cofilin and either Xpress-actopaxin 223–372 or Xpress--galactosidase, as a control, and respread on 10 g/ml fibronectin-coated culture dishes. Lysates were collected in suspension and at 30, 60, and 120 min post-re- spreading. Phospho-GFP cofilin levels were then measured by Western blotting with an antibody specific for phosphorylation on serine 3, the site phosphorylated by TESK1 that inactivates cofilin. Phosphoserine 3 GFP cofilin levels are diminished in the Xpress-actopaxin 223–372-expressing cells during spread- FIG.3. TESK1 associates with actopaxin in vivo. A, HeLa cells ing on fibronectin (Fig. 6A). This assay was repeated following were co-transfected with Myc-TESK1 and Xpress full-length actopaxin the co-transfection of Myc-TESK1 to determine whether the then lysed in co-immunoprecipitation buffer. Xpress-actopaxin immu- observed deficit of cofilin phosphorylation could be rescued. noprecipitations were performed with Omni-probe polyclonal antibody Indeed, introduction of TESK1 was found to rescue the cofilin (which recognizes a region of the actopaxin epitope tag) and subjected to Western blotting with FAK, 9E10, and Xpress monoclonal antibodies. phosphorylation defect observed in the Xpress-actopaxin 223– Myc-TESK1 is specifically co-immunoprecipitated with actopaxin. FAK 372 cells at the 90-min time point (Fig. 6B, lanes 4 and 5). served as a negative control. B, HeLa cells were co-transfected with TESK1 Rescues the Spreading Defect in Cells Expressing the Myc-TESK1 and either amino-terminal (1–222) or carboxyl-terminal Actopaxin Carboxyl Terminus—The importance of the TESK1/ (223–372) Xpress-actopaxin constructs. Immunoprecipitations were performed as above. FAK served as a negative control. As with the in actopaxin association in regulating cell morphology was exam- vitro experiments, TESK1 interacted with the carboxyl-terminal region ined by evaluating effects on cell spreading. Expression of of actopaxin. ILK also bound to the carboxyl terminus of actopaxin, as Xpress-actopaxin 223–372 impaired spreading of HeLa cells on has been reported. fibronectin at the 90-min time point, as has been reported previously (Fig. 7A) (18). Co-expression of Myc-TESK1 par- sion and then were allowed to adhere to 10 g/ml fibronectin tially rescued this spreading defect, while TESK1 had no ap- for 90 min, followed by immunoprecipitations of the actopaxin parent effect on spreading when overexpressed by itself, al- constructs. Binding of TESK1 to the carboxyl terminus of ac- though there was a mild increase in cortical actin as visualized topaxin was retained, while full-length actopaxin displayed a by rhodamine-phalloidin staining (Fig. 7A). Equivalent expres- diminished association upon adhesion (Fig. 4C). In contrast, sion of proteins in these experiments was confirmed by West- both Xpress-actopaxin constructs were able to precipitate ILK ern blotting (Fig. 7B). (Fig. 4C). These data suggest that an adhesion-dependent mod- We next performed quantitative analysis of spreading in ification of the actopaxin amino terminus, such as phosphoryl- these cells to confirm the TESK1-dependent rescue of the ob- ation, serves to regulate TESK1 binding to the carboxyl termi- served spreading defect caused by expression of Xpress-acto- nus of actopaxin. paxin 223–372. Quantitation of cell areas showed that Xpress- Actopaxin Binding Inhibits TESK1 Kinase Activity—Cell ad- actopaxin 223–372 reduced the spreading of HeLa cells to 37% hesion stimulates TESK1 kinase activity (3), which inversely of that observed in GFP control cells (Fig. 7C). Co-expression of correlates with actopaxin binding. Thus, we tested whether Myc-TESK1 significantly rescued the spreading of cells ex- actopaxin binding affected TESK1 kinase activity, as has been pressing Xpress-actopaxin 223–372 (p  0.01) (Fig. 7C). How- observed previously for the associations between TESK1 and ever, this rescue was incomplete, as cells expressing Myc- 14-3-3 and Sprouty4 (7, 8). Myc-TESK1 was immunoprecipi- tated, and in vitro kinase assays were performed in the pres- TESK1 with Xpress-actopaxin 223–372 were still significantly less well spread than GFP control cells (Fig. 7C). ence of GST-actopaxin fusion proteins using MBP as a sub- strate. The ability of Myc-TESK1 to phosphorylate MBP was An actopaxin mutant with an altered paxillin binding site reduced by 40–50% in the presence of both full-length (aa has previously been shown to be mislocalized and to also inhibit 1–372) and carboxyl-terminal (aa 223–372) GST-actopaxin fu- cell spreading when expressed in HeLa cells (18). We confirmed 21684 Actopaxin Regulation of TESK1 Function FIG.5. Actopaxin binding negatively regulates TESK1 kinase activity. A, HeLa cells expressing Myc-TESK1 were lysed, and Myc (9E10) immunoprecipitations were performed. Myc-TESK1 immune complexes were subjected to in vitro kinase assays in the presence of GST or GST-actopaxin fusion proteins using 10 g of MBP as a sub- strate. Lane 1, control immunoprecipitation using control mouse IgG (MOPC) and GST fusion protein; lane 2, Myc IP and GST; lane 3, Myc IP and GST-actopaxin 1–372; lane 4, Myc IP and GST-actopaxin 1–222; and lane 5, Myc IP and GST-actopaxin 223–372. Samples were resolved by SDS-PAGE followed by Coomassie Blue staining (A) and autoradi- ography (B). B, Coomassie staining of MBP (total MBP) and autora- diograph of MBP bands (phospho-MBP) following exposure to film. Quantitation of phospho-MBP was performed using a Storm Phosphor- Imager. The relative intensity of each signal was compared with the TESK1 kinase reaction in the presence of GST (lane 2) and is displayed as a percentage thereof. The results displayed are from a representative experiment. TESK1 kinase activity is inhibited in the presence of GST fusion proteins composed of 1–372 (lane 3) and 223–372 (lane 5) acto- paxin, both of which bind TESK1. These data show that TESK1 kinase activity is negatively regulated by actopaxin binding. C, autoradiograph of Coomassie-stained gel shown in A. Both the full-length and amino terminus actopaxin GST constructs are phosphorylated by TESK1. There is no apparent phosphorylation of the carboxyl terminus of acto- paxin or the GST control fusion proteins. this finding and also determined that overexpression of TESK1 is unable to rescue this phenotype, thereby indicating the spec- ificity of the rescue of the Xpress-actopaxin 223–372 spreading defect by TESK1 (Fig. 7C). The spreading defect of the paxillin binding site mutant caused by loss of actopaxin/paxillin asso- ciation therefore operates through some alternate pathway, possibly involving mislocalization of other actopaxin binding partners. Taken together, these results suggested that the spreading defect in Xpress-actopaxin 223–372-expressing cells is depend- ent upon altered cofilin signaling. Previous reports have dem- onstrated that phosphocofilin is localized in a region of the cell closely juxtaposed to the extending lamellipodia in a transition zone where actin filaments become stabilized (13). Thus, we examined phosphocofilin localization in cells actively spreading FIG.4. The association between TESK1 and actopaxin is de- upon fibronectin. GFP-transfected control cells exhibited mod- creased during cell spreading. A, HeLa cells were co-transfected est phosphocofilin immunostaining concentrated toward the with Xpress-actopaxin and Myc-TESK1. Omni-probe immunoprecipita- cell periphery, while Myc-TESK1 expression increased this tions for Xpress-actopaxin were then performed either from cells grow- ing in culture or from cells that had been spread on 10 g/ml fibronectin for 90 min. Actopaxin more readily precipitates TESK1 from asynchro- nously growing cells than from those spreading on fibronectin. -Acti- each actopaxin construct. C, HeLa cells were co-transfected with Myc- nin does not bind actopaxin in either condition. B, HeLa cells were TESK1 and either Xpress-actopaxin 1–372 (full-length) or 223–372 co-transfected with Myc-TESK1 and either phosphomimetic S4/8D or (carboxyl terminus). These cells were then spread on 10 g/ml fibronec- wild-type Xpress-actopaxin constructs. Xpress-actopaxin constructs tin for 90 min followed by Omni-probe immunoprecipitations. The as- were immunoprecipitated with Omni-probe polyclonal antibody, re- sociation between full-length actopaxin and TESK1 is diminished dur- solved using SDS-PAGE, and transferred to nitrocellulose. Myc-TESK1 ing spreading. However, the carboxyl terminus of actopaxin retains its co-immunoprecipitated with the wild-type but not the phosphomimetic association with TESK1. Conversely, both Xpress-actopaxin constructs S4/8D Xpress-actopaxin construct. Equivalent amounts of ILK bound to still precipitate ILK. Actopaxin Regulation of TESK1 Function 21685 FIG.6. Expression of the carboxyl terminus of actopaxin in- hibits adhesion-dependent cofilin phosphorylation. A, HeLa cells were co-transfected with GFP-cofilin and either -galactosidase (-Gal), as control, or the actopaxin carboxyl terminus (Xpress-actopaxin 223– 372). Cells were then placed in suspension for 1 h and either lysed or spread onto plates coated with 10 g/ml fibronectin. Samples were then collected at 30, 60, and 120 min and analyzed by SDS-PAGE for levels of phosphoserine 3 GFP-cofilin and total GFP-cofilin. Cells expressing the carboxyl terminus of actopaxin displayed reduced phosphocofilin levels upon cell attachment and spreading on fibronectin. B, to deter- mine whether TESK1 could rescue the observed cofilin phosphorylation defect, HeLa cells were transfected and respread as in A, with the exception that samples were collected solely at 90 min post-respread- ing. HeLa cells were transfected with: Xpress -galactosidase (lane 1); GFP-cofilin (lane 2), GFP-cofilin and Myc-TESK1 (lane 3), GFP-cofilin and Xpress-actopaxin 223–372 (lane 4), or GFP-cofilin, Myc-TESK1, and Xpress actopaxin 223–372 (lane 5). Total amounts of transfected cDNA were standardized with Xpress -galactosidase. Myc-TESK1 is able to rescue the defect in cofilin phosphorylation displayed in the cells FIG.7. TESK1 rescues the spreading defect in cells expressing expressing Xpress-actopaxin 223–372. the actopaxin carboxyl terminus. A, HeLa cells transfected with the indicated constructs were spread on 10 g/ml fibronectin-coated cover- phosphocofilin signal (Fig. 8). Consistent with our biochemical slips for 90 min and then processed for immunofluorescence. Trans- fected cells were determined by co-transfection with GFP and are indi- evaluation (Fig. 6), cells expressing Xpress-actopaxin 223–372 cated by asterisks. Cells expressing the actopaxin carboxyl terminus had diminished phosphocofilin staining, which was notably (Xpress-actopaxin 223–372) displayed impaired spreading upon fi- absent from the periphery when compared with an adjacent bronectin that was rescued by co-expression of Myc-TESK1. Bar,10 m. non-transfected cell. Importantly, phosphocofilin staining was B, lysates from experiments in A were blotted to demonstrate equiva- lent expression of proteins under each condition. C, cells were respread restored by co-expression of Myc-TESK1 with Xpress-actopaxin as in A, followed by area measurement and analysis as detailed under 223–372 (Fig. 8). No apparent difference in total cofilin cell “Experimental Procedures.” Error bars represent standard deviation. A staining was observed (data not shown). These data support a minimum of 40 cells was quantified per condition per trial (n was a role for actopaxin in regulating TESK1 signaling to cofilin. minimum of three per condition). * indicates a condition significantly different from GFP (p  0.01). The combination of Myc-TESK1 and We further tested a role for cofilin by evaluating the ability of Xpress-actopaxin 223–372 significantly increases spreading as com- phosphomimetic (S3E) cofilin constructs to rescue the spreading pared with cells expressing Xpress-actopaxin 223–372 (p  0.01). An on fibronectin in these cells. Significantly, co-expression of a Xpress-actopaxin construct containing a mutated paxillin binding site non-active S3E phosphomimetic cofilin construct with Xpress- (PBS) also displays impaired spreading. However, this defect is not actopaxin 223–372 was able to partially rescue spreading, simi- reversed by TESK1, indicating the specificity of the rescue of the Xpress-actopaxin 223–372 construct. lar to that seen with Myc-TESK1 (Fig. 9). Conversely, expression of an S3A construct, which mimics a non-phosphorylated, active cofilin, decreased spreading to levels comparable with those seen A TESK1 Construct That Does Not Bind Actopaxin Increases in HeLa cells expressing Xpress-actopaxin 223–372 (Fig. 9). Co- Cell Spreading on Fibronectin—To further examine the impor- expression of the S3A cofilin construct with Xpress-actopaxin tance of the actopaxin/TESK1 interaction in the regulation of 223–372 did not result in further inhibition of spreading (Fig. 9). cell spreading, we created a GFP-TESK1 1–528 construct, These data demonstrate that expression of the carboxyl ter- which lacks the actopaxin binding site but contains the kinase minus of actopaxin severely inhibits cell spreading on fibronec- domain and the autophosphorylation and previously character- tin in part through alteration of cofilin phosphorylation, most ized regulatory sites. GST binding assays confirmed the lack of likely mediated through the TESK1 kinase. The ability of binding of actopaxin to this construct (Fig. 10A). We then TESK1 to only partially rescue this phenotype indicates the expressed this construct in HeLa cells and respread them on 10 possible perturbation of other actopaxin interactions, for in- g/ml fibronectin for 90 min. The cells were then processed for stance the association with actin. immunofluorescence microscopy. In contrast to full-length 21686 Actopaxin Regulation of TESK1 Function FIG.9. The actopaxin carboxyl terminus creates a spreading defect that is cofilin-dependent. HeLa cells were transfected with the indicated constructs, resuspended, and then spread on 10 g/ml fibronectin-coated coverslips for 90 min. Coverslips were subsequently fixed and processed for immunofluorescence followed by area quantifi- cation as detailed under “Experimental Procedures.” Co-transfection with a phosphomimetic cofilin construct (S3E) reverted the actopaxin 223–372 spreading defect, although it does not significantly affect spreading by itself. Expression of a non-phosphorylatable cofilin con- struct (S3A) significantly decreases spreading but does not further decrease spreading of the actopaxin 223–372 cells. * indicates a group FIG.8. Cells expressing the actopaxin carboxyl terminus dis- that is significantly different from GFP (p  0.01). ** indicates S3E- play aberrant phosphocofilin localization during spreading. cofilin able to diminish the spreading defect in Xpress-actopaxin 223– HeLa cells were transfected and spread on fibronectin as in figure 7. 372 cells (p  0.01). However, the combination of S3E and Xpress Cells were then fixed after 90 min and stained for phosphocofilin. GFP actopaxin 223–372 is significantly less well spread than GFP cells (p control cells exhibited an enrichment of phosphocofilin toward the cell 0.01). These results confirm that the defect in spreading in cells ex- periphery. Myc-TESK1-expressing cells showed slightly elevated phos- pressing actopaxin 223–372 is partially cofilin-dependent. phocofilin levels. In contrast, cells expressing the actopaxin carboxyl terminus (Xpress-actopaxin 223–372) displayed substantially reduced mimetic S4/8D actopaxin construct that promotes cell spread- levels of phosphocofilin as compared with adjacent non-transfected cells. Localized phosphocofilin staining and cell spreading are restored ing in osteosarcoma cells (27) was found to exhibit impaired when Myc-TESK1 is co-transfected with the Xpress-actopaxin 223–372 TESK1 binding, while the carboxyl terminus (Xpress-actopaxin construct. Transfected cells are indicated by GFP co-transfection. Bar, 223–372) alone bound constitutively to TESK1. 10 m. Actopaxin phosphorylation is stimulated upon cell adhesion and has been demonstrated to be MEK/Erk2-dependent (27). TESK1, which was found not to have an effect on spreading by However, since actopaxin was also weakly phosphorylated by itself, the GEF-TESK 1–528 increased spreading above that TESK1 in vitro, this kinase may also contribute to integrin-de- seen in GFP control cells (Figs. 10B and 7C). An increase in pendent phosphorylation of actopaxin perhaps via a feed-forward cortical actin structures was observed, consistent with elevated mechanism in which adhesion-stimulated phosphorylation of ac- TESK1 activity (Fig. 10B). We confirmed this effect by quanti- topaxin results in TESK1 dissociation and activation and thus fying areas of respread cells. GFP-TESK1 1–528 significantly additional actopaxin phosphorylation. It is currently uncertain increased spreading to 1.34 times that seen in control cells. how phosphorylation of the actopaxin amino terminus regulates Furthermore, this result was blocked by co-expression of cofilin TESK1 binding to the carboxyl terminus but likely involves long S3A, indicating the effect is dependent on cofilin phosphoryla- distance allosteric changes. This model is supported by the loss of tion. Finally, expression of TESK1 1–528 lacking the actopaxin adhesion-dependent regulation of the TESK1 association exhib- binding site completely rescued spreading in HeLa cells ex- ited by the carboxyl terminus of actopaxin. pressing Xpress actopaxin 223–372, confirming a role for Although both LIMK, which is activated downstream of Rho- TESK1 downstream of actopaxin in cell spreading (Fig. 10C). ROCK or Cdc42/Rac-PAK pathways, and TESK can phospho- DISCUSSION rylate cofilin (1), TESK1 has been suggested to be the primary Actopaxin performs a critical, evolutionarily conserved, role regulator of adhesion-dependent cofilin phosphorylation, as in stabilizing integrin-actin interactions at sites of cell adhe- supported by a significant loss of this signaling event in cells sion to the extracellular matrix in muscle and non-muscle cells expressing kinase-dead TESK1 (3). This translates into an (18, 26). Herein, we detail a functional interaction between inability of these cells to spread efficiently on fibronectin (8). actopaxin and the serine/threonine kinase TESK1, which is an Interestingly, overexpression of the carboxyl terminus of acto- important modulator of integrin-mediated actin dynamics due paxin, which, as opposed to full-length actopaxin, maintains to its ability to phosphorylate and thereby regulate the activity binding to TESK1 during adhesion, inhibits both cell spreading and cofilin phosphorylation. While it has previously been sug- of the F-actin-severing protein cofilin (3). Using a combination of yeast two-hybrid analysis, GST pull-down, and co-immuno- gested that this spreading defect is due to perturbation of the F-actin binding site on actopaxin and thus disruption of the precipitation assays we have localized the sites of interaction to within the carboxyl terminus of each molecule. In experiments integrin-actin linkage (18), our current results, showing that the spreading defect can be partially rescued following overex- designed to evaluate how the interaction between actopaxin and TESK1 may contribute to the regulation of actin dynamics pression of TESK1, provide evidence that cell spreading can also be controlled through actopaxin-mediated regulation of we have determined that the association between TESK1 and actopaxin negatively regulates TESK1 kinase activity and fur- TESK signaling to cofilin. The introduction of an S3E phospho- mimetic cofilin construct also reverts the spreading defect. As ther that the interaction is reduced in cells actively spreading on fibronectin. Dissociation of the actopaxin-TESK1 complex is this mutant does not bind actin, its rescue is likely mediated through competition for binding partners with endogenous co- likely to be regulated via a mechanism involving the phospho- rylation of the amino terminus of actopaxin, since a phospho- filin (9). This may potentially act through binding and seques- Actopaxin Regulation of TESK1 Function 21687 FIG. 11. Model for the adhesion-dependent regulation of TESK1 by actopaxin. Actopaxin and TESK1 are associated in asyn- chronously growing cells, which serves to inhibit TESK1 kinase activ- ity. Upon integrin stimulation, actopaxin becomes phosphorylated and TESK1 dissociates, relieving inhibition of its kinase activity. Active TESK1 then phosphorylates cofilin on serine 3, thereby inhibiting the association of cofilin with F-actin. This inactivates the F-actin severing activity of cofilin, allowing adhesion-dependent actin reorganization and facilitating spreading. thus activation, is necessary for growth factor-initiated lamel- lipodia formation (34, 35). Precise compartmentalization of the pools of active and inactive cofilin explains these seemingly conflicting observations. Thus, the non-phosphorylated pool of active cofilin has been localized to the extreme leading edge of a cell where it facilitates lamellipodial extension through the creation of free F-actin barbed ends that are conducive to branching via the activity of the Arp2/3 complex (17). In con- trast, a pool of phosphorylated cofilin is enriched a short dis- tance away from the edge of the lamellipodia, where it func- tions to stabilize actin structures necessary to support further membrane protrusions (13). Consistent with a role for phos- phocofilin in lamellipodia extension during cell spreading, we have found phosphocofilin to be enriched toward the cell pe- riphery following integrin ligation. Importantly, the phospho- cofilin staining was reduced in cells expressing Xpress-acto- paxin 223–372, consistent with the ability of this mutant to interfere with TESK1 activity and thus phosphorylation of cofilin. Incorporating our data into the model of TESK1 regulation of cofilin function, we propose (Fig. 11) that actopaxin and TESK1 associate with one another in the cytosol of asynchronously growing adherent cells or cells held in suspension, and this serves to inhibit TESK1 kinase activity. Upon integrin-medi- ated attachment to the ECM, as occurs during cell spreading or FIG. 10. A TESK1 mutant defective in actopaxin binding in- at sites immediately proximal to the leading edge of an extend- creases cell spreading. A, HeLa cells were transfected with GFP- TESK1 1–528 followed by GST pull-down binding assays. GST-acto- ing lamellipodium, actopaxin is recruited to the nascent focal paxin fails to bind this portion of TESK1, confirming that the carboxyl- complexes and becomes phosphorylated within its amino ter- terminal TESK1 residues 529–626 are the site of interaction with minus. This likely promotes an intramolecular rearrangement actopaxin. ILK was used as a positive control for binding, while -ac- between the amino and carboxyl terminus of actopaxin that tinin served as a negative control. B, HeLa cells were transfected with either GFP or GFP-TESK1 1–528 and respread on 10 g/ml fibronectin- reduces TESK1 binding, thereby relieving an inhibition of coated coverslips for 90 min. Coverslips were then fixed and processed TESK1 kinase activity. TESK1 then phosphorylates the local for immunofluorescence using rhodamine phalloidin to visualize F- pool of cofilin and thereby stabilizes actin filaments adjacent to actin. Cells expressing GFP-TESK1 1–528 are more spread than control focal complexes. Interestingly, nascent focal complexes have cells and exhibit increased cortical actin. Bar,10 m. C, GFP-TESK1 1–528 was co-expressed with cofilin S3A or Xpress-actopaxin 223–372 been shown to form at the transition zone between the extend- and respread on 10 g/ml fibronectin for 90 min. Cell areas were ing lamellipodium, which is rich in cofilin and Arp2/3 activity quantified as described under “Experimental Procedures.” Expression and the more stable actin network of the lamella (36–39). of GFP-TESK1 1–528 significantly increases spreading as compared The phosphoserine binding adaptor protein 14-3-3 has previ- with GFP control cells. This effect is blocked by S3A cofilin. The com- bination of GFP-TESK1 1–528 and Xpress-actopaxin 223–372 spreads ously been shown to regulate adhesion-dependent activation of comparably with GFP control cells. * indicates significantly different TESK1 via a similar mechanism (8). Interestingly, we found that from GFP (p  0.01). expression of GFP-TESK1 1–528, which lacks actopaxin binding but retains serine 439, the site of 14-3-3 binding, promoted tering the cofilin phosphatases slingshot or chronophin, with aberrant cell spreading and cortical actin structure formation, subsequent alteration of endogenous cofilin phosphorylation while wild-type TESK1 overexpression exerted minimal effects. levels (10, 11). This likely suggests that 14-3-3 and actopaxin contribute to the The morphologic changes that occur when cells are spreading regulation of TESK1 function via overlapping as well as distinct on extracellular matrix are considered analogous to lamellipo- mechanisms. For instance, it will be important to determine dia formation at the leading edge of motile cells. Interestingly, whether actopaxin binding influences TESK1 phosphorylation while TESK1 promotes cell spreading via cofilin phosphoryla- on serine 439 and thus 14-3-3 binding. tion, it has been shown that cofilin dephosphorylation, and Finally, it has previously been suggested that a stable asso- 21688 Actopaxin Regulation of TESK1 Function Cell 108, 233–246 ciation between actopaxin, PINCH, and ILK is obligatory and 11. Gohla, A., Birkenfeld, J., and Bokoch, G. M. (2005) Nat. Cell Biol. 7, 21–29 serves to stabilize these proteins (32). Interestingly, TESK1 12. Ghosh, M., Song, X., Mouneimne, G., Sidani, M., Lawrence, D. S., and Con- binds a population of actopaxin distinct from that which binds deelis, J. S. (2004) Science 304, 743–746 13. Dawe, H. R., Minamide, L. S., Bamburg, J. R., and Cramer, L. P. (2003) Curr. ILK, indicating an alternate pool of actopaxin within the cell. Biol. 13, 252–257 Following recruitment to focal adhesions and release of TESK1, 14. Desmarais, V., Ghosh, M., Eddy, R., and Condeelis, J. (2005) J. Cell Sci. 118, 19–26 this pool of actopaxin may be more susceptible to degradation 15. DesMarais, V., Macaluso, F., Condeelis, J., and Bailly, M. (2004) J. Cell Sci. which, in turn, could contribute to focal adhesion turnover and 117, 3499–3510 cell migration (40, 41). It has also been shown that actopaxin 16. Pollard, T. D., and Borisy, G. G. (2003) Cell 112, 453–465 17. Ichetovkin, I., Grant, W., and Condeelis, J. (2002) Curr. Biol. 12, 79–84 (-parvin) and affixin (-parvin) compete for ILK binding (42). 18. Nikolopoulos, S. N., and Turner, C. E. (2000) J. Cell Biol. 151, 1435–1448 These interactions exert opposing effects on ILK kinase activity 19. Gimona, M., Djinovic-Carugo, K., Kranewitter, W. J., and Winder, S. J. (2002) (25, 43). Due to the negative regulation of TESK1 activity by FEBS Lett. 513, 98–106 20. Olski, T. M., Noegel, A. A., and Korenbaum, E. (2001) J. Cell Sci. 114, 525–538 actopaxin reported herein, it will be important to determine 21. Brown, M. C., and Turner, C. E. (2004) Physiol. Rev. 84, 1315–1339 whether -parvin binds TESK1 and if so how this may affect its 22. Nikolopoulos, S. N., and Turner, C. E. (2002) J. Biol. Chem. 277, 1568–1575 23. Rosenberger, G., Jantke, I., Gal, A., and Kutsche, K. (2003) Hum. Mol. Genet. activity. These possibilities are made more intriguing by the 12, 155–167 ability of TESK1 to facilitate cell spreading on fibronectin, 24. Zhang, Y., Chen, K., Tu, Y., Velyvis, A., Yang, Y., Qin, J., and Wu, C. (2002) while ILK has been shown to negatively affect spreading on J. Cell Sci. 115, 4777–4786 25. Attwell, S., Mills, J., Troussard, A., Wu, C., and Dedhar, S. (2003) Mol. Biol. this matrix (3, 31). The varying interactions between acto- Cell 14, 4813–4825 paxin, ILK, TESK1, and possibly -parvin indicate the possi- 26. Lin, X., Qadota, H., Moerman, D. G., and Williams, B. D. (2003) Curr. Biol. 13, bility of multiple signaling cassettes composed of these mole- 922–932 27. Clarke, D. M., Brown, M. C., LaLonde, D. P., and Turner, C. E. (2004) J. Cell cules that could be assembled to differentially regulate actin Biol. 166, 901–912 dynamics and cell morphology. In view of the changes in ex- 28. Meberg, P. J., Ono, S., Minamide, L. S., Takahashi, M., and Bamburg, J. R. (1998) Cell Motil. Cytoskeleton 39, 172–190 pression of actopaxin/parvin family members and ILK observed 29. Curtis, M., Nikolopoulos, S. N., and Turner, C. E. (2002) Biochem. J. 363, in certain tumors (44, 45), it will be important to establish how 233–242 changes in the balance of these multiple interactions may con- 30. Bernstein, B. W., Painter, W. B., Chen, H., Minamide, L. S., Abe, H., and Bamburg, J. R. (2000) Cell Motil. Cytoskeleton 47, 319–336 tribute to cell transformation and/or metastasis. 31. Khyrul, W. A., Lalonde, D. P., Brown, M. C., Levinson, H., and Turner, C. E. (2004) J. 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Actopaxin Interacts with TESK1 to Regulate Cell Spreading on Fibronectin *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2005 Elsevier Inc.
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0021-9258
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10.1074/jbc.m500752200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 22, Issue of June 3, pp. 21680–21688, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Actopaxin Interacts with TESK1 to Regulate Cell Spreading on Fibronectin* Received for publication, January 20, 2005, and in revised form, March 24, 2005 Published, JBC Papers in Press, April 6, 2005, DOI 10.1074/jbc.M500752200 David P. LaLonde, Michael C. Brown, Brian P. Bouverat‡, and Christopher E. Turner§ From the Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York 13210 proteins, including the LIM kinases (LIMK) (1). Closely re- The focal adhesion protein actopaxin contributes to integrin-actin associations and is involved in cell adhe- lated to the LIM family of kinases are TESK1 and TESK2 sion, spreading, and motility. Herein, we identify and (testicular protein kinases), which were originally identified characterize an association between actopaxin and the in testicular cells but have since been found to be ubiqui- serine/threonine kinase testicular protein kinase 1 tously expressed (2–5). Unlike LIMK, regulation of TESK1 (TESK1), a ubiquitously expressed protein previously activity by Rho GTPases has not been confirmed, although reported to regulate cellular spreading and focal adhe- recent studies in Drosophila implicate a role in the Rac sion formation via phosphorylation of cofilin. The inter- pathway associated with both eye development and spermat- action between actopaxin and TESK1 is direct and the ogenesis (6). However, this kinase has been shown to be binding sites were mapped to the carboxyl terminus of activated upon matrix adhesion and is regulated by binding both proteins. The association between actopaxin and of 14-3-3 and Sprouty4 (7, 8). TESK1 is negatively regulated by adhesion to fibronec- TESK1, as is the case with LIMK, regulates integrin-depend- tin, and a phosphomimetic actopaxin mutant that pro- motes cell spreading also exhibits impaired binding to ent focal adhesion assembly and actin organization through TESK1. Binding of actopaxin to TESK1 inhibits TESK1 phosphorylation of the amino terminus of the F-actin-severing kinase activity in vitro. Expression of the carboxyl ter- protein cofilin (3). Phosphorylation on serine 3, which is re- minus of actopaxin has previously been reported to re- versed by the serine phosphatases slingshot and chronophin, tard cell spreading. This effect was reversed following has been shown to decrease cofilin activity by interfering with overexpression of TESK1 and was found to be depend- its ability to bind F-actin (9–11). Cofilin is a critical regulator ent on an inability of actopaxin carboxyl terminus ex- of both growth factor and matrix-dependent actin reorganiza- pressing cells to promote cofilin phosphorylation upon tion, affecting lamellipodia formation, cell spreading, motility, matrix adhesion and caused by retention of TESK1 by and polarity (12–16). For instance, cofilin’s F-actin severing this actopaxin mutant. Thus, the association between activity potentiates Arp2/3-mediated actin assembly that is actopaxin and TESK1, which is likely regulated by phos- phorylation of actopaxin, regulates TESK1 activity and required for epidermal growth factor-induced lamellipodia for- subsequent cellular spreading on fibronectin. mation (17). The focal adhesion protein actopaxin is the -isoform of the parvin family and binds actin through a pair of calponin homol- Integrin-mediated adhesion to the extracellular matrix ogy (CH) domains (18–20). It interacts with the integrin-linked (ECM) leads to extensive actin reorganization that is regu- kinase (ILK) and the focal adhesion scaffolding proteins paxillin lated predominantly by the Rho family of GTPases, Cdc42, Rac, and Hic-5 (18, 21, 22). Affixin, the -isoform of the parvin family, and Rho, to stimulate formation of the actin-dependent struc- has been found to bind the putative Rac/Cdc42 guanine nucleo- tures filopodia, lamellipodia, and stress fibers, respectively (1). tide exchange factor PIX suggesting the possibility of this inter- Activated Rho family members interact with numerous effec- action for actopaxin as well (23). The associations between acto- tors, including the p21-associated kinase (PAK), the Wiskott- paxin, ILK, and paxillin constitute an evolutionarily conserved Aldrich Syndrome protein (WASP), and the Rho-associated integrin-actin linkage important in muscle cytoarchitecture and kinase (ROCK). PAK and ROCK share some common target contribute to the regulation of cellular spreading and adhesion in mesenchymal cells (18, 22, 24–26). Recent evidence suggests that * This work was supported by National Institutes of Health Grant Erk-dependent phosphorylation of the actopaxin amino terminus RO1 HL070244 (to C. E. T.). The costs of publication of this article were regulates cellular spreading and motility via modulation of Rho defrayed in part by the payment of page charges. This article must family signaling (27). therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. In this study we performed a yeast two-hybrid screen of a ‡ Current address: Dept. of Pharmacology and Physiology, University human placenta library using actopaxin as bait and identified of Rochester Medical Center, Rochester, NY 14642. TESK1 as a direct binding partner. We have established that § To whom correspondence should be addressed: Dept. of Cell and Developmental Biology, State University of New York Upstate Medical an association between actopaxin and TESK1 negatively regu- University, 750 East Adams St., Syracuse, NY 13210. Tel.: 315-464- lates TESK1 kinase activity and thus phosphorylation of cofi- 8598; Fax: 315-464-8535; E-mail: [email protected]. lin. Furthermore, this association is negatively modulated by The abbreviations used are: ECM, extracellular matrix; aa, amino adhesion to fibronectin, most likely through phosphorylation of acids; CH, calponin homology; Erk, extracellular signal-regulated ki- nase; FAK, focal adhesion kinase; ILK, integrin-linked kinase; LIMK, the amino terminus of actopaxin. Consequently, the association LIM kinase; MBP, myelin basic protein; TESK1 and TESK2, testicular between TESK1 and actopaxin provides a mechanism for the protein kinases 1 and 2; XAC, Xenopus actin depolymerizing factor/ regulation of cell spreading and potentially cell migration via cofilin; PAK, p21-associated kinase; ROCK, Rho-associated kinase; GFP, green fluorescent protein. modulation of cofilin activity. 21680 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Actopaxin Regulation of TESK1 Function 21681 EXPERIMENTAL PROCEDURES Myc-TESK1 was then resuspended in reaction buffer and incubated at room temperature for 30 min in the presence of 5 Ci of [- P]ATP, 10 Antibodies and Materials—Human plasma fibronectin was purchased g of focal adhesion kinase (MBP), and various GST-actopaxin con- from Sigma or BD Biosciences. Monoclonal antibody to the Xpress tag was structs as indicated. The reaction was terminated by boiling in sample purchased from Invitrogen. -Actinin and MOPC monoclonal antibodies buffer and the samples resolved on a 15% SDS-PAGE gel, followed were obtained from Sigma. Focal adhesion kinase (FAK) and ILK mono- by Coomassie Blue staining and autoradiography. Phosphorylation clonal antibodies were purchased from BD Transduction Laboratories. levels were quantitated with a Storm PhosphorImager (Amersham The 9E10 anti-Myc monclonal antibody developed by J. M. Bishop was Biosciences). obtained from the Developmental Studies Hybridoma Bank developed Respreadings and Immunofluorescence—These assays were per- under the auspices of the NICHD and maintained by The University of formed as described previously (27, 31). Area values obtained were Iowa, Department of Biological Sciences, Iowa City, IA. Omni-probe (di- compared with GFP-expressing control cells in the same experiment rected against region of Xpress epitope tag) and GFP polyclonal antibodies and further normalized against attached, unspread cells to quantitate were obtained from Santa Cruz Biotechnology. Polyclonal antibody to comparative spreading capacities. For phosphocofilin staining, cover- cofilin phosphorylated upon serine 3 was provided by Dr. James Bamburg slips were fixed in cytoskeletal stabilization buffer (3.7% formaldehyde, (Colorado State University) (28). 0.32 M sucrose, 10 mM MES, pH 6.1, 3 mM MgCl , 138 mM KCl, 2 mM Yeast Two-hybrid Assay—The MoBiTec Grow’n’Glow Two-Hybrid EGTA) for 15 min, then processed as described (27, 31). System (MoBiTec, Goettingen, Germany) based on the Brent LexA Interaction Trap System was used to identify proteins that interact RESULTS with actopaxin. Full-length rat actopaxin and the “nonspecific” protein bait, mouse p53 (aa 72–390), were cloned into pEG202 (His selectable Actopaxin Interacts with TESK1 in Vitro—To identify acto- marker) as a COOH-terminal fusion to LexA. To moderate the inherent paxin-binding proteins, a yeast two-hybrid screen was per- transactivation potential of full-length actopaxin, we utilized the low formed with full-length rat actopaxin fused to LexA as a bait. sensitivity Saccharomyces cerevisiae strain EGY188 that contains two Ten positive clones were isolated from the screening of 1  10 copies of the LexA operator upstream of the LEU2 reporter integrated recombinant colonies. To confirm the observed interactions, in the genome. A GFP reporter plasmid (pGNG1) with a URA3 select- selected clones were subjected to an additional round of screen- able marker was used in place of a -galactosidase reporter, allowing for simple screening by UV transillumination. A human placenta cDNA ing using either p53 as a nonspecific protein bait or actopaxin. library in pJG4-5 (TRP1 selectable marker), fused to the activation Two of the plasmids that were positive for actopaxin binding domain B42, was employed in the screen. Plasmids were sequentially and at the same time negative for p53 association encoded transformed by lithium acetate, into EGY188. Positive pJG4-5 library/ sequence representing the serine/threonine kinase testicular prey plasmids were isolated, and a directed interaction of the positive, protein kinase 1 (TESK1) (data not shown). isolated library/prey with nonspecific protein bait, p53, was performed. One plasmid insert contained DNA encoding aa 529–626, A directed interaction with actopaxin plasmid was also performed to confirm the association. True positive clones were sequenced at the comprising the carboxyl terminus of TESK1, as well as 80 BioResource Center of Cornell University (Ithaca, NY). nucleotides from the 3-untranslated region. The second plas- Plasmids—To generate full-length human TESK1, PCR primer pairs mid contained aa 544–626 and 74 nucleotides from the 3- were synthesized representing the 5 and 3 termini of the published untranslated region. It is notable that the principal difference TESK1 coding sequence incorporating EcoRI and XbaI restriction sites. between TESK1 and its family member TESK2 is the presence A human heart cDNA library (Clontech) was used for PCR amplifica- of this proline-rich carboxyl terminus extension within tion. The product obtained was identical to that reported previously, except that nucleotides 31–111, representing amino acids 11–37, were TESK1 (5). absent. Notably this stretch of coding sequence represents human exon An association between actopaxin and TESK1 was first con- 2 suggesting the existence of a new TESK1 splice isoform. Full-length firmed using GST binding assays. These studies were re- rat TESK1 and TESK2 Myc-tagged constructs were generously pro- stricted to the analysis of exogenous TESK1 due to the lack of vided by Dr. Kensaku Mizuno (Tohoku University, Sendai, Japan) (2, an available antibody to the endogenous form. Myc-tagged 4). A pGEX-4T TESK1 carboxyl-terminal (aa 529–626) fusion protein TESK1 and TESK2 were expressed in HeLa cells and actopaxin was generated for use in precipitation assays. A construct encoding binding tested using GST-actopaxin fusion proteins. TESK1 TESK1 1–528 with an amino-terminal GFP tag was created via PCR amplification. Actopaxin constructs were as described previously (18, was found to specifically bind full-length GST-actopaxin (Fig. 22, 27, 29). GFP-XAC (Xenopus actin depolymerizing factor/cofilin), 1A). In contrast, GST-actopaxin did not precipitate TESK2, hereafter referred to as GFP-cofilin, constructs were provided by Dr. consistent with the absence of the carboxyl-terminal extension James Bamburg (Colorado State University) (30). of TESK1 in this isoform (Fig. 1A). The binding of ILK served Cell Culture and Transfections—HeLa cells were maintained in as a positive control. Additional GST pull-down assays estab- Dulbecco’s modified Eagle’s medium (Mediatech) supplemented with lished the carboxyl-terminal actopaxin amino acids 223–372 as 10% fetal bovine serum (Atlanta Biologicals). Transfections were performed with FuGENE 6 (Roche Applied Science) according to the binding region for TESK1 (Fig. 1, B and C). This region of manufacturer’s protocols. actopaxin consists of a portion of the intra-CH linker domain Binding Assays—GST binding assays were performed essentially as and the second CH domain. It also contains the binding sites described previously (18). For in vivo binding experiments, cells were for paxillin and ILK (18, 22). lysed in co-immunoprecipitation buffer (50 mM Tris-HCl, pH 7.6, 0.5% The yeast two-hybrid screen identified the carboxyl-terminal Nonidet P-40, 100 mM NaCl, 10% glycerol, 1 mM MgCl ,10 g/ml 98 residues of TESK1 as the site of interaction with actopaxin. leupeptin, 1 mM NaVO ,and1mM NaF). Lysates were centrifuged to remove cellular debris, and then Xpress-actopaxin was precipitated A construct was created consisting of this portion of TESK1 using Omni-probe antibody (anti-epitope tag of Xpress-actopaxin) and (529–626) fused to the carboxyl terminus of GFP. Pull-down protein A/G beads (Santa Cruz Biotechnology). Immunoprecipitated assays confirmed the binding of GFP-TESK1 529–626 to GST- proteins were subsequently solubilized in sample buffer and analyzed actopaxin (Fig. 2A). Furthermore, a GST fusion construct of by Western blotting. this region of TESK1 was created to verify binding with acto- In Vitro Kinase Assays—Kinase assays were performed essentially as paxin. GST binding assays performed using HeLa cell lysates described previously (3). HeLa cells expressing Myc-TESK1 were lysed in kinase immunoprecipitation buffer (50 mM Tris-HCl, pH 7.6, 150 mM demonstrated binding of endogenous actopaxin to GST-TESK1 NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl flu- 529–626, while the GST-paxillin LD4 motif served as a posi- oride, 10 g/ml leupeptin), incubated on ice, and centrifuged to remove tive control for actopaxin binding, as reported previously (Fig. cellular debris. The resulting supernatant was incubated at 4 °C for 2 h 2B) (18). It has been suggested that ILK and actopaxin are with protein A/G beads and either control antibody (MOPC) or 9E10 obligate binding partners (32). However, ILK was not present anti-Myc antibody. Immune complexes were then washed three times in in the GST-TESK1 precipitate, indicating that TESK1 can bind wash buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% Nonidet P-40) a pool of actopaxin that is not concurrently bound to ILK (Fig. and twice in reaction buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM NaF, 1 mM NaVO ,5mM MnCl ,5mM MgCl ). Immunoprecipitated 2B). Conversely, the presence of ILK and actopaxin in the LD4 4 2 2 21682 Actopaxin Regulation of TESK1 Function FIG.2. The carboxyl terminus of TESK1 binds a pool of acto- paxin that is not associated with ILK. A, HeLa cells were trans- fected with GFP-TESK1 amino acids 529–626 and then lysed and incubated with either GST or GST-actopaxin fusion proteins. GFP- TESK1 529–626 was precipitated by GST-actopaxin but not by the GST control. GST-actopaxin also bound paxillin, as described previously, but not -actinin. B, HeLa lysates were incubated with GST, GST-TESK1 529–626, or GST-paxillin LD4. Bound proteins were analyzed by SDS- PAGE and blotted with antibodies to ILK, actopaxin, and -actinin. Actopaxin was precipitated by both GST-paxillin LD4 and GST-TESK1 529–626. However, ILK was only co-precipitated in the GST-paxillin FIG.1. TESK1 binds the carboxyl terminus of actopaxin in LD4 lane. These results indicate that TESK1 binds to a pool of acto- vitro. A, HeLa cells were transfected with either Myc-TESK1 or Myc- paxin, which does not concurrently bind ILK. TESK2 then lysed in GST binding buffer and subjected to pull-down assays with both GST and GST full-length actopaxin. Samples were analyzed by Western blotting with antibodies to -actinin, ILK, and lamellipodia extension associated with cell migration. To de- Myc (9E10). GST-actopaxin precipitated TESK1 but not TESK2, while termine whether the interaction between TESK1 and acto- -actinin served as a negative control. ILK served as a positive control paxin might be modulated during cell spreading, we performed for GST-actopaxin binding. B, to delineate the region of actopaxin co-immunoprecipitations of co-transfected proteins in HeLa involved in TESK1 binding, GST fusion protein pull-down binding assays were performed as above, with the addition of amino-terminal cells that were growing asynchronously in culture versus cells (amino acids 1–222) and carboxyl-terminal (amino acids 223–372) GST- that had been actively spreading upon 10 g/ml fibronectin for actopaxin fusion proteins. TESK1 was precipitated with full-length and 90 min. Actopaxin precipitated Myc-TESK1 less efficiently in carboxyl-terminal actopaxin, while -actinin served as a negative con- spreading cells than unstimulated cells (Fig. 4A). trol. C, Coomassie Blue staining of GST fusion proteins. Lane 1, GST; lane 2, GST-actopaxin 1–372; lane 3, GST-actopaxin 1–222; and lane 4, Adhesion-dependent phosphorylation of actopaxin affects GST-actopaxin 223–372. cell spreading and migration. This post-translational modifica- tion of actopaxin may exert its effects by altering its association pull-down shows that existing actopaxin/ILK associations were with various binding partners. To test whether this is the case not disrupted in these lysates as paxillin LD4 only binds ILK for TESK1, HeLa cells were co-transfected with Myc-TESK1 indirectly through actopaxin (22, 33). and either wild type or phosphomimetic (S4/8D) Xpress-acto- Actopaxin and TESK1 Interact in Vivo—The association be- paxin constructs (27). Immunoprecipitations using an antibody tween TESK1 and actopaxin was demonstrated in vivo using to the actopaxin epitope-tag showed that the phosphomimetic co-immunoprecipitation experiments. Epitope-tagged versions of S4/8D Xpress-actopaxin failed to precipitate Myc-TESK1, these proteins were co-expressed in HeLa cells and immunopre- while the wild-type Xpress-actopaxin displayed robust binding cipitated using a polyclonal antibody (Omni-probe) to the epitope- (Fig. 4B). The specificity of this result was confirmed by the tag of actopaxin. Myc-TESK1, but not FAK, was co-immunopre- observation that ILK was precipitated by both Xpress-acto- cipitated with full-length Xpress-actopaxin (Fig. 3A). Consistent paxin constructs (Fig. 4B). with the GST pull-down assays, TESK1 co-immunoprecipitated As the sites of actopaxin phosphorylation are on the amino with the carboxyl terminus of actopaxin (aa 223–372) (Fig. 3B). terminus of the protein, while the carboxyl terminus mediates FAK served as a negative control, while ILK binding to the TESK1 binding, the actopaxin phosphorylation-dependent ab- actopaxin carboxyl terminus was used as a positive control (Fig. rogation of TESK1 binding may involve allosteric regulation 3B). These data demonstrate that actopaxin and TESK1 are between the amino and carboxyl terminus of actopaxin. Thus, associated in asynchronously growing cells. we next co-transfected Myc-TESK1 with either full-length (aa The Association between TESK1 and Actopaxin Is Negatively 1–372) or carboxyl-terminal (aa 223–372) Xpress-actopaxin Regulated during Adhesion to Fibronectin—Cell attachment constructs. Notably, the carboxyl terminus of actopaxin con- and spreading on the ECM leads to the activation of integrins tains the TESK1 binding site but lacks the phosphorylation and, in turn, Rho GTPases and is a widely accepted model for sites (Fig. 1B). Both populations of cells were placed in suspen- Actopaxin Regulation of TESK1 Function 21683 sion proteins as compared with GST alone (Fig. 5A,B). Both of these actopaxin fusion proteins bind TESK1 (Fig. 1B). Con- versely, a GST fusion protein consisting of the amino terminus (aa 1–222) of actopaxin, which does not bind TESK1, failed to inhibit TESK1 kinase activity (Fig. 1B and Fig. 5, A and B). Therefore, Myc-TESK1 kinase activity is reduced by interac- tion with actopaxin. Interestingly, the difference in activity of TESK1 in the presence versus the absence of actopaxin that we observed is equivalent to the relative changes in TESK1 activ- ity that occurred upon adhesion to fibronectin (3), with a near doubling of kinase activity. A low level of phosphorylation of the actopaxin amino terminus (aa 1–222) and full-length GST fusion proteins was also observed in these assays, suggesting TESK1 kinase activity may, in turn, regulate actopaxin func- tion in vivo via direct phosphorylation (Fig. 5C). Expression of the Carboxyl Terminus of Actopaxin Inhibits Adhesion-dependent Cofilin Phosphorylation—Since the car- boxyl terminus of actopaxin retains binding to TESK1 during adhesion and inhibits its kinase activity in vitro, this mutant can be used as a tool to evaluate the effects on cell function of alteration of the physiologic association between TESK1 and actopaxin. Specifically, we evaluated how introduction of this mutant influenced phosphorylation of the TESK1 substrate cofilin during cell spreading on fibronectin. HeLa cells were co-transfected with GFP-cofilin and either Xpress-actopaxin 223–372 or Xpress--galactosidase, as a control, and respread on 10 g/ml fibronectin-coated culture dishes. Lysates were collected in suspension and at 30, 60, and 120 min post-re- spreading. Phospho-GFP cofilin levels were then measured by Western blotting with an antibody specific for phosphorylation on serine 3, the site phosphorylated by TESK1 that inactivates cofilin. Phosphoserine 3 GFP cofilin levels are diminished in the Xpress-actopaxin 223–372-expressing cells during spread- FIG.3. TESK1 associates with actopaxin in vivo. A, HeLa cells ing on fibronectin (Fig. 6A). This assay was repeated following were co-transfected with Myc-TESK1 and Xpress full-length actopaxin the co-transfection of Myc-TESK1 to determine whether the then lysed in co-immunoprecipitation buffer. Xpress-actopaxin immu- observed deficit of cofilin phosphorylation could be rescued. noprecipitations were performed with Omni-probe polyclonal antibody Indeed, introduction of TESK1 was found to rescue the cofilin (which recognizes a region of the actopaxin epitope tag) and subjected to Western blotting with FAK, 9E10, and Xpress monoclonal antibodies. phosphorylation defect observed in the Xpress-actopaxin 223– Myc-TESK1 is specifically co-immunoprecipitated with actopaxin. FAK 372 cells at the 90-min time point (Fig. 6B, lanes 4 and 5). served as a negative control. B, HeLa cells were co-transfected with TESK1 Rescues the Spreading Defect in Cells Expressing the Myc-TESK1 and either amino-terminal (1–222) or carboxyl-terminal Actopaxin Carboxyl Terminus—The importance of the TESK1/ (223–372) Xpress-actopaxin constructs. Immunoprecipitations were performed as above. FAK served as a negative control. As with the in actopaxin association in regulating cell morphology was exam- vitro experiments, TESK1 interacted with the carboxyl-terminal region ined by evaluating effects on cell spreading. Expression of of actopaxin. ILK also bound to the carboxyl terminus of actopaxin, as Xpress-actopaxin 223–372 impaired spreading of HeLa cells on has been reported. fibronectin at the 90-min time point, as has been reported previously (Fig. 7A) (18). Co-expression of Myc-TESK1 par- sion and then were allowed to adhere to 10 g/ml fibronectin tially rescued this spreading defect, while TESK1 had no ap- for 90 min, followed by immunoprecipitations of the actopaxin parent effect on spreading when overexpressed by itself, al- constructs. Binding of TESK1 to the carboxyl terminus of ac- though there was a mild increase in cortical actin as visualized topaxin was retained, while full-length actopaxin displayed a by rhodamine-phalloidin staining (Fig. 7A). Equivalent expres- diminished association upon adhesion (Fig. 4C). In contrast, sion of proteins in these experiments was confirmed by West- both Xpress-actopaxin constructs were able to precipitate ILK ern blotting (Fig. 7B). (Fig. 4C). These data suggest that an adhesion-dependent mod- We next performed quantitative analysis of spreading in ification of the actopaxin amino terminus, such as phosphoryl- these cells to confirm the TESK1-dependent rescue of the ob- ation, serves to regulate TESK1 binding to the carboxyl termi- served spreading defect caused by expression of Xpress-acto- nus of actopaxin. paxin 223–372. Quantitation of cell areas showed that Xpress- Actopaxin Binding Inhibits TESK1 Kinase Activity—Cell ad- actopaxin 223–372 reduced the spreading of HeLa cells to 37% hesion stimulates TESK1 kinase activity (3), which inversely of that observed in GFP control cells (Fig. 7C). Co-expression of correlates with actopaxin binding. Thus, we tested whether Myc-TESK1 significantly rescued the spreading of cells ex- actopaxin binding affected TESK1 kinase activity, as has been pressing Xpress-actopaxin 223–372 (p  0.01) (Fig. 7C). How- observed previously for the associations between TESK1 and ever, this rescue was incomplete, as cells expressing Myc- 14-3-3 and Sprouty4 (7, 8). Myc-TESK1 was immunoprecipi- tated, and in vitro kinase assays were performed in the pres- TESK1 with Xpress-actopaxin 223–372 were still significantly less well spread than GFP control cells (Fig. 7C). ence of GST-actopaxin fusion proteins using MBP as a sub- strate. The ability of Myc-TESK1 to phosphorylate MBP was An actopaxin mutant with an altered paxillin binding site reduced by 40–50% in the presence of both full-length (aa has previously been shown to be mislocalized and to also inhibit 1–372) and carboxyl-terminal (aa 223–372) GST-actopaxin fu- cell spreading when expressed in HeLa cells (18). We confirmed 21684 Actopaxin Regulation of TESK1 Function FIG.5. Actopaxin binding negatively regulates TESK1 kinase activity. A, HeLa cells expressing Myc-TESK1 were lysed, and Myc (9E10) immunoprecipitations were performed. Myc-TESK1 immune complexes were subjected to in vitro kinase assays in the presence of GST or GST-actopaxin fusion proteins using 10 g of MBP as a sub- strate. Lane 1, control immunoprecipitation using control mouse IgG (MOPC) and GST fusion protein; lane 2, Myc IP and GST; lane 3, Myc IP and GST-actopaxin 1–372; lane 4, Myc IP and GST-actopaxin 1–222; and lane 5, Myc IP and GST-actopaxin 223–372. Samples were resolved by SDS-PAGE followed by Coomassie Blue staining (A) and autoradi- ography (B). B, Coomassie staining of MBP (total MBP) and autora- diograph of MBP bands (phospho-MBP) following exposure to film. Quantitation of phospho-MBP was performed using a Storm Phosphor- Imager. The relative intensity of each signal was compared with the TESK1 kinase reaction in the presence of GST (lane 2) and is displayed as a percentage thereof. The results displayed are from a representative experiment. TESK1 kinase activity is inhibited in the presence of GST fusion proteins composed of 1–372 (lane 3) and 223–372 (lane 5) acto- paxin, both of which bind TESK1. These data show that TESK1 kinase activity is negatively regulated by actopaxin binding. C, autoradiograph of Coomassie-stained gel shown in A. Both the full-length and amino terminus actopaxin GST constructs are phosphorylated by TESK1. There is no apparent phosphorylation of the carboxyl terminus of acto- paxin or the GST control fusion proteins. this finding and also determined that overexpression of TESK1 is unable to rescue this phenotype, thereby indicating the spec- ificity of the rescue of the Xpress-actopaxin 223–372 spreading defect by TESK1 (Fig. 7C). The spreading defect of the paxillin binding site mutant caused by loss of actopaxin/paxillin asso- ciation therefore operates through some alternate pathway, possibly involving mislocalization of other actopaxin binding partners. Taken together, these results suggested that the spreading defect in Xpress-actopaxin 223–372-expressing cells is depend- ent upon altered cofilin signaling. Previous reports have dem- onstrated that phosphocofilin is localized in a region of the cell closely juxtaposed to the extending lamellipodia in a transition zone where actin filaments become stabilized (13). Thus, we examined phosphocofilin localization in cells actively spreading FIG.4. The association between TESK1 and actopaxin is de- upon fibronectin. GFP-transfected control cells exhibited mod- creased during cell spreading. A, HeLa cells were co-transfected est phosphocofilin immunostaining concentrated toward the with Xpress-actopaxin and Myc-TESK1. Omni-probe immunoprecipita- cell periphery, while Myc-TESK1 expression increased this tions for Xpress-actopaxin were then performed either from cells grow- ing in culture or from cells that had been spread on 10 g/ml fibronectin for 90 min. Actopaxin more readily precipitates TESK1 from asynchro- nously growing cells than from those spreading on fibronectin. -Acti- each actopaxin construct. C, HeLa cells were co-transfected with Myc- nin does not bind actopaxin in either condition. B, HeLa cells were TESK1 and either Xpress-actopaxin 1–372 (full-length) or 223–372 co-transfected with Myc-TESK1 and either phosphomimetic S4/8D or (carboxyl terminus). These cells were then spread on 10 g/ml fibronec- wild-type Xpress-actopaxin constructs. Xpress-actopaxin constructs tin for 90 min followed by Omni-probe immunoprecipitations. The as- were immunoprecipitated with Omni-probe polyclonal antibody, re- sociation between full-length actopaxin and TESK1 is diminished dur- solved using SDS-PAGE, and transferred to nitrocellulose. Myc-TESK1 ing spreading. However, the carboxyl terminus of actopaxin retains its co-immunoprecipitated with the wild-type but not the phosphomimetic association with TESK1. Conversely, both Xpress-actopaxin constructs S4/8D Xpress-actopaxin construct. Equivalent amounts of ILK bound to still precipitate ILK. Actopaxin Regulation of TESK1 Function 21685 FIG.6. Expression of the carboxyl terminus of actopaxin in- hibits adhesion-dependent cofilin phosphorylation. A, HeLa cells were co-transfected with GFP-cofilin and either -galactosidase (-Gal), as control, or the actopaxin carboxyl terminus (Xpress-actopaxin 223– 372). Cells were then placed in suspension for 1 h and either lysed or spread onto plates coated with 10 g/ml fibronectin. Samples were then collected at 30, 60, and 120 min and analyzed by SDS-PAGE for levels of phosphoserine 3 GFP-cofilin and total GFP-cofilin. Cells expressing the carboxyl terminus of actopaxin displayed reduced phosphocofilin levels upon cell attachment and spreading on fibronectin. B, to deter- mine whether TESK1 could rescue the observed cofilin phosphorylation defect, HeLa cells were transfected and respread as in A, with the exception that samples were collected solely at 90 min post-respread- ing. HeLa cells were transfected with: Xpress -galactosidase (lane 1); GFP-cofilin (lane 2), GFP-cofilin and Myc-TESK1 (lane 3), GFP-cofilin and Xpress-actopaxin 223–372 (lane 4), or GFP-cofilin, Myc-TESK1, and Xpress actopaxin 223–372 (lane 5). Total amounts of transfected cDNA were standardized with Xpress -galactosidase. Myc-TESK1 is able to rescue the defect in cofilin phosphorylation displayed in the cells FIG.7. TESK1 rescues the spreading defect in cells expressing expressing Xpress-actopaxin 223–372. the actopaxin carboxyl terminus. A, HeLa cells transfected with the indicated constructs were spread on 10 g/ml fibronectin-coated cover- phosphocofilin signal (Fig. 8). Consistent with our biochemical slips for 90 min and then processed for immunofluorescence. Trans- fected cells were determined by co-transfection with GFP and are indi- evaluation (Fig. 6), cells expressing Xpress-actopaxin 223–372 cated by asterisks. Cells expressing the actopaxin carboxyl terminus had diminished phosphocofilin staining, which was notably (Xpress-actopaxin 223–372) displayed impaired spreading upon fi- absent from the periphery when compared with an adjacent bronectin that was rescued by co-expression of Myc-TESK1. Bar,10 m. non-transfected cell. Importantly, phosphocofilin staining was B, lysates from experiments in A were blotted to demonstrate equiva- lent expression of proteins under each condition. C, cells were respread restored by co-expression of Myc-TESK1 with Xpress-actopaxin as in A, followed by area measurement and analysis as detailed under 223–372 (Fig. 8). No apparent difference in total cofilin cell “Experimental Procedures.” Error bars represent standard deviation. A staining was observed (data not shown). These data support a minimum of 40 cells was quantified per condition per trial (n was a role for actopaxin in regulating TESK1 signaling to cofilin. minimum of three per condition). * indicates a condition significantly different from GFP (p  0.01). The combination of Myc-TESK1 and We further tested a role for cofilin by evaluating the ability of Xpress-actopaxin 223–372 significantly increases spreading as com- phosphomimetic (S3E) cofilin constructs to rescue the spreading pared with cells expressing Xpress-actopaxin 223–372 (p  0.01). An on fibronectin in these cells. Significantly, co-expression of a Xpress-actopaxin construct containing a mutated paxillin binding site non-active S3E phosphomimetic cofilin construct with Xpress- (PBS) also displays impaired spreading. However, this defect is not actopaxin 223–372 was able to partially rescue spreading, simi- reversed by TESK1, indicating the specificity of the rescue of the Xpress-actopaxin 223–372 construct. lar to that seen with Myc-TESK1 (Fig. 9). Conversely, expression of an S3A construct, which mimics a non-phosphorylated, active cofilin, decreased spreading to levels comparable with those seen A TESK1 Construct That Does Not Bind Actopaxin Increases in HeLa cells expressing Xpress-actopaxin 223–372 (Fig. 9). Co- Cell Spreading on Fibronectin—To further examine the impor- expression of the S3A cofilin construct with Xpress-actopaxin tance of the actopaxin/TESK1 interaction in the regulation of 223–372 did not result in further inhibition of spreading (Fig. 9). cell spreading, we created a GFP-TESK1 1–528 construct, These data demonstrate that expression of the carboxyl ter- which lacks the actopaxin binding site but contains the kinase minus of actopaxin severely inhibits cell spreading on fibronec- domain and the autophosphorylation and previously character- tin in part through alteration of cofilin phosphorylation, most ized regulatory sites. GST binding assays confirmed the lack of likely mediated through the TESK1 kinase. The ability of binding of actopaxin to this construct (Fig. 10A). We then TESK1 to only partially rescue this phenotype indicates the expressed this construct in HeLa cells and respread them on 10 possible perturbation of other actopaxin interactions, for in- g/ml fibronectin for 90 min. The cells were then processed for stance the association with actin. immunofluorescence microscopy. In contrast to full-length 21686 Actopaxin Regulation of TESK1 Function FIG.9. The actopaxin carboxyl terminus creates a spreading defect that is cofilin-dependent. HeLa cells were transfected with the indicated constructs, resuspended, and then spread on 10 g/ml fibronectin-coated coverslips for 90 min. Coverslips were subsequently fixed and processed for immunofluorescence followed by area quantifi- cation as detailed under “Experimental Procedures.” Co-transfection with a phosphomimetic cofilin construct (S3E) reverted the actopaxin 223–372 spreading defect, although it does not significantly affect spreading by itself. Expression of a non-phosphorylatable cofilin con- struct (S3A) significantly decreases spreading but does not further decrease spreading of the actopaxin 223–372 cells. * indicates a group FIG.8. Cells expressing the actopaxin carboxyl terminus dis- that is significantly different from GFP (p  0.01). ** indicates S3E- play aberrant phosphocofilin localization during spreading. cofilin able to diminish the spreading defect in Xpress-actopaxin 223– HeLa cells were transfected and spread on fibronectin as in figure 7. 372 cells (p  0.01). However, the combination of S3E and Xpress Cells were then fixed after 90 min and stained for phosphocofilin. GFP actopaxin 223–372 is significantly less well spread than GFP cells (p control cells exhibited an enrichment of phosphocofilin toward the cell 0.01). These results confirm that the defect in spreading in cells ex- periphery. Myc-TESK1-expressing cells showed slightly elevated phos- pressing actopaxin 223–372 is partially cofilin-dependent. phocofilin levels. In contrast, cells expressing the actopaxin carboxyl terminus (Xpress-actopaxin 223–372) displayed substantially reduced mimetic S4/8D actopaxin construct that promotes cell spread- levels of phosphocofilin as compared with adjacent non-transfected cells. Localized phosphocofilin staining and cell spreading are restored ing in osteosarcoma cells (27) was found to exhibit impaired when Myc-TESK1 is co-transfected with the Xpress-actopaxin 223–372 TESK1 binding, while the carboxyl terminus (Xpress-actopaxin construct. Transfected cells are indicated by GFP co-transfection. Bar, 223–372) alone bound constitutively to TESK1. 10 m. Actopaxin phosphorylation is stimulated upon cell adhesion and has been demonstrated to be MEK/Erk2-dependent (27). TESK1, which was found not to have an effect on spreading by However, since actopaxin was also weakly phosphorylated by itself, the GEF-TESK 1–528 increased spreading above that TESK1 in vitro, this kinase may also contribute to integrin-de- seen in GFP control cells (Figs. 10B and 7C). An increase in pendent phosphorylation of actopaxin perhaps via a feed-forward cortical actin structures was observed, consistent with elevated mechanism in which adhesion-stimulated phosphorylation of ac- TESK1 activity (Fig. 10B). We confirmed this effect by quanti- topaxin results in TESK1 dissociation and activation and thus fying areas of respread cells. GFP-TESK1 1–528 significantly additional actopaxin phosphorylation. It is currently uncertain increased spreading to 1.34 times that seen in control cells. how phosphorylation of the actopaxin amino terminus regulates Furthermore, this result was blocked by co-expression of cofilin TESK1 binding to the carboxyl terminus but likely involves long S3A, indicating the effect is dependent on cofilin phosphoryla- distance allosteric changes. This model is supported by the loss of tion. Finally, expression of TESK1 1–528 lacking the actopaxin adhesion-dependent regulation of the TESK1 association exhib- binding site completely rescued spreading in HeLa cells ex- ited by the carboxyl terminus of actopaxin. pressing Xpress actopaxin 223–372, confirming a role for Although both LIMK, which is activated downstream of Rho- TESK1 downstream of actopaxin in cell spreading (Fig. 10C). ROCK or Cdc42/Rac-PAK pathways, and TESK can phospho- DISCUSSION rylate cofilin (1), TESK1 has been suggested to be the primary Actopaxin performs a critical, evolutionarily conserved, role regulator of adhesion-dependent cofilin phosphorylation, as in stabilizing integrin-actin interactions at sites of cell adhe- supported by a significant loss of this signaling event in cells sion to the extracellular matrix in muscle and non-muscle cells expressing kinase-dead TESK1 (3). This translates into an (18, 26). Herein, we detail a functional interaction between inability of these cells to spread efficiently on fibronectin (8). actopaxin and the serine/threonine kinase TESK1, which is an Interestingly, overexpression of the carboxyl terminus of acto- important modulator of integrin-mediated actin dynamics due paxin, which, as opposed to full-length actopaxin, maintains to its ability to phosphorylate and thereby regulate the activity binding to TESK1 during adhesion, inhibits both cell spreading and cofilin phosphorylation. While it has previously been sug- of the F-actin-severing protein cofilin (3). Using a combination of yeast two-hybrid analysis, GST pull-down, and co-immuno- gested that this spreading defect is due to perturbation of the F-actin binding site on actopaxin and thus disruption of the precipitation assays we have localized the sites of interaction to within the carboxyl terminus of each molecule. In experiments integrin-actin linkage (18), our current results, showing that the spreading defect can be partially rescued following overex- designed to evaluate how the interaction between actopaxin and TESK1 may contribute to the regulation of actin dynamics pression of TESK1, provide evidence that cell spreading can also be controlled through actopaxin-mediated regulation of we have determined that the association between TESK1 and actopaxin negatively regulates TESK1 kinase activity and fur- TESK signaling to cofilin. The introduction of an S3E phospho- mimetic cofilin construct also reverts the spreading defect. As ther that the interaction is reduced in cells actively spreading on fibronectin. Dissociation of the actopaxin-TESK1 complex is this mutant does not bind actin, its rescue is likely mediated through competition for binding partners with endogenous co- likely to be regulated via a mechanism involving the phospho- rylation of the amino terminus of actopaxin, since a phospho- filin (9). This may potentially act through binding and seques- Actopaxin Regulation of TESK1 Function 21687 FIG. 11. Model for the adhesion-dependent regulation of TESK1 by actopaxin. Actopaxin and TESK1 are associated in asyn- chronously growing cells, which serves to inhibit TESK1 kinase activ- ity. Upon integrin stimulation, actopaxin becomes phosphorylated and TESK1 dissociates, relieving inhibition of its kinase activity. Active TESK1 then phosphorylates cofilin on serine 3, thereby inhibiting the association of cofilin with F-actin. This inactivates the F-actin severing activity of cofilin, allowing adhesion-dependent actin reorganization and facilitating spreading. thus activation, is necessary for growth factor-initiated lamel- lipodia formation (34, 35). Precise compartmentalization of the pools of active and inactive cofilin explains these seemingly conflicting observations. Thus, the non-phosphorylated pool of active cofilin has been localized to the extreme leading edge of a cell where it facilitates lamellipodial extension through the creation of free F-actin barbed ends that are conducive to branching via the activity of the Arp2/3 complex (17). In con- trast, a pool of phosphorylated cofilin is enriched a short dis- tance away from the edge of the lamellipodia, where it func- tions to stabilize actin structures necessary to support further membrane protrusions (13). Consistent with a role for phos- phocofilin in lamellipodia extension during cell spreading, we have found phosphocofilin to be enriched toward the cell pe- riphery following integrin ligation. Importantly, the phospho- cofilin staining was reduced in cells expressing Xpress-acto- paxin 223–372, consistent with the ability of this mutant to interfere with TESK1 activity and thus phosphorylation of cofilin. Incorporating our data into the model of TESK1 regulation of cofilin function, we propose (Fig. 11) that actopaxin and TESK1 associate with one another in the cytosol of asynchronously growing adherent cells or cells held in suspension, and this serves to inhibit TESK1 kinase activity. Upon integrin-medi- ated attachment to the ECM, as occurs during cell spreading or FIG. 10. A TESK1 mutant defective in actopaxin binding in- at sites immediately proximal to the leading edge of an extend- creases cell spreading. A, HeLa cells were transfected with GFP- TESK1 1–528 followed by GST pull-down binding assays. GST-acto- ing lamellipodium, actopaxin is recruited to the nascent focal paxin fails to bind this portion of TESK1, confirming that the carboxyl- complexes and becomes phosphorylated within its amino ter- terminal TESK1 residues 529–626 are the site of interaction with minus. This likely promotes an intramolecular rearrangement actopaxin. ILK was used as a positive control for binding, while -ac- between the amino and carboxyl terminus of actopaxin that tinin served as a negative control. B, HeLa cells were transfected with either GFP or GFP-TESK1 1–528 and respread on 10 g/ml fibronectin- reduces TESK1 binding, thereby relieving an inhibition of coated coverslips for 90 min. Coverslips were then fixed and processed TESK1 kinase activity. TESK1 then phosphorylates the local for immunofluorescence using rhodamine phalloidin to visualize F- pool of cofilin and thereby stabilizes actin filaments adjacent to actin. Cells expressing GFP-TESK1 1–528 are more spread than control focal complexes. Interestingly, nascent focal complexes have cells and exhibit increased cortical actin. Bar,10 m. C, GFP-TESK1 1–528 was co-expressed with cofilin S3A or Xpress-actopaxin 223–372 been shown to form at the transition zone between the extend- and respread on 10 g/ml fibronectin for 90 min. Cell areas were ing lamellipodium, which is rich in cofilin and Arp2/3 activity quantified as described under “Experimental Procedures.” Expression and the more stable actin network of the lamella (36–39). of GFP-TESK1 1–528 significantly increases spreading as compared The phosphoserine binding adaptor protein 14-3-3 has previ- with GFP control cells. This effect is blocked by S3A cofilin. The com- bination of GFP-TESK1 1–528 and Xpress-actopaxin 223–372 spreads ously been shown to regulate adhesion-dependent activation of comparably with GFP control cells. * indicates significantly different TESK1 via a similar mechanism (8). Interestingly, we found that from GFP (p  0.01). expression of GFP-TESK1 1–528, which lacks actopaxin binding but retains serine 439, the site of 14-3-3 binding, promoted tering the cofilin phosphatases slingshot or chronophin, with aberrant cell spreading and cortical actin structure formation, subsequent alteration of endogenous cofilin phosphorylation while wild-type TESK1 overexpression exerted minimal effects. levels (10, 11). This likely suggests that 14-3-3 and actopaxin contribute to the The morphologic changes that occur when cells are spreading regulation of TESK1 function via overlapping as well as distinct on extracellular matrix are considered analogous to lamellipo- mechanisms. For instance, it will be important to determine dia formation at the leading edge of motile cells. Interestingly, whether actopaxin binding influences TESK1 phosphorylation while TESK1 promotes cell spreading via cofilin phosphoryla- on serine 439 and thus 14-3-3 binding. tion, it has been shown that cofilin dephosphorylation, and Finally, it has previously been suggested that a stable asso- 21688 Actopaxin Regulation of TESK1 Function Cell 108, 233–246 ciation between actopaxin, PINCH, and ILK is obligatory and 11. Gohla, A., Birkenfeld, J., and Bokoch, G. M. (2005) Nat. Cell Biol. 7, 21–29 serves to stabilize these proteins (32). Interestingly, TESK1 12. Ghosh, M., Song, X., Mouneimne, G., Sidani, M., Lawrence, D. S., and Con- binds a population of actopaxin distinct from that which binds deelis, J. S. (2004) Science 304, 743–746 13. Dawe, H. R., Minamide, L. S., Bamburg, J. R., and Cramer, L. P. (2003) Curr. ILK, indicating an alternate pool of actopaxin within the cell. 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Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Jun 3, 2005

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