Pseudopodium-Enriched Atypical Kinase 1 Regulates the Cytoskeleton and Cancer Progres- Sion.” the Title Has Been Corrected Online

Corrections

ANTHROPOLOGY Correction for “Y chromosome diversity, human expansion, drift, and cultural evolution,” by Jacques Chiaroni, Peter A. Underhill, and Luca L. Cavalli-Sforza, which appeared in issue 48, December 1, 2009, of Proc Natl Acad Sci USA (106:20174–20179; ﬁrst published November 17, 2009; 10.1073/pnas.0910803106). The authors note the following statement should be added to the Acknowledgments: “This work was supported by ANR Pro- gram AFGHAPOP N° BLAN07-3_222301.”

www.pnas.org/cgi/doi/10.1073/pnas.1008738107

CELL BIOLOGY Correction for “Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progession,” by Yingchun Wang, Jonathan A. Kelber, Hop S. Tran Cao, Greg T. Cantin, Rui Lin, Wei Wang, Sharmeela Kaushal, Jeanne M. Bristow, Thomas S. Edgington, Robert M. Hoffman, Michael Bouvet, John R. Yates III, and Richard L. Klemke, which appeared in issue 24, June 15, 2010, of Proc Natl Acad Sci USA (107:10920–10925; ﬁrst published June 1, 2010; 10.1073/pnas.0914776107). The authors note that the title of their manuscript appeared incorrectly. The title should appear as “Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression.” The title has been corrected online.

www.pnas.org/cgi/doi/10.1073/pnas.1008849107

DEVELOPMENTAL BIOLOGY Correction for “TIF1β regulates the pluripotency of embryonic stem cells in a phosphorylation-dependent manner,” by Yasuhiro Seki, Akira Kurisaki, Kanako Watanabe-Susaki, Yoshiro Nakajima, Mio Nakanishi, Yoshikazu Arai, Kunio Shiota, Hiromu Sugino, and Makoto Asashima, which appeared in issue 24, June 15, 2010, of Proc Natl Acad Sci USA (107:10926–10931; ﬁrst published May 27, 2010; 10.1073/pnas.0907601107). The authors note that the gene name “Smarcad1” appeared incorrectly throughout the article and supporting information. The correct spelling of the gene is “Smarcd1.”

www.pnas.org/cgi/doi/10.1073/pnas.1008651107

13556 | PNAS | July 27, 2010 | vol. 107 | no. 30 www.pnas.org Downloaded by guest on October 3, 2021 Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression

Yingchun Wanga,b,1,2, Jonathan A. Kelbera,b,1, Hop S. Tran Caoc, Greg T. Cantind, Rui Line, Wei Wanga,b, Sharmeela Kaushalb, Jeanne M. Bristowa,b, Thomas S. Edgingtone, Robert M. Hoffmanc,f, Michael Bouvetb,c, John R. Yates IIId, and Richard L. Klemkea,b,3

Departments of aPathology and cSurgery and bMoores Cancer Center, University of California, La Jolla, CA 92093; Departments of dChemical Physiology and eImmunology, The Scripps Research Institute, La Jolla, CA 92037; and fAntiCancer, Inc., San Diego, CA 92111

Edited by Joan S. Brugge, Harvard Medical School, Boston, MA, and approved April 27, 2010 (received for review December 24, 2009) Regulation of the actin-myosin cytoskeleton plays a central role in cell novel kinases involved in cell migration and cancer cell invasion migration and cancer progression. Here, we report the discovery of (14). This approach uncovered a unique 190-kDa nonreceptor a cytoskeleton-associated kinase, pseudopodium-enriched atypical atypical tyrosine kinase family member KIAA2002 (sgk269) that is kinase 1 (PEAK1). PEAK1 is a 190-kDa nonreceptor tyrosine kinase enriched by 2.6-fold in the pseudopodium. We have named this that localizes to actin filaments and focal adhesions. PEAK1 under- protein pseudopodium-enriched atypical kinase 1 (PEAK1). Our goes Src-induced tyrosine phosphorylation, regulates the p130Cas- biochemical and biological findings indicate that PEAK1 is a pre- Crk-paxillin and Erk signaling pathways, and operates downstream viously undescribed member of the canonical Src-p130Cas-Crk-II- of integrin and epidermal growth factor receptors (EGFR) to control Paxillin and Erk cytoskeletal signaling pathways. Furthermore, we cell spreading, migration, and proliferation. Perturbation of PEAK1 observed that PEAK1 localizes to focal adhesions, strongly asso- levels in cancer cells alters anchorage-independent growth and ciates with the actin cytoskeleton, and plays an important role in tumor progression in mice. Notably, primary and metastatic samples cancer cell migration and proliferation in vitro and in vivo. from colon cancer patients display amplified PEAK1 levels in 81% of the cases. Our findings indicate that PEAK1 is an important cytoskel- Results etal regulatory kinase and possible target for anticancer therapy. Proteomic and Bioinformatic Analyses of the Pseudopodial pY Proteome. The pseudopodium is highly enriched with pY pro- cancer | cell migration | phosphoproteomics teins involved in the regulation of actin polymerization and focal adhesion dynamics (Fig. S1A) (9). To identify kinases and their substrates that spatially regulate these processes, we used ll cells have the fundamental ability to change shape and to fi Aextend membrane projections from the cell surface (1). This a strategy that allows for the large-scale puri cation of pseudopodia actively extending toward an LPA gradient (9–11). The process is mediated by the actin-myosin cytoskeleton and is relative differences in pY proteins from pseudopodia were critical for sensing and adapting to changes in the extracellular compared with pY proteins isolated from purified cell bodies by environment. Although proper cytoskeletal regulation is impor- using pY immunoaffinity purification followed by MudPIT (14). tant for numerous physiological processes including axon/den- Importantly, many tyrosine-phosphorylated proteins bind drite formation, migration, differentiation, and proliferation, its strongly to the actin cytoskeleton and focal adhesions in migra- deregulation can also contribute to human diseases including tory cells and, thus, are largely insoluble in detergents like cancer metastasis (1–5). Therefore, it is crucial to understand the Nonidet P-40 and TX-100 (15). Therefore, to increase the yield mechanisms that control the cytoskeleton and how it contributes of pY proteins, cellular extracts were prepared by using de- to cancer progression. naturing conditions that maximize protein solubility by boiling the samples in 1% SDS lysis buffer. These conditions also Tyrosine phosphorylation of cytoskeleton-associated proteins – plays a central role in pseudopodium formation (6). A pseudo- eliminate non-pY to pY protein protein interactions. After di- lution of the SDS/protein extract, pY proteins were then selec- podium (or invadopodium in a cancer cell) is a highly specialized, tively purified with antiphosphotyrosine antibodies and identified actin-rich structure that protrudes from the cell surface in mi- by MudPIT (14). This method significantly enhances the specific grating cells. It serves to tether the extending membrane to the purification and coverage of the pseudopodial pY proteome. underlying substrate via the formation of focal adhesions and to Using this approach, a total of 309 pY proteins were identified in mediate traction forces that propel the cell forward (7, 8). The the cell body and pseudopodial fractions (Dataset S1 and Table pseudopodium also plays an important role in steering the cell by S1). Of these, 211 proteins were enriched by at least 1.5-fold in degrading extracellular matrix protein barriers and by sensing the pseudopodium compared with the cell body (Fig. S1B). changes in chemokine and adhesive gradients that serve as guid- Functional annotation and statistical analysis of these pseudo- ance cues. Therefore, understanding how this structure is regu- podial-enriched pY proteins using the Fatigo search engine (16) lated is crucial to understanding how cells migrate and invade and the KEGG pathway databases revealed that the highest percentage of pY proteins mapped to signaling pathways that tissues during cancer progression. To this end, we described a novel method for purifying the pseudopodia from cells for signal transduction and proteomic studies (9–11). Using this fraction- Author contributions: Y.W., J.A.K., G.T.C., W.W., J.M.B., and R.L.K. designed research; Y.W., ation method and quantitative mass spectrometry (MS), we have J.A.K., H.S.T.C., G.T.C., R.L., W.W., S.K., J.M.B., and R.M.H. performed research; Y.W., M.B., profiled the relative differences in the pseudopodium and cell J.R.Y., and R.L.K. contributed new reagents/analytic tools; Y.W., J.A.K., H.S.T.C., G.T.C., R.L., body proteomes (10). Although this approach has identified im- W.W., J.M.B., T.S.E., and R.L.K. analyzed data; and Y.W., J.A.K., and R.L.K. wrote the paper. portant new pseudopodial proteins, it did not provide a robust The authors declare no conflict of interest. method for detailed analysis of phosphotyrosine (pY) proteins or This article is a PNAS Direct Submission. kinases present in this subcellular structure (10). pY proteins are 1Y.W. and J.A.K. contributed equally to this work. fi dif cult to detect because of their low abundance and typically 2Present address: Institute of Genetics and Developmental Biology, Chinese Academy of require an enrichment step before MS analysis (12, 13). Sciences, No.1 West Beichen Road, Beijing 100101, China. In this report, we use pY immunoaffinity enrichment and Mul- 3To whom correspondence should be addressed. E-mail: [email protected]. fi tidimensional Protein Identi cation Technology (MudPIT) to de- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. fine the pseudopodial phosphotyrosine proteome and to search for 1073/pnas.0914776107/-/DCSupplemental.

10920–10925 | PNAS | June 15, 2010 | vol. 107 | no. 24 www.pnas.org/cgi/doi/10.1073/pnas.0914776107 regulate the actin cytoskeleton and focal adhesions (Fig. S1C). There are also several important protein interaction sites present Protein network analysis using the Ingenuity pathway analysis in PEAK1, including an SH2 binding motif for Src kinase (Y665), software (10) also revealed a well-connected cytoskeletal net- a proline rich motif for binding to the Crk SH3 domain (P1153), work of pY proteins known to regulate the pseudopodium and a Shc binding site (Y1188), and an ERK binding motif (P228). focal adhesion dynamics including paxillin, cortactin, talin, shc, The kinase domain spans aa 1330–1664, and the ATP binding site GIT1/2, p130Cas, FAK, and Src family kinases (9, 17–26) (Fig. spans aa 1133–1140. Overall, our bioinformatic analyses predict S1D). As expected, signaling pathways involved in regulation of that PEAK1 is a phosphotyrosine protein and an atypical tyrosine cell cycle and calcium signaling were highly represented in the kinase that associates with the actin cytoskeleton. cell body fraction, which contains the nuclear compartment. Thus, the combined methods of pseudopodium purification, pY PEAK1 Shows in Vitro Kinase Activity. All active kinases are pre- protein enrichment, and MudPIT provided a representative dicted to contain three motifs, VAIK,HRD, and DFG, within sample of the pseudopodium pY proteome and its spatial or- the kinase domain (33). Each of these motifs contain one highly ganization in migrating cells. conserved residue (VAIK: K,HRD: D, DFG: D) that is predicted to be important for full catalytic activity. Sequence analysis Bioinformatic Analyses Indicate That PEAK1 Contains Multiple Motifs, revealed that PEAK1 contains all of the three motifs YAVK, Domains and Consensus Phosphorylation Sites That Couple It to the HCD, and NFS. The YAVK and HCD motif are highly con- Cytoskeleton. Twelve unique phosphoproteins were found to be served on the critical K and D residues, but the D residue in the enriched in the pseudopodium (Table S1). Although these pro- NFS motif has been replaced by N, which classifies it as an teins have not been previously studied, their function can be atypical kinase (33). However, it is not yet known whether this inferred from bioinformatic software, which identify known pro- amino acid substitution can affect kinase catalytic activity or tein interaction domains and consensus kinase phosphorylation whether this site may be mutated in human cancers to confer motifs that are based on amino acid sequence homologies (27). Of full catalytic activity (33). Nonetheless, to investigate whether these 12 proteins, PEAK1 was chosen for detailed analysis. PEAK1 displays kinase activity, we first determined whether PEAK1’s complete domain structure, its known and predicted PEAK1 can autophosphorylate in vitro because many kinases phosphorylation sites, and its predicted signaling pathway are phosphorylate themselves (34, 35). For these experiments, GFP- shown schematically in Fig. 1A. Previous work has shown that PEAK1 was purified from HEK 293T cells as described in Ex- PEAK1 is ubiquitously expressed in tissues with the highest level of perimental Procedures and its purity was confirmed by silver expression in brain and kidney (28) and is highly conserved from staining (Fig. S3A). Autophosphorylation was determined by zebrafish to human. The complete gene structure and cloning using an in vitro kinase assay (35) and antiphosphotyrosine strategy for obtaining full-length PEAK1 is shown in Fig. S2. Western blotting. PEAK1 displayed significant ability to auto- Using the bioinformatic software Scansite (27), PEAK1 is phosphorylate on tyrosine residues (Fig. S3A). To determine predicted to be phosphorylated by Abl (Y797), Src (Y665), and whether PEAK1 can phosphorylate an exogenous substrate, we performed an in-gel kinase assay by using myelin basic protein ERK (S779, T783) kinases (27) (Fig. 1A). All of these enzymes are 32 known to regulate the cytoskeleton and contribute to cancer (MBP) as a generic substrate and γ-[ P]ATP (36). Under these progression (18, 29–31). Although we have not yet mapped the conditions PEAK1 was able to weakly, but consistently, phos- sites of phosphorylation on PEAK1, at least 13 phosphotyrosine phorylate MBP (Fig. S3B). Finally, to rule out the possibility sites have been identified in previous phosphoproteomic studies that a coprecipitating kinase or cofactor from mammalian cell including Y665 (32) (see also the phosphosites database for the extracts may account for the observed kinase activity of PEAK1 complete list of PEAK1 phosphosites: www.phosphosite.org). in these experiments, we purified PEAK1’s kinase domain (aa 1289–1746) from Escherichia coli by using a Smt3 tag and size exclusion chromatography (Fig. S3C) and then tested it for tyrosine kinase activity toward MBP in vitro. MBP was phosphorylated by the purified kinase domain under these conditions (Fig. S3D). Together, these results demonstrate that PEAK1 has tyrosine kinase activity.

PEAK1 Localizes to the Actin Cytoskeleton and Focal Adhesions. PEAK1 subcellular localization was determined using ﬂuores- cence microscopy in NIH 3T3 cells transfected with full-length PEAK1 fused with GFP (GFP-PEAK1). PEAK1 strongly coloc- alizes with the F-actin cytoskeleton and vinculin-positive focal adhesions, whereas control cells expressing GFP showed only diffuse cytoplasmic staining (Fig. 1 B and C). In the majority of cells (>80%), PEAK1 displayed punctate staining along actin cables and cortical actin structures. To map which region of PEAK1 is responsible for its F-actin localization, we expressed a series of truncated forms of PEAK1 fused to GFP at the N terminus in NIH 3T3 cells (Fig. S4A). Using this mutagenesis approach, the actin targeting region was mapped to residues 338– 727 (Fig. S4B). Interestingly, this region of PEAK1 also displays the majority of known and predicted tyrosine phosphorylation sites, suggesting that upstream kinases and phosphatases may regulate cytoskeletal localization of PEAK1 (Fig. 1A). In support of this notion, serum starvation reduced PEAK1 localization to F-

actin, whereas stimulation of cells with serum or PDGF-BB in- CELL BIOLOGY duced strong PEAK1 colocalization with F-actin, and this re- Fig. 1. PEAK1 localizes to the actin cytoskeleton and focal adhesions. (A) sponse required residues 338–727 (Fig. S4C). In addition, using Schematic showing predicted PEAK1 protein domains, motifs, and known pY immunofluorescence staining and a monoclonal antibody to sites. (B and C) Immunofluorescence images showing GFP-PEAK1 colocali- PEAK1, we assessed whether endogenous PEAK1 protein lo- zation with the actin cytoskeleton and vinculin-positive focal adhesions (red) calized to these cytoskeletal structures. Endogenous PEAK1 was in NIH 3T3 fibroblast cells. White boxes (Inset) show the respective zoomed observed to localize to actin and focal adhesions (Fig. S4D), images. The fluorescence intensity of GFP-PEAK1 and vinculin along the in- whereas cells depleted of PEAK1 by siRNA did not show PEAK1 dicated line were scanned by using MetaMorph imaging software, and their immunostaining (data not shown). Although the focal adhesion colocalization was determined by using the Pearson correlation coefficient targeting domain in PEAK1 has not yet been ellucidated, our (r = 0.91) method. (Scale bars: 15 μm.) preliminary data suggest that this process will be complex and

Wang et al. PNAS | June 15, 2010 | vol. 107 | no. 24 | 10921 multifaceted, requiring phosphorylation of PEAK1 and binding of multiple proteins that target PEAK1 to these structures. Never- theless, these ﬁndings indicate that PEAK1 associates with the actin cytoskeleton and localizes to cell-matrix focal adhesions.

PEAK1 Expression in Cells Alters Phosphorylation of Cytoskeleton- Associated Proteins. Activation of integrin and growth factor receptors promotes tyrosine phosphorylation and the molecular scaffolding of cytoskeleton effector proteins including Src/ p130Cas/Crk/Paxillin/Erk (9, 17, 18). Interestingly, exogenous expression of PEAK1 in cells increased the phosphorylation of paxillin (Y31), p130Cas (Y249), and the Erk activation sites (T185/Y187) in response to cell adhesion to fibronectin (Fig. 2A). Paxillin Y31 phosphorylation was shown to regulate the assembly and formation of cell-matrix adhesions, and p130Cas Y249 phosphorylation by Src provides a docking site for the Crk cytoskeleton adaptor protein (37), whereas Erk phosphorylation on T185/Y187 is necessary for its kinase activity. Importantly, the phosphorylation of these proteins at these specific amino acid residues are known to play important roles in modulation of the cytoskeleton and cell migration (17, 18, 30, 38–40). In contrast, depletion of endogenous PEAK1 using a specific siRNA (siPEAK1) decreased the phosphorylation of paxillin (Y31) and Erk under these conditions (Fig. 2B). No obvious change was Fig. 2. PEAK1 regulates cytoskeletal and focal adhesion proteins and observed in the phosphorylation of p130Cas (Y249), indicating undergoes Src kinase-dependent tyrosine phosphorylation in response to that PEAK1 can enhance phosphorylation, but is not necessary cell adhesion or EGF stimulation. (A) Protein lysates of 293T cells expressing for phosphorylation at this specific site. Furthermore, we found GFP-PEAK1 (PEAK1) in suspension (Sus) or attached (Atta) to fibronectin (FN) that p130Cas and Crk coimmunoprecipitated with PEAK1. Un- were immunoblotted with indicated total protein and pY site-specific anti- der these conditions, Crk coprecipitated only with full-length bodies. (B) Lysates from PEAK1-specific siRNA (siPEAK1) or scrambled control PEAK1 or the C-terminal truncated forms of PEAK1 (C1 and C2) siRNA (siCtrl)-treated cells in suspension or attached to FN for 30 min were that contain the predicted Crk-SH3 binding motif (P-X-L-P-X-K) immunoblotted as in A.(C) GFP, GFP-PEAK1, and GFP-PEAK1 truncated (41) and not the N-terminal truncations that lack this domain forms (N1–N3, C1, and C2; Fig. S4A) were immunoprecipitated then immu- (Fig. 2C). On the other hand, p130Cas coprecipitated with wild- noblotted for associated Crk and Cas proteins. Whole-cell lysates were also type and all mutant forms of PEAK1, suggesting that it may in- immunoblotted for the indicated proteins. (D and E) pY Western blots of teract with PEAK1 at multiple sites or may associate with other GFP-PEAK1 immunoprecipitated from cells stimulated or not stimulated with proteins that interact at multiple sites with PEAK1. Taken to- EGF for 10 min (D) or stimulated with EGF and in the absence or presence of gether, these data indicate that the modulation of PEAK1 protein the Src kinase inhibitor PP2 (E). (F and G) pY Western blots of GFP-PEAK1 levels altered the phosphorylation/activation of known cytoskel- immunoprecipitated from cells in suspension (Sus) or reattached to 5 μg/mL eton-associated proteins. of poly-L-lysine (PLL), FN, or laminin (LN), respectively, (F) or cells reattached to FN in the absence or presence of PP2 (G). Total PEAK1 levels in E and F Src Kinase Activity Is Necessary for PEAK1 Tyrosine Phosphorylation were detected by Ponceau staining. (H) pY immunoprecipitation of total +/+ −/− Induced by Integrin and Growth Factor Receptor Activation. We next lysates from wild-type ( ) or Src/Fyn/Yes (SYF) knockout ( ) MEFs and tested whether PEAK1 is phosphorylated in response to growth Western blots for PEAK1 after EGF stimulation for 10 min. factor stimulation or integrin engagement and the role of Src kinase in this response. PEAK1 was maximally tyrosine phosphor- toward a fibronectin gradient. For these experiments, we used ylated in HEK 293T cells exposed to EGF for 10 min, and this a specific shRNA to stably deplete PEAK1 from XPA-1 pancre- response was inhibited by the Src kinase inhibitor, PP2 (Fig. 2 D atic cancer cells (Fig. S5D). These cells showed significantly re- and E). Similar findings were obtained in Src/Yes/Fyn-deficient duced migration toward fibronectin compared with the control MEF cells exposed to EGF (Fig. 2H). Cell adhesion to fibronectin cells stably expressing a nontargeting shRNA (Fig. 3E). Again, also induced PEAK1 tyrosine phosphorylation, and this was depletion of PEAK1 in XPA-1 cells did not significantly change inhibited by PP2 (Fig. 2 F and G). Together, these findings in- adhesion to the ECM (Fig. S6A Right). Additionally, we analyzed dicate that cell adhesion and growth factor stimulation induce the migration tracks of these cells over 24 h using time-lapse video PEAK1 tyrosine phosphorylation in a Src-dependent manner. microscopy in combination with Metamorph software. This test revealed that PEAK1 promotes a more persistent type of migra- PEAK1 Expression Modulates Cell Spreading and Migration. PEAK1 tion of cancer cells (Fig. 3 F and G). Finally, because Src can localization to the cytoskeleton and its ability to modulate known regulate PEAK1 phosphorylation (Fig. 2 E, G, and H), we wanted focal adhesion proteins suggest that it regulates spreading and to determine whether PEAK1 played a role in Src-induced cell migration. To investigate this possibility, Cos-7 or HEK 293T cells migration. In this case, exogenous expression of Src-enhanced were depleted of PEAK1 using siRNA and tested for their ability detectable Src activity (Fig. S5E), leading to an increase in the to spread and migrate in vitro on fibronectin. Knockdown of velocity of cell migration that depended on endogenous PEAK1 PEAK1 in Cos-7 cells (Fig. S5C) delayed the initial stages of cell protein (Fig. 3H). Taken together, these data show that PEAK1 is spreading (<90 min) as indicated by a decrease in cell area (Fig. sufficient and necessary for proper cell spreading and migration in 3A). However, by 180 min there was no difference in the size of response to fibronectin and growth factors as well as Src kinase control and PEAK1-depleted cells, indicating that other spread- activity. Although the mechanism through which PEAK1 modu- ing signals can compensate for the loss of PEAK1 (Fig. 3A). On lates cell spreading and migration is not yet understood, PEAK1 the other hand, exogenous expression of PEAK1 increased cell expression in cells was observed to significantly increase focal spreading at an early stage (<30 min) (Fig. 3B). Also, cells de- adhesion length (Fig. S4E), suggesting that it may play a role in pleted of PEAK1 showed significantly reduced chemotaxis com- regulating adhesion dynamics. pared to control cells (Fig. 3C). Importantly, PEAK1 depletion did not alter cell attachment to fibronectin (Fig. S6A Left). Con- PEAK1 Promotes Anchorage-Independent Cancer Cell Growth in Vitro versely, Cos-7 cells expressing exogenous PEAK1 showed in- and Tumor Progression in Mice. The deregulation of kinases and creased cell chemotaxis toward LPA (Fig. 3D), and this did not cytoskeletal proteins often contribute to cancer progression (42). alter cell attachment to fibronectin Fig. S6A Center). We further Therefore, we wanted to determine whether PEAK1 plays a role in tested whether PEAK1 is necessary for haptotactic migration cancer progression. The ability of cells to grow in soft agar in the

10922 | www.pnas.org/cgi/doi/10.1073/pnas.0914776107 Wang et al. levels in cancer cells alters tumor growth prompted us to ex- amine whether expression of PEAK1 mRNA is altered in human cancer. In situ hybridization was performed to analyze expression of PEAK1 mRNA in healthy colon tissues and the corresponding colon tumors and liver metastases by using human colon cancer tissue array that included samples from 22 patients. Of these, 18 patients (81.8%; P < 0.001) showed elevated PEAK1 staining in the primary tumor compared with the normal colon tissue (Fig. 4G and Fig. S6B). Twelve of these patients (54.5%) showed positive staining in their corresponding liver metastases (P < 0.05) (Fig. 4G and Fig. S6B). In contrast, only 5 patients (22.7%) showed positive PEAK1 staining in normal colon tissues. Taken together these data indicate that PEAK1 is a unique cytoskeleton-associated kinase and a member of the Src/p130Cas/Crk/ paxillin/Erk signaling pathways that regulate cell migration and promote cancer progression. Discussion Our ability to affinity purify pY proteins from isolated pseudopodia proved to be a robust system to identify proteins involved in cell migration. Also, the solubilization of pseudopodial proteins in SDS buffer significantly improved our yield of pY pro- Fig. 3. PEAK1 regulates cell spreading and directional cell migration. Cos-7 teins, which can be tightly associated with the insoluble cells treated with siPEAK1 or siCtrl (A) or HEK 293T cells expressing exoge- cytoskeleton (15). This approach allowed us to identify many low nous GFP-PEAK1 or GFP (B) only were allowed to attach and spread on 5 μg/ abundant pY proteins involved in cell migration and led to the mL fibronectin for the indicated times. Cell areas were measured by using discovery of PEAK1. Collectively, our findings demonstrate that MetaMorph. Bars indicate mean ± SD in all figures unless indicated other- PEAK1 is a unique nonreceptor tyrosine kinase that operates wise. *P < 0.01; **P < 0.001. Cos-7 cell chemotaxis toward LPA after treat- within the Src-p130Cas-Crk-Paxillin signaling pathway to regu- ment with siPEAK1 or siCtrl (C) or overexpression of GFP-PEAK1 or GFP (D). late cell spreading, migration, and cancer progression. (E) Cell migration toward FN of XPA-1 pancreatic cancer cells treated with Modulation of PEAK1 affected the phosphorylation level of shRNA specific for PEAK1 (shPEAK1) or a scrambled control shRNA (shCtrl). (F several known cytoskeleton regulatory proteins including paxillin, and G) Cells (5 × 104)asinE were plated onto FN-coated six-well plates, and p130Cas, and Erk, and it was found to associate with the Crk >70 cells were tracked in each population by using phase-contrast micros- adaptor protein. PEAK1 has a proline-rich sequence (P-X-L-P-X- copy and MetaMorph software during the subsequent 24 h. The resulting K) that conforms to the predicted binding site for the N-terminal endpoint displacement (F) and cell migration persistance (G) were plotted. SH3 domain of Crk (41). Our finding that Crk coprecipitates with (H) After cell migration tracking as in F and G, cell velocity was quantified for the C-terminal region of PEAK1 has important implications for shCtrl and shPEAK1 cell populations that were transiently transfected with how PEAK1 could regulate the cytoskeleton. Crk is an adaptor empty vector or Src overexpression vector. ***P < 0.0001. protein that regulates cells spreading and migration by coupling critical signaling proteins such as EGFR, PDGFR, and C-Abl to absence of integrin adhesions to the ECM is a hallmark of cancer the cytoskeleton and focal adhesions including the scaffolding (43–45). To determine whether PEAK1 modulates cancer cell protein p130Cas (47). p130Cas and its family members are necessary for cell migration and cancer cell invasion and have been growth, human MDA-MB-435 cancer cells stably expressing ex- – ogenous PEAK1 were cultured in soft agar and the number and associated with cancer progression in patients (48 51). In- size of colonies measured after 14 d. Exogenous expression of terestingly, p130Cas coprecipitates with PEAK1 and PEAK1 PEAK1 increased the number and size of soft agar colonies and modulates p130Cas-Y249 phosphorylation (Fig. 2 B and C). Y249 is known to be phosphorylated by Src family kinases and to was associated with increased Erk kinase activity (Fig. 4 A and C). – In contrast, depletion of exogenous PEAK1 protein with an provide a binding site for the Crk SH2 domain (38, 47, 52 55). shRNA construct inhibited this response (Fig. 4 B and C and Fig. The Src/p130Cas/Crk complex has been shown to modulate Rac fi activity, pseudopodium protrusion, cell migration, and cancer S5 A and B). These ndings indicate that PEAK1 provides a growth – fi advantage to tumor cells independent of integrin adhesion signals. progression (9, 17, 48, 56 59). Together, these ndings suggest Having established that PEAK1 is important for anchorage-in- a possible scenario in which integrins and growth factors activate dependent tumor cell growth, we wanted to determine whether Src that, in turn, phosphorylates p130Cas Y249, leading to Crk binding through its SH2 domain. PEAK1 is bound to Crk’s SH3 PEAK1 could contribute to tumor formation in vivo. MDA-MB- domain and, thus, is recruited to the p130Cas/Crk scaffold, where 435 cells stably expressing GFP (MDA-435-GFP) or GFP-PEAK1 fl it is phosphorylated by Src at Y665, the Src consensus site (Fig. (MDA-435-PEAK1) were injected s.c. into the anks of nude 1A and Fig. S5E). Consistent with this idea, Src kinase activity is mice. Tumor progression was monitored weekly by using whole- necessary for PEAK1 tyrosine phosphorylation in response to body fluorescence imaging as described (46). MDA-435-PEAK1 – fi growth factor and integrin receptor activation (Fig. 2 D H). showed a signi cant advantage in tumor formation as indicated by Under these conditions, PEAK1 could serve several important the increase in tumor size over the 9-wk period compared with purposes. First, it may modulate protein–protein interactions by MDA-435-GFP (Fig. 4D). At the end of the 9 wk, the animals were fi directly phosphorylating components of the p130Cas/Crk/Paxillin sacri ced; the tumors were removed and weighed. Consistent with scaffold via its tyrosine kinase activity (Fig. S3). Second, given our in vitro data, cells overexpressing PEAK1 formed tumors that that PEAK1 translocates to focal adhesions and the actin cyto- showed increased area and weight compared with GFP control skeleton after growth factor stimulation (Fig. 1 and Fig. S4), it tumors (Fig. 4 D and E). We also used shRNA to stably knock- could provide a mechanism to transport the Src/p130Cas/Crk CELL BIOLOGY down endogenous PEAK1 expression in human XPA-1 pancreatic scaffold to these structures. Third, its strong association with the cancer cells and determined their ability to form tumors when actin cytoskeleton suggests that it may tether the Src/p130Cas/Crk transplanted orthotopically into the pancreas of nude mice. In this complex to the cytoskeleton. Finally, it may deliver unique ef- case, PEAK1-depleted tumor cells showed a reduction in tumor fector proteins to focal adhesions and the cytoskeleton that, in growth as indicated by reduced tumor size compared with control turn, modulate cell migration and/or proliferation. It is intriguing cells (Fig. 4F). These findings indicate that PEAK1 is necessary for that the majority of the known and predicted phosphorylation tumor growth in vitro and in vivo. sites cluster in the actin localization region of PEAK1. It seems plausible then that PEAK1’s association with the cytoskeleton is PEAK1 Expression Is Up-Regulated in Human Colon Cancer and Liver regulated by phosphorylation of one or more of these sites. In any Metastases. The observation that modulation of PEAK1 protein case, our observations that PEAK1 can interact with and modu-

Wang et al. PNAS | June 15, 2010 | vol. 107 | no. 24 | 10923 Fig. 4. PEAK1 promotes oncogenic growth of cancer cells in vitro and in vivo and is up-regulated in human colon cancer. Comparison of soft agar colony size and number in MDA-435 cells expressing GFP-PEAK1 or GFP only (A)ordepletedofGFP-PEAK1byshRNA(B)(seealsoFig. S5). The area of the colonies is shown as mean ± SEM. (C)CellsfromA and B were cultured under suspension conditions for 30 min followed by lysis and Western blot analysis of P-Erk and total Erk proteins. (D) Whole-animal fluorescent images of MDA-435-GFP (GFP) and MDA-435-PEAK1 (PEAK1) cancer cells allowed to grow s.c. in nude mice for 9 wk. (E)Tumordatafrom mice shown in D: Upper, average tumor weight after tumor excision; Lower, average tumor area as measured by whole-body fluorescence imaging throughout the course of the experiment. (F) Average tumor size of XPA-1 human pancreatic cancer cells stably expressing control scrambled or PEAK1 shRNA and growing orthotopically in the pancreas of nude mice for 4 and 5 wk. The data are shown as mean ± SEM for E and F.(G) Representative in situ hybridization images of PEAK1 expression levels (red stain) in normal colon tissue (NC), primary colon cancer tissue (CC), and a liver metastasis (LM) taken from the same patient. late the Src/p130Cas/Crk/paxillin and Erk signaling pathways nomic screens have also revealed that PEAK1 is involved in cancer point to a central role for this unique protein in mediating cell cell proliferation (62). Together, these findings suggest that PEAK1 migration and proliferation in normal and cancer cells. and sgk223 are cytoskeleton-associated kinases that regulate cell Although we directly study the function of PEAK1, several in- migration and cancer progression in response to Src kinase activity. dependent lines of evidence also suggested that PEAK1 and its only The fact that Src is a major contributor to human cancers and our family member sgk223 (33% overall homology to PEAK1) play an findings that PEAK1 levels are amplified in >80% of colon cancer integral role in regulation of cell motility and tumor progression patients underscore the importance of this protein in human cancer (60, 61). For example, sgk223 has been reported to be a unique and warrants further investigation. effector of Rnd2 GTPase. It has also been shown to stimulate RhoA activity in HeLa cells and mediated cancer cell invasion in a Src- Experimental Procedures dependent manner (61). More recently, sgk223 and PEAK1 were Protein Sample Preparation and Identification. Pseudopodia andcell bodieswere both identified as the potential targets that mediate Src invasive purified from Cos-7 cells as described (9–11). PY proteins were purified by immu- activity in advanced colon carcinoma cells in a quantitative phos- noprecipitation and identified by MudPIT and mass spectrometry (see SI Experi- phoproteomics study (60). Here, we demonstrate that PEAK1 mental Procedures for details). modulates anchorage-independent growth in soft agar and tumor formation in nude mice. Similar to sgk223, Src mediates PEAK1 SiRNA and shRNA Assay. The siRNA pool specific for PEAK1 was purchased tyrosine phosphorylation (Fig. 2 D–H). Finally, large shRNA ge- from Dharmacon, and transfection was performed according to manufacturer’s

10924 | www.pnas.org/cgi/doi/10.1073/pnas.0914776107 Wang et al. instructions. The shRNA was designed using the software RNAi OligoRetriever ACKNOWLEDGMENTS. We thank Drs. Olivier Pertz, and Hisashi Kato for (63) and cloned into the lentiviral vector FG12. The depletion of PEAK1 was assistance in lentivirus production, Spencer Wei for imaging assistance, and conﬁrmed by measuring the mRNA level using RT-PCR and Western blot anal- Tiffany Taylor, Ryan Matson, and Elizabeth Hampton for assisting with the molecular biology and biochemistry work. We also thank Dr. Stephen K. ysis. See Table S2 for the sequence information of all siRNAs and shRNAs. Burley and his research team at New York SGX Research Center for Structural Genomics (J. Michael Sauder, Shawn S. Chang, Kevin Bain, Tumor Progression Assays. The soft agar assay was performed as described Jacqueline Freeman, and Tarun Gheyi) for the construct design, cloning, (64) by using lentivirus infected, and FACS sorted MDA-MB-435 cells that expression, puriﬁcation, and MS analysis of the PEAK1 kinase domain (a.a. – stably express GFP or GFP/PEAK1. An aliquot of these cells were injected s.c. 1289 1746) for our in vitro kinase assays. Finally, we thank Drs. Beverley Emerson and Matthias Kaeser (Salk Institute for Biological Studies) for their in nude mice for tumor formation. A second in vivo experiment consisted of kind gift of the CMV-MCS lentiviral expression vector. This work was sup- establishing orthotopic human pancreatic cancer xenografts in nude mice by ported by the Susan G. Komen Foundation Grant PDF0503999 (to Y.W.), direct injection of XPA-1-GFP shCtrl and XPA-1-GFP shPEAK-1 into their National Institutes of Health Grants GM068487 (to R.L.K.) and CA097022 pancreas (65) (see SI Experimental Procedures for further details). (to R.L.K.), and Cell Migration Consortium Grant GM064346 (to R.L.K.).

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