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• W1996688457 abstract "Differential phosphorylation of transcription factors by signal transduction pathways play an important role in regulation of gene expression and functions. ESE-1 is an epithelium-specific ETS transcription factor that transforms human breast epithelial cells through a serine- and aspartic acid-rich domain (SAR) by an unknown cytoplasmic mechanism. Here we found that a signaling kinase, p21-activated kinase-1 (Pak1), interacts with and phosphorylates ESE-1. Interestingly, Pak1 selectively phosphorylates ESE-1 at Ser207, which is located within the SAR domain. A S207A substitution in ESE-1 reduced its ability to transform breast cancer cells. We also found that ESE-1 is a labile protein and by interacting with F-box-binding protein β-TrCP, undergoes ubiquitin-dependent proteolysis. Intriguingly, Pak1 phosphorylation inactive mutant ESE1-S207A is more unstable than either wild-type ESE-1 or its Pak1 phosphorylation mimetic mutant, i.e. ESE1-S207E. These findings provide novel insights into the mechanism of transformation potential of ESE-1 and discovered that ESE-1 functions are coordinately regulated by Pak1 phosphorylation and β-TrCP-dependent ubiquitin-proteasome pathways. Differential phosphorylation of transcription factors by signal transduction pathways play an important role in regulation of gene expression and functions. ESE-1 is an epithelium-specific ETS transcription factor that transforms human breast epithelial cells through a serine- and aspartic acid-rich domain (SAR) by an unknown cytoplasmic mechanism. Here we found that a signaling kinase, p21-activated kinase-1 (Pak1), interacts with and phosphorylates ESE-1. Interestingly, Pak1 selectively phosphorylates ESE-1 at Ser207, which is located within the SAR domain. A S207A substitution in ESE-1 reduced its ability to transform breast cancer cells. We also found that ESE-1 is a labile protein and by interacting with F-box-binding protein β-TrCP, undergoes ubiquitin-dependent proteolysis. Intriguingly, Pak1 phosphorylation inactive mutant ESE1-S207A is more unstable than either wild-type ESE-1 or its Pak1 phosphorylation mimetic mutant, i.e. ESE1-S207E. These findings provide novel insights into the mechanism of transformation potential of ESE-1 and discovered that ESE-1 functions are coordinately regulated by Pak1 phosphorylation and β-TrCP-dependent ubiquitin-proteasome pathways. The ETS family of transcription factors plays critical roles in the normal and pathologic processes important for growth and development (1Oikawa T. Yamada T. Gene (Amst). 2003; 303: 11-34Crossref PubMed Scopus (518) Google Scholar). In addition to regulating cell proliferation, differentiation, apoptosis, migration, and epithelial mesenchymal transitions during normal development, ETS proteins, when their levels and activity are deregulated, also contribute to the initiation and progression of human cancers (2Sharrocks A.D. Nat. Rev. Mol. Cell. Biol. 2001; 2: 827-837Crossref PubMed Scopus (803) Google Scholar, 3Hsu T. Trojanowska M. Watson D.K. J. Cell. Biochem. 2004; 91: 896-903Crossref PubMed Scopus (221) Google Scholar, 4Dittmer J. Nordheim A. Biochim. Biophys. Acta. 1998; 1377: F1-11PubMed Google Scholar). In general, ETS family transcription factors are defined by a highly conserved 85-amino acid motif called the ETS domain that binds to a core recognition sequence (GGA(A/T)) located on promoters of their target genes (5Graves B.J. Petersen J.M. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar). The epithelium-specific ETS (ESE) family, which is part of the ETS family and includes ESE-1, ESE-2, ESE-3, and PDEF, has been implicated in epithelial cell differentiation (6Feldman R.J. Sementchenko V.I. Watson D.K. Anticancer Res. 2003; 23: 2125-2131PubMed Google Scholar). ESE-1 (also known as ERT, Jen, ESX, or Elf3) was initially identified as a regulator of epithelial cell differentiation because of its ability to regulate expression of epithelial cell markers in keratinocytes (7Cabral A. Fischer D.F. Vermeij W.P. Backendorf C. J. Biol. Chem. 2003; 278: 17792-17799Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 8Oettgen P. Alani R.M. Barcinski M.A. Brown L. Akbarali Y. Boltax J. Kunsch C. Munger K. Libermann T.A. Mol. Cell. Biol. 1997; 17: 4419-4433Crossref PubMed Scopus (197) Google Scholar), bronchial (9Reddy S.P. Vuong H. Adiseshaiah P. J. Biol. Chem. 2003; 278: 21378-21387Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), and retina (10Jobling A.I. Fang Z. Koleski D. Tymms M.J. Investig. Ophthalmol. Vis. Sci. 2002; 43: 3530-3537PubMed Google Scholar). As a transcription factor, ESE-1 regulates a variety of genes, such as HER-2 in breast epithelial cells (11Scott G.K. Chang C.H. Erny K.M. Xu F. Fredericks W.J. Rauscher III, F.J. Thor A.D. Benz C.C. Oncogene. 2000; 19: 6490-6502Crossref PubMed Scopus (67) Google Scholar), MIP3α in colon (12Kwon J.H. Keates S. Simeonidis S. Grall F. Libermann T.A. Keates A.C. J. Biol. Chem. 2003; 278: 875-884Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and transforming growth factor-β type II receptor (13Choi S.G. Yi Y. Kim Y.S. Kato M. Chang J. Chung H.W. Hahm K.B. Yang H.K. Rhee H.H. Bang Y.J. Kim S.J. J. Biol. Chem. 1998; 273: 110-117Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). ESE-1 also regulates genes involved in inflammation, such as angiopoietin-1 and cyclooxygenase-2 (14Brown C. Gaspar J. Pettit A. Lee R. Gu X. Wang H. Manning C. Voland C. Goldring S.R. Goldring M.B. Libermann T.A. Gravallese E.M. Oettgen P. J. Biol. Chem. 2004; 279: 12794-12803Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 15Grall F.T. Prall W.C. Wei W. Gu X. Cho J.Y. Choy B.K. Zerbini L.F. Inan M.S. Goldring S.R. Gravallese E.M. Goldring M.B. Oettgen P. Libermann T.A. FEBS J. 2005; 272: 1676-1687Crossref PubMed Scopus (51) Google Scholar). Interestingly, the HER-2-mediated pathway regulates ESE-1 expression, and ESE-1 regulates HER-2 expression by binding at the HER-2 promoter (11Scott G.K. Chang C.H. Erny K.M. Xu F. Fredericks W.J. Rauscher III, F.J. Thor A.D. Benz C.C. Oncogene. 2000; 19: 6490-6502Crossref PubMed Scopus (67) Google Scholar, 16Neve R.M. Ylstra B. Chang C.H. Albertson D.G. Benz C.C. Oncogene. 2002; 21: 3934-3938Crossref PubMed Google Scholar). Accumulating evidence suggests that overexpression of several ETS genes, including PEA3, ESE-1, ETS-1, and platelet-derived growth factor, is associated with breast cancer (17Kurpios N.A. Sabolic N.A. Shepherd T.G. Fidalgo G.M. Hassell J.A. J. Mammary Gland Biol. Neoplasia. 2003; 8: 177-190Crossref PubMed Scopus (55) Google Scholar). Recent reports suggest that ESE-1 transforms human breast epithelial MCF12A cells through the serine- and aspartic acid-rich (SAR) 2The abbreviations used are: SAR, serine- and aspartic acid-rich; siRNA, small interfering RNA; DLC1, dynein light chain 1; MAPK, mitogen-activated protein kinase; GST, glutathione S-transferase; RT, reverse transcriptase; CHX, cycloheximide; Pak1, p21-activated kinase 1; SCF, Skp1/Cul1/F-box protein. 2The abbreviations used are: SAR, serine- and aspartic acid-rich; siRNA, small interfering RNA; DLC1, dynein light chain 1; MAPK, mitogen-activated protein kinase; GST, glutathione S-transferase; RT, reverse transcriptase; CHX, cycloheximide; Pak1, p21-activated kinase 1; SCF, Skp1/Cul1/F-box protein. domain by an unknown cytoplasmic mechanism (18Prescott J.D. Koto K.S. Singh M. Gutierrez-Hartmann A. Mol. Cell. Biol. 2004; 24: 5548-5564Crossref PubMed Scopus (50) Google Scholar). Furthermore, overexpression of ESE-1 in MCF12A cells confers a motile phenotype to these cells similar to Rho, Rac and Cdc42 GTPases displayed motile phototypes (19Schedin P.J. Eckel-Mahan K.L. McDaniel S.M. Prescott J.D. Brodsky K.S. Tentler J.J. Gutierrez-Hartmann A. Oncogene. 2004; 23: 1766-1779Crossref PubMed Scopus (54) Google Scholar). In general, unstable eukaryotic proteins are encoded by short destruction sequences called “degrons,” including the PEST domain, DSGXXG motif, which can be recognized and targeted for ubiquitin proteolysis (20Muratani M. Tansey W.P. Nat. Rev. Mol. Cell. Biol. 2003; 4: 192-201Crossref PubMed Scopus (664) Google Scholar, 21Rechsteiner M. Rogers S.W. Trends Biochem. Sci. 1996; 21: 267-271Abstract Full Text PDF PubMed Scopus (1390) Google Scholar). The Skp1/Cul1/F-box protein (SCF) complex E3-ubiquitin ligase targets many proteins for proteolysis in diverse cellular context (22Cardozo T. Pagano M. Nat. Rev. Mol. Cell. Biol. 2004; 5: 739-751Crossref PubMed Scopus (860) Google Scholar). β-TrCP, a WD40 repeat-containing F-box protein of SCFβ-TrCP, recognizes the doubly phosphorylated DSG motif (DpSGφXpS, where φ represents a hydrophobic amino acid and X represents any amino acid) in various SCFβ-TrCP target proteins (23Fuchs S.Y. Spiegelman V.S. Kumar K.G. Oncogene. 2004; 23: 2028-2036Crossref PubMed Scopus (256) Google Scholar). The phosphoserine residues within the DSG motif are essential for target proteins to interact with β-TrCP (22Cardozo T. Pagano M. Nat. Rev. Mol. Cell. Biol. 2004; 5: 739-751Crossref PubMed Scopus (860) Google Scholar). Although ETS transcription factors are unstable proteins with short half-lives, the molecular mechanisms underlying the degradation of ETS proteins and the influence of phosphorylation on this process remain elusive (24Li R. Pei H. Watson D.K. Oncogene. 2000; 19: 6514-6523Crossref PubMed Scopus (185) Google Scholar). In recent years, it has become increasingly evident that ETS transcription factors are targets of signaling pathways (25Wasylyk B. Hagman J. Gutierrez-Hartmann A. Trends Biochem. Sci. 1998; 23: 213-216Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 26Yordy J.S. Muise-Helmericks R.C. Oncogene. 2000; 19: 6503-6513Crossref PubMed Scopus (266) Google Scholar). In particular, the MAPK pathway has been intimately linked with diverse regulatory events involving ETS-domain proteins (25Wasylyk B. Hagman J. Gutierrez-Hartmann A. Trends Biochem. Sci. 1998; 23: 213-216Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). Phosphorylation of ETS proteins at particular serine or threonine residues has been shown to affect their transactivation potential, DNA binding activity, interaction with coregulatory partners, or subcellular localization (2Sharrocks A.D. Nat. Rev. Mol. Cell. Biol. 2001; 2: 827-837Crossref PubMed Scopus (803) Google Scholar). In brief, signaling pathways may regulate the activity and functions of ETS proteins in an important manner. p21-activated kinase 1 (Pak1), an evolutionary conserved family of serine/threonine kinases, was initially identified as an effector of Rac1 and Cdc42, and as being involved in formation of lamellipodia and membrane ruffles and required for cell motility (27Bagrodia S. Cerione R.A. Trends Cell Biol. 1999; 9: 350-355Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 28Kumar R. Vadlamudi R.K. J. Cell. Physiol. 2002; 193: 133-144Crossref PubMed Scopus (97) Google Scholar, 29Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (869) Google Scholar, 30Kumar R. Gururaj A.E. Barnes C.J. Nat. Rev. Cancer. 2006; 6: 459-471Crossref PubMed Scopus (485) Google Scholar). It has been shown that expression of a catalytically active Pak1 mutant stimulates anchorage-independent growth of breast cancer cells (31Vadlamudi R.K. Adam L. Wang R.A. Mandal M. Nguyen D. Sahin A. Chernoff J. Hung M.C. Kumar R. J. Biol. Chem. 2000; 275: 36238-36244Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Recent findings suggest that Pak1 promotes the process of tumorigenesis by phosphorylating DLC1 (30Kumar R. Gururaj A.E. Barnes C.J. Nat. Rev. Cancer. 2006; 6: 459-471Crossref PubMed Scopus (485) Google Scholar, 32Vadlamudi R.K. Bagheri-Yarmand R. Yang Z. Balasenthil S. Nguyen D. Sahin A.A. den Hollander P. Kumar R. Cancer Cell. 2004; 5: 575-585Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) and epithelial to mesenchymal transition by phosphorylating Snail (33Yang Z.B. Rayala S. Nguyen D. Vadlamudi R.K. Chen S. Kumar R. Cancer Res. 2005; 65: 3179-3184Crossref PubMed Scopus (215) Google Scholar). Furthermore, Pak1 hyperactivation is sufficient for tumor formation in the murine mammary gland (34Wang R.A. Zhang H. Balasenthil S. Medina D. Kumar R. Oncogene. 2005; 11: 2931-2936Google Scholar). Because ESE-1 and Pak1 exhibit overlapping functions such as cell motility, epithelial mesenchymal transitions, and tumorigenesis, and because the mechanism of ESE-1-mediated transformation remains unknown, here we investigated the possibilities that Pak1 is an upstream kinase of ESE-1 and that Pak1 phosphorylation of ESE-1 regulates the stability and transforming ability of ESE-1. Plasmid Construction—The following plasmids were constructed for expressing glutathione S-transferase (GST) fusion proteins in Escherichia coli DE3. pGST-ESE-1 (encoding GST-ESE-1; ESE-1 amino acids 1-371), pED1 (encoding GST-ESE-1-(1-159); ESE-1 amino acids 1-159), pED2 (encoding GST-ESE-1-(128-159); ESE-1 amino acids 128-159)), pED3 (encoding GST-ESE-1-(274-371); ESE-1 amino acids 274-371), pED4 (encoding GST-ESE-1-(159-371); ESE-1 amino acids 159-371), and pED5 (encoding GST-ESE-1-(1-259); ESE-1 amino acids 1-259) were constructed by inserting a EcoRI-NotI-cleaved PCR-derived fragment, using pCGN2-ESX as a template, to the same sites of pGEX5X1 (Amersham Biosciences). pGST-ESE1-S207A (encoding serine 207 to the alanine mutant of GST-ESE-1) and pGST-ESE1-S254A (encoding serine 254 to the alanine mutant of GST-ESE-1) with site-directed mutations at the indicated amino acids were mutated by site-directed mutagenesis using a QuikChange mutagenesis kit (Stratagene). The following plasmids were constructed for use in mammalian cell transfections. T7-tagged ESE-1 (encoding T7-ESE-1; ESE-1 amino acids 1-371) was constructed by inserting a EcoRI-NotI-cleaved PCR-derived fragment into the same sites of pcDNA3.1C (Invitrogen). pcDNA-ESE1-S207A (encoding the serine 207 to alanine mutant of T7-ESE-1) and pcDNA-ESE1-S207E (encoding serine 207 to the aspartic acid mutant of T7-ESE-1) were derived from pcDNA-ESE-1, with site-directed mutations at the indicated amino acids by site-directed mutagenesis using a QuikChange mutagenesis kit (Stratagene). Cell Culture and Reagents—Human breast cancer cell lines MCF-7 and ZR75 were obtained from the American Type Culture Collection (Manassas, VA) and were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium and F-12 medium supplemented with 10% fetal calf serum. MCF12A human breast epithelial cells (American Type Culture Collection) were maintained in Ham's F-12/Dulbecco's modified Eagle's medium containing 100 ng/ml cholera toxin (Invitrogen), 0.5 μg/ml hydrocortisone (Sigma), 10 μg/ml bovine insulin (Sigma), 20 ng/ml epidermal growth factor (Sigma), and 5% horse serum (Invitrogen). Polyclonal ESE-1 antibody was obtained from Abcam, MA. Mouse antibody against T7 was purchased from Novagen (Madison, WI), and rabbit antibody against T7 was purchased from Bethyl Lab (Montgomery, TX). Vinculin antibody was purchased from Sigma. Antibody against Pak1 was purchased from Cell Signaling Technology (Beverly, MA). In Vitro Pak1 Kinase Assay—Using GST-ESE-1 fusion protein as a substrate and bacterially purified GST-Pak1 as an enzyme, we performed in vitro kinase assays in HEPES buffer (50 mm HEPES, 10 mm MgCl2, 2 mm MnCl2, 1 mm dithiothreitol) containing 100 ng of purified GST-Pak1 enzyme, 10 μCi of [γ-32P]ATP, and 25 μm cold ATP. Dynein light chain 1 (DLC1) was used as positive control and GST as negative control. GST proteins were purified using glutathione-Sepharose (Amersham Biosciences) according to the manufacturer's instructions. The reaction was carried out in a volume of 30 μl for 30 min at 30 °C and then stopped by the addition of 10 μl of 4× SDS buffer. We resolved the reaction products by SDS-PAGE and analyzed the results by autoradiography. Stable Cell Lines—Nontransformed but immortalized MCF12A human mammary epithelial cells were transfected by using the FuGENE reagent (Roche Molecular Biochemicals) with 5 μg of pcDNA3.1C, pcDNA-ESE-1, pcDNA-ESE1-S207A, or pcDNA-ESE1-S207E in 60-mm dishes. Twenty-four hours after transfection, the medium was replaced with fresh Ham's medium as well as 500 μg/ml of G418 to select for geneticin-resistant cells. After 12 days of selection, pools of G418-resistant cells, denoted as pcDNA3.1C, WT-ESE-1 (wild-type), ESE1-S207A, and ESE1-S207E, were replated, expanded, and maintained in complete medium containing 200 μg/ml G418. For experiments, log phase cells between passages 4 and 10 were utilized. Cell Extracts, Immunoblotting, and Immunoprecipitation—Cells were grown in Ham's F-12/Dulbecco's modified Eagle's medium containing all components already described. To prepare cell extracts, cells were washed three times with phosphate-buffered saline and then subjected to lysis in Nonidet P-40 lysis buffer (50 mm Tris-HCl, pH 8.0, 137 mm NaCl, 10% glycerol, 1% Nonidet P-40, 50 mm NaF, 1× protease inhibitor mixture (Roche Biochemicals), 1 mm sodium vanadate) for 15 min on ice. The lysates were subjected to centrifugation in an Eppendorf centrifuge at 4 °C for 15 min. Cell lysates containing equal amounts of protein (∼100 μg) were resolved by SDS-PAGE (10-12% acrylamide), transferred to nitrocellulose membranes, probed with the appropriate antibodies, and developed using the enhanced chemiluminescence method. Immunoprecipitation was performed for 4 h at 4 °C using 1 μg of antibody/mg of protein as previously reported (35Manavathi B. Nair S.S. Wang R.A. Kumar R. Vadlamudi R.K. Cancer Res. 2005; 65: 5571-5577Crossref PubMed Scopus (51) Google Scholar). Glutathione S-Transferase Pull-down Assay—In vitro transcription and translation of Pak1 was done using a T7 TnT kit (Promega), where 1 μg of cDNA in pcDNA3.1 vector was translated in the presence of [35S]methionine in a reaction volume of 50 μl. The reaction mixture was diluted to 1 ml with GST binding buffer (25 mm Tris-HCl, pH 8.0, 50 mm NaCl, 10% glycerol, 0.1% Nonidet P-40). An equal aliquot was used for each GST pull-down assay. Translation and product size were verified by subjecting 2 μl of the reaction mixture to SDS-PAGE and autoradiography. The GST pull-down assays were done by incubating equal amounts of GST, GST-tagged full-length proteins, and GST-tagged deletion constructs immobilized on glutathione-Sepharose beads with in vitro translated 35S-labeled protein to which the binding was being tested. Bound proteins were isolated by incubating the mixture for 2 h at 4 °C in GST binding buffer, washing five times with GST binding buffer, eluting the proteins with 2× SDS buffer, and separating them by SDS-PAGE. The bound proteins were then visualized by autoradiography. siRNA Transfection and RT-PCR—Pak1-specific small interfering RNA (siRNA; Cell Signaling Technology), ESE-1 siRNA, and control nonspecific siRNA (Dharmacon, Lafayette, CO) were purchased. siRNA transfections were performed using 100 nm of a pool of four individual RNA interference against Pak1, ESE-1, or control nonspecific siRNA and 4 μl of Oligofectamine (Invitrogen) according to the manufacturer's protocol in 6-well plates. Forty-eight hours were allowed to elapse after transfection to allow efficient silencing of the gene. Reverse transcription-PCR (RT-PCR) for ESE-1 was done using the Access RT-PCR kit (Promega) using specific primers: FP, 5′-AAAAGAACAGCAACATGACCTACGA-3′; RP, 5′-GTTCCAACCCTCAGTTCCGAC-3′; and T7-FP, 5′-CAGCAAATGGGTCGGGATC-3′. Soft Agar Assays—Soft agar colony growth assays were done as previously described (36Vadlamudi R.K. Manavathi B. Balasenthil S. Nair S.S. Yang Z.B. Sahin A.A. Kumar R. Cancer Res. 2005; 65: 7724-7732Crossref PubMed Scopus (89) Google Scholar). Briefly, 1 ml of 0.6% Difco agar in Dulbecco's modified Eagle's medium was layered onto tissue culture plates. Test cells (1 × 104) mixed with 1 ml of 0.36% bactoagar solution in Dulbecco's modified Eagle's medium were layered on top of the 0.6% bactoagar layer and plates were incubated at 37 °C in 5% CO2 for 21 days and colonies were counted. Immunofluorescence and Confocal Microscopy Studies—We determined the cellular localization of proteins by indirect immunofluorescence using an Olympus FV300 laser scanning confocal microscope as described previously (36Vadlamudi R.K. Manavathi B. Balasenthil S. Nair S.S. Yang Z.B. Sahin A.A. Kumar R. Cancer Res. 2005; 65: 7724-7732Crossref PubMed Scopus (89) Google Scholar). Briefly, cells grown on glass coverslips were fixed in 4% paraformaldehyde at 30 °C for 4 min. The cells were incubated with primary antibodies for 1 h, washed three times in phosphate-buffered saline, and then incubated with secondary antibodies conjugated with Alexa 546 (red), Alexa 633 (blue), or Alexa 488 (green) dye from Molecular Probes (Eugene, OR). The DNA dye Topro-3 (Molecular Probes) was used to co-stain the DNA (blue). Cells treated only with the secondary antibody served as controls. Pak1 Phosphorylates ESE-1 Both in Vitro and in Vivo—To explore the possibility that Pak1 phosphorylates ESE-1, we first performed an in vitro Pak1 kinase assay by incubating recombinant GST-ESE-1 and purified Pak1 enzyme. The DLC1 protein, an established Pak1 substrate (32Vadlamudi R.K. Bagheri-Yarmand R. Yang Z. Balasenthil S. Nguyen D. Sahin A.A. den Hollander P. Kumar R. Cancer Cell. 2004; 5: 575-585Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), and GST were used as positive and negative controls, respectively. The results showed that the Pak1 enzyme could phosphorylate GST-ESE-1 but not GST alone (Fig. 1A). Furthermore, we also observed a strong dose-dependent enhancement of ESE-1 phosphorylation by Pak1 (Fig. 1B), suggesting that Pak1 effectively phosphorylates ESE-1 in vitro. Next we examined the status of ESE-1 phosphorylation in vivo. To that end, MCF7 cells were metabolically labeled with [32P]orthophosphoric acid, and the status of ESE-1 phosphorylation was determined after treatment with the physiological signal that activates Pak1 (29Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (869) Google Scholar), such as serum. Signal (serum) that activates Pak1 increased the phosphorylation of transfected epitope-tagged ESE-1 (Fig. 1C). To further confirm the direct involvement of Pak1 in ESE-1 phosphorylation in vivo, we cotransfected either control siRNA or Pak1 siRNA, along with T7-ESE-1, into MCF7 cells, and then cells were labeled metabolically with [32P]orthophosphoric acid. Under modest knockdown conditions of Pak1 (Fig. 1D, lower panel), phosphorylation of ESE-1 was reduced significantly (Fig. 1D). Together, these results suggest that Pak1 efficiently phosphorylates ESE-1 both in vitro and in vivo. Pak1 Phosphorylates ESE-1 at Serine 207 in the SAR Domain—After confirming that Pak1 phosphorylates ESE-1 both in vitro and in vivo, we next mapped the potential Pak1 phosphorylation sites in ESE-1. ESE-1 protein contains several functional domains, including the pointed, transactivation, SAR, AT hook, and ETS domains (Fig. 2A). It is known that Pak1 is a serine/threonine kinase that phosphorylates a number of substrates on serine/threonine residues, preferring preceding basic residues such as (K/R)(R/X)X(S/T) (28Kumar R. Vadlamudi R.K. J. Cell. Physiol. 2002; 193: 133-144Crossref PubMed Scopus (97) Google Scholar, 29Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (869) Google Scholar). Analysis of the ESE-1 amino acid sequence identified several potential putative Pak1 phosphorylation sites (Fig. 2A). To map the functional Pak1 phosphorylation site in ESE-1, we next generated a series of GST-fused ESE-1 deletions and assayed each one for its phosphorylation by the Pak1 enzyme. Results indicated that the Pak1 phosphorylation site on ESE-1 is located in the region between amino acids 159 and 259 (Fig. 2A). Examination of this region of ESE-1 revealed the presence of two potential Pak1 phosphorylation sites, i.e. serine 207 and serine 254. Next, single point mutation of serine 254 to alanine did not affect the phosphorylation of ESE-1 by Pak1, whereas point mutation of ESE-1 at serine 207 to alanine completely abolished Pak1 phosphorylation of ESE-1 as demonstrated by in vitro Pak1 kinase assay, isolating serine 207 as the only Pak1 phosphorylation site in ESE-1 (Fig. 2B). To further confirm the in vitro kinase assay results in vivo, we subcloned the Pak1 phosphorylation mutant of ESE-1, such as GST-ESE1-S207A, into mammalian expression vector pcDNA3.1C, resulting in pcDNA-ESE1-S207A, which expresses T7-tagged ESE1-S207A in mammalian cells. MCF7 cells were transfected with either wild-type T7-ESE-1 or T7-ESE1-S207A, or control pcDNA vector and then were metabolically labeled with [32P]orthophosphoric acid. Immunoprecipitation of the cell lysates with a T7 monoclonal antibody indicated a substantial reduction of phosphorylation of ESE1-S207A as compared with intense phosphorylation of the wild-type T7-ESE-1, and thus strengthening serine 207 as the Pak1 phosphorylation site in ESE-1 (Fig. 2C). To further confirm that phosphorylation at Ser207 on ESE-1 is Pak1 dependent or not, we have silenced Pak1 using Pak1 siRNA in either wild-type ESE-1 or ESE1-S207A expressing MCF7 cells. Under effective knockdown of Pak1, substantial reduction in the [32P] signal of wild-type ESE-1 but not ESE1-S207A indicates that phosphorylation at serine 207 is indeed Pak1 dependent (Fig. 2D). Interestingly, we found that ESE-1 serine 207 is well conserved in human, mouse, and rat (Fig. 2E). All together these results suggest that Pak1 phosphorylate ESE-1 at serine 207 both in vivo and in vitro. Pak1 Interacts with ESE-1 in Breast Cancer Cells—Although ESE-1 is a transcriptional factor, it localizes predominantly in the cytoplasm in breast cancer cells (18Prescott J.D. Koto K.S. Singh M. Gutierrez-Hartmann A. Mol. Cell. Biol. 2004; 24: 5548-5564Crossref PubMed Scopus (50) Google Scholar) and because Pak1 is also localized predominantly in the cytoplasm (28Kumar R. Vadlamudi R.K. J. Cell. Physiol. 2002; 193: 133-144Crossref PubMed Scopus (97) Google Scholar), we next explored the possibility of interaction between ESE-1 and Pak1 in breast cancer cells. Results from the GST pull-down assay showed that in vitro translated ESE-1 efficiently interacted with GST-Pak1 (Fig. 3A). Conversely, in vitro translated Pak1 protein also interacted with GST-ESE-1 (Fig. 3B). To further confirm this interaction in a physiologic setting, lysates of ZR75 cells grown in 10% serum were subjected to immunoprecipitation using a Pak1 antibody. Results showed that ESE-1 is coprecipitated with Pak1 but not with control IgG (Fig. 3C). Because the available ESE-1 antibodies and the one used here are not good for immunoprecipitation, we were unable to conduct a reverse immunoprecipitation using an ESE-1 antibody. To further strengthen this observation, we carried out confocal microscopy studies using ZR75 breast cancer cells by double staining for transiently transfected myc-Pak1 and endogenous ESE-1. As shown in Fig. 3D, transfected myc-Pak1 colocalizes with endogenous ESE-1, which is predominantly localized in the cytoplasm. Next, we defined the minimal region of Pak1 required for its interaction with ESE-1. Pak1 has several important domains that include N-terminal domain (amino acids 1-132), Cdc42/Rac1 interactive binding domain (amino acids 52-132), PIX binding domain (amino acids 132-270), and C-terminal kinase domain (amino acids 270-545) (28Kumar R. Vadlamudi R.K. J. Cell. Physiol. 2002; 193: 133-144Crossref PubMed Scopus (97) Google Scholar). These C- and N-terminal Pak1 deletion constructs were used and expressed as GST fusion proteins; they were then subjected to GST pull-down assays with the 35S-labeled ESE-1. The results suggested that ESE-1 binds to the region comprising amino acids 1-270, which include the N-terminal domain and the PIX binding domain, but did not binds to the C-terminal kinase domain (amino acids 270-545) of Pak1 (Fig. 3E). Conversely, to define the binding region(s) of ESE-1 that are important for Pak1 interaction, we used a series of GST-fused ESE-1 deletion constructs. Results of the GST pull-down assays indicated that ESE-1 uses amino acids 159-371, which contain the SAR, AT hook, and ETS domains, to interact with Pak1 (Fig. 3F). Taken together, these results suggest that ESE-1 interacts with Pak1 both in vitro and in vivo. Pak1 Phosphorylation Promotes ESE-1 Transforming Ability—Because overexpression of either ESE-1 or the SAR domain of ESE-1 alone in MCF12A mammary epithelial cells has been shown to be transforming in nature (18Prescott J.D. Koto K.S. Singh M. Gutierrez-Hartmann A. Mol. Cell. Biol. 2004; 24: 5548-5564Crossref PubMed Scopus (50) Google Scholar) and the fact that Pak1 phosphorylates ESE-1 at serine 207, which is located in the SAR domain, raised the possibility that phosphorylation of ESE-1 by Pak1 may be required for its transformation ability. To address this question, we generated stable MCF12A cells expressing either wild-type T7-ESE-1 or its Pak1 phosphorylation mutants. Then the expression of T7-tagged wild-type ESE-1 and its phosphorylation mutants such as T7-ESE1-S207A and T7-ESE1-S207E mutants was determined by Western analysis (Fig. 4A) and also by RT-PCR analysis (Fig. 4B). Next we assayed the transformation ability of each of these stable clones by an anchorage independent-based colony formation assay under control siRNA or Pak1 siRNA knockdown conditions. Typical colonies were photographed after 21 days and the numbers of colonies counted (Fig. 4C). In agreement with previous reports (18Prescott J.D. Koto K.S. Singh M. Gutierrez-Hartmann A. Mo" @default.
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