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• W2110546905 abstract "The Rac1/Cdc42 effector, p21-activated kinase (PAK), is activated by various signaling cascades, including receptor-tyrosine kinases and integrins, and regulates a number of processes such as cell proliferation and motility. PAK activity has been shown to be required for maximal activation of the canonical RAF-MEK-MAPK signaling cascade, possibly because of PAK co-activation of RAF and MEK. Here we have shown that trihydrophobin 1 (TH1), originally identified as a negative regulator of A-RAF kinase, also interacted with PAK1 in cultured cells. Confocal microscopy assay indicated that TH1 colocalized with PAK1 in both the cytoplasm and nucleus, which is consistent with our previous results. GST pulldown and coimmunoprecipitation experiments demonstrated that TH1 interacted directly with PAK1 and bound selectively to the carboxyl-terminal kinase domain of PAK1, and the ability of the binding was enhanced along with activation of PAK1. The binding pattern of PAK1 implies that this interaction was mediated in part by PAK1 kinase activity. As indicated by in vitro kinase activity assays and Western blot detections, TH1 inhibited PAK1 kinase activity and negatively regulated MAPK signal transduction. Interestingly, TH1 bound with MEK1/ERK in cells and in vitro without directly suppressing their kinase activity. Furthermore, we observed that TH1 localized to focal adhesions and filopodia in the leading edge of cells, where TH1 reduced cell migration through affecting actin and adhesion dynamics. Based on these observations, we propose a model in which TH1 interacts with PAK1 and specifically restricts the activation of MAPK modules through the upstream region of the MAPK pathway, thereby influencing cell migration. The Rac1/Cdc42 effector, p21-activated kinase (PAK), is activated by various signaling cascades, including receptor-tyrosine kinases and integrins, and regulates a number of processes such as cell proliferation and motility. PAK activity has been shown to be required for maximal activation of the canonical RAF-MEK-MAPK signaling cascade, possibly because of PAK co-activation of RAF and MEK. Here we have shown that trihydrophobin 1 (TH1), originally identified as a negative regulator of A-RAF kinase, also interacted with PAK1 in cultured cells. Confocal microscopy assay indicated that TH1 colocalized with PAK1 in both the cytoplasm and nucleus, which is consistent with our previous results. GST pulldown and coimmunoprecipitation experiments demonstrated that TH1 interacted directly with PAK1 and bound selectively to the carboxyl-terminal kinase domain of PAK1, and the ability of the binding was enhanced along with activation of PAK1. The binding pattern of PAK1 implies that this interaction was mediated in part by PAK1 kinase activity. As indicated by in vitro kinase activity assays and Western blot detections, TH1 inhibited PAK1 kinase activity and negatively regulated MAPK signal transduction. Interestingly, TH1 bound with MEK1/ERK in cells and in vitro without directly suppressing their kinase activity. Furthermore, we observed that TH1 localized to focal adhesions and filopodia in the leading edge of cells, where TH1 reduced cell migration through affecting actin and adhesion dynamics. Based on these observations, we propose a model in which TH1 interacts with PAK1 and specifically restricts the activation of MAPK modules through the upstream region of the MAPK pathway, thereby influencing cell migration. p21-activated kinases (PAKs), 2The abbreviations used are: PAK, p21-activated kinase; TH1, trihydrophobin 1; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; shRNA, short hairpin RNA; shTH1, short hairpin RNA against human TH1; GST, glutathione S-transferase; HA, hemagglutinin; EGF, epidermal growth factor; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; IP, immunoprecipitation; PBS, phosphate-buffered saline; WT, wild type; RFP, red fluorescent protein; aa, amino acid(s). mammalian homologues of Ste20-like Ser/Thr protein kinases, are the effector for Rac1 and Cdc42 (1Knaus U.G. Bokoch G.M. Int. J. Biochem. Cell Biol. 1998; 30: 857-862Crossref PubMed Scopus (158) Google Scholar, 2Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (869) Google Scholar, 3Kumar R. Vadlamudi R.K. J. Cell. Physiol. 2002; 193: 133-144Crossref PubMed Scopus (97) Google Scholar). Members of the PAK family have been implicated in a variety of intracellular signaling events, including cell cytoskeleton rearrangement, proliferation, differentiation, transformation, apoptosis, cell cycle progression, and cell migration (2Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (869) Google Scholar). Based on their structures, the PAK family can be grouped into two subfamilies: group A (PAK1-3), which can be activated by small GTPases such as Rac-GTP or Cdc42-GTP binding (2Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (869) Google Scholar); and group B (PAK4-6), which can interact with Cdc42-GTP but cannot be activated by this binding (2Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (869) Google Scholar, 4Jaffer Z.M. Chernoff J. Int. J. Biochem. Cell Biol. 2002; 34: 713-717Crossref PubMed Scopus (309) Google Scholar). Outside of the kinase- and GTPase-binding domains, group B PAKs are quite different from group A, and their regulation may be distinct (5Dan C. Nath N. Liberto M. Minden A. Mol. Cell. Biol. 2002; 22: 567-577Crossref PubMed Scopus (133) Google Scholar). From the crystal structure of PAK1 (6Lei M. Lu W. Meng W. Parrini M.C. Eck M.J. Mayer B.J. Harrison S.C. Cell. 2000; 102: 387-397Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar), it appears that the inactive state exists as autoinhibited dimers. Upon GTPase binding, PAK1 undergoes a conformational change that separates the autoinhibitory domain from the kinase domain (7Parrini M.C. Lei M. Harrison S.C. Mayer B.J. Mol. Cell. 2002; 9: 73-83Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). This induces kinase activity and autophosphorylation at several sites, including the Thr-423 site in the activation loop to stabilize the active state (8Chong C. Tan L. Lim L. Manser E. J. Biol. Chem. 2001; 276: 17347-17353Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 9Buchwald G. Hostinova E. Rudolph M.G. Kraemer A. Sickmann A. Meyer H.E. Scheffzek K. Wittinghofer A. Mol. Cell. Biol. 2001; 21: 5179-5189Crossref PubMed Scopus (86) Google Scholar). In resting cells, PAK1 is localized primarily to the membrane structure within the cytoplasm (10Dharmawardhane S. Sanders L.C. Martin S.S. Daniels R.H. Bokoch G.M. J. Cell Biol. 1997; 138: 1265-1278Crossref PubMed Scopus (195) Google Scholar); however, activated PAK1 translocates to focal adhesions and membrane ruffles (11Sells M.A. Pfaff A. Chernoff J. J. Cell Biol. 2000; 151: 1449-1458Crossref PubMed Scopus (133) Google Scholar). Overexpression of constitutively activated PAK1 mutants induces dissolution of actin stress fibers and focal adhesions and increases membrane protrusions, cell polarization, and cell motility (12Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, 13Sells M.A. Boyd J.T. Chernoff J. J. Cell Biol. 1999; 145: 837-849Crossref PubMed Scopus (326) Google Scholar, 14Manser E. Huang H.Y. Loo T.H. Chen X.Q. Dong J.M. Leung T. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar, 15Frost J.A. Khokhlatchev A. Stippec S. White M.A. Cobb M.H. J. Biol. Chem. 1998; 273: 28191-28198Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Conversely, in endothelial cells, overexpression of active PAK1 results in decreased cell migration and stabilization of actin stress fibers and focal adhesions (16Kiosses W.B. Daniels R.H. Otey C. Bokoch G.M. Schwartz M.A. J. Cell Biol. 1999; 147: 831-844Crossref PubMed Scopus (250) Google Scholar). These effects are mediated through the action of PAK1 on cytoskeletal regulatory proteins such as LIM kinase (17Edwards D.C. Sanders L.C. Bokoch G.M. Gill G.N. Nat. Cell Biol. 1999; 1: 253-259Crossref PubMed Scopus (824) Google Scholar), myosin light chain kinase (18Sanders L.C. Matsumura F. Bokoch G.M. de Lanerolle P. Science. 1999; 283: 2083-2085Crossref PubMed Scopus (496) Google Scholar), filamin A (19Vadlamudi R.K. Li F. Adam L. Nguyen D. Ohta Y. Stossel T.P. Kumar R. Nat. Cell Biol. 2002; 4: 681-690Crossref PubMed Scopus (258) Google Scholar), and Op18/stathmin (20Wittmann T. Bokoch G.M. Waterman-Storer C.M. J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (213) Google Scholar). In addition, the activation of the ERK/MAPK is considered to direct the migration of numerous cell types (21Krueger J.S. Keshamouni V.G. Atanaskova N. Reddy K.B. Oncogene. 2001; 20: 4209-4218Crossref PubMed Scopus (146) Google Scholar, 22Jo M. Thomas K.S. Somlyo A.V. Somlyo A.P. Gonias S.L. J. Biol. Chem. 2002; 277: 12479-12485Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 23Huang C. Jacobson K. Schaller M.D. J. Cell Sci. 2004; 117: 4619-4628Crossref PubMed Scopus (872) Google Scholar, 24Webb D.J. Donais K. Whitmore L.A. Thomas S.M. Turner C.E. Parsons J.T. Horwitz A.F. Nat. Cell Biol. 2004; 6: 154-161Crossref PubMed Scopus (1051) Google Scholar). Recent studies indicate that PAK1 activity plays important roles for activation of the MAPK signaling pathways (25Slack-Davis J.K. Eblen S.T. Zecevic M. Boerner S.A. Tarcsafalvi A. Diaz H.B. Marshall M.S. Weber M.J. Parsons J.T. Catling A.D. J. Cell Biol. 2003; 162: 281-291Crossref PubMed Scopus (208) Google Scholar, 26Jin S. Zhuo Y. Guo W. Field J. J. Biol. Chem. 2005; 280: 24698-24705Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 27Sundberg-Smith L.J. Doherty J.T. Mack C.P. Taylor J.M. J. Biol. Chem. 2005; 280: 2055-2064Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). However, the exact mechanism of PAK1 regulation in these pathways is not so clear. The human trihydrophobin 1 (TH1) gene is the homologue of Drosophila TH1, which was originally identified during the positional cloning of mei-41 (28Banga S.S. Yamamoto A.H. Mason J.M. Boyd J.B. Mol. Gen. Genet. 1995; 246: 148-155Crossref PubMed Scopus (16) Google Scholar, 29Hari K.L. Santerre A. Sekelsky J.J. McKim K.S. Boyd J.B. Hawley R.S. Cell. 1995; 82: 815-821Abstract Full Text PDF PubMed Scopus (245) Google Scholar). The TH1 gene lies adjacent to mei-41 and was characterized further by Bonthron et al. (30Bonthron D.T. Hayward B.E. Moran V. Strain L. Hum. Genet. 2000; 107: 165-175Crossref PubMed Scopus (21) Google Scholar) in 2000. According to their studies, TH1 is highly conserved from Drosophila to human, as shown by sequence comparison, and is located in chromosome 20q13, which had a transcript product of 2.4 kb. Northern blots showed that TH1 is widely expressed in multiple tissues. The human TH1 protein has been predicted to have a molecular mass of 65.8 kDa and display high levels of expression in cardiac and skeletal muscle, kidney, adrenal, and thyroid. Although highly conserved and ubiquitously expressed, human TH1 is not well understood in terms of its function. Recently, data from our laboratory indicate that TH1, as a new negative regulator of A-RAF kinase in MAPK signal transduction pathways, can specifically bind to A-RAF and inhibit its kinase activity (31Liu W. Shen X. Yang Y. Yin X. Xie J. Yan J. Jiang J. Liu W. Wang H. Sun M. Zheng Y. Gu J. J. Biol. Chem. 2004; 279: 10167-10175Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). TH1 also interacts with human papilloma virus E6-associated protein (E6AP) and induces ubiquitin-dependent proteolysis (32Yang Y. Liu W. Zou W. Wang H. Zong H. Jiang J. Wang Y. Gu J. J. Cell. Biochem. 2007; 101: 167-180Crossref PubMed Scopus (8) Google Scholar). In addition, it is also found to be identical to NELF-C/D, an integral subunit of the human negative transcription elongation factor (NELF) complex (33Narita T. Yamaguchi Y. Yano K. Sugimoto S. Chanarat S. Wada T. Kim D.K. Hasegawa J. Omori M. Inukai N. Endoh M. Yamada T. Handa H. Mol. Cell. Biol. 2003; 23: 1863-1873Crossref PubMed Scopus (156) Google Scholar). Although our understanding of the function of TH1 has advanced, little is known about the relationship between TH1 and PAK1. We report here that TH1 interacted with PAK1 in cell and in vitro, and the ability of the binding was enhanced along with the activation of PAK1. TH1 bound selectively to the carboxyl-terminal kinase domain of PAK1 and inhibited the ability of PAK1 to phosphorylate substrate, thereby reducing MAPK signal transduction. Finally, our wound healing assay and Boyden chamber assay showed that overexpression of TH1 inhibited ERK/MAPK-driven cell migration. This suggests that TH1 specifically restricted the activation of MAPK modules through the upstream of MAPK pathway, thereby influencing cell migration. Cell Culture-293T, HeLa, NIH3T3, and COS-1 cells were obtained from the Institute of Cell Biology Academic Sinica and cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% CO2. Reagents and Antibodies-Protein G-agarose and anti-GFP antibody were purchased from Roche Applied Science. [γ-32P]ATP (>3000 Ci/mm) and the ECL assay kit were from Amersham Biosciences. Leupeptin, aprotinin, and phenylmethylsulfonyl fluoride were purchased from Sigma. Antibodies against PAK1, ERK1/2, phospho-MEK1 (Ser-298), phospho-ERK (E-4), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and HA peptide were from Santa Cruz Biotechnology. Antibody against phospho-PAK1 (Thr-423) was from Cell Signaling. Antibodies against paxillin were from Abcam. Antibodies against Myc, recombinant human epidermal growth factor (EGF), the vector pcDNA3.0, myelin basic protein, and Lipofectamine™ 2000 reagents were from Invitrogen. TH1 antiserum was raised against GST-TH1 protein purified from Escherichia coli. Other reagents were commercially available in China. Plasmids-Full-length TH1 expression plasmids were constructed as described previously (31Liu W. Shen X. Yang Y. Yin X. Xie J. Yan J. Jiang J. Liu W. Wang H. Sun M. Zheng Y. Gu J. J. Biol. Chem. 2004; 279: 10167-10175Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Myc-PAK1 WT, Myc-PAK1 T423E, and Myc-PAK1 K299R (provided by J. Chernoff) were amplified by PCR and cloned into the SalI/BamHI site of pEGFP-N3 vector or the SacI/BamHI site of pDsRed-Express-C1 vector (Clontech). AU5-cdc42 and Myc-tagged Rac1 (T17N) were gifts from G. Bokoch. All of the generated sequences and plasmids were confirmed by sequencing. Immunoprecipitation and Immunoblot Assays-Twenty-four h after transfection, cells were washed three times with ice-cold PBS and lysed with 0.5 ml of IP lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% Nonidet P-40, 5 mm EDTA, 5 mm EGTA, 15 mm MgCl2, 60 mm β-glycerophosphate, 0.1 mm sodium orthovanadate, 0.1 mm NaF, 0.1 mm benzamide, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml phenylmethylsulfonyl fluoride) for 1.5 h at 4 °C. Detergent-insoluble materials were removed by centrifugation at 12,000 rpm for 10 min at 4 °C. The whole cell lysates were incubated with control rabbit normal IgG (Santa Cruz Biotechnology) or the relevant antibody at 4 °C for 1 h. Pre-equilibrated protein G-agarose beads (Roche Applied Science) were then added, collected by centrifugation after 1 h of incubation, and then gently washed three times with the lysis buffer. The cell lysates containing recombinant protein were incubated with specific antibodies and protein G-agarose beads at 4 °C. The precipitates were washed three times with ice-cold lysis buffer. The bound proteins were eluted by boiling in SDS sample buffer and resolved on a 10% SDS-polyacrylamide gel. The proteins were transferred onto a polyvinylidene difluoride membrane and probed with a 1:1000 dilution of the relevant antibodies. Then the proteins were detected using the ECL assay kit. GST Pulldown Assay-GST-TH1 was purified from bacterial lysates using glutathione-agarose beads (Amersham Biosciences). His-tagged PAK1 protein was purified from E. coli using a nickel-Sepharose high performance column (GE Healthcare). HA-tagged PAK1 and its deletion mutations were expressed in 293T cells for 24 h. Cells were lysed with 0.5 ml of IP lysis buffer for 1.5 h at 4 °C. Detergent-insoluble materials were removed by centrifugation at 12,000 rpm for 10 min at 4 °C. The whole cell lysates were incubated with 10 μl (about 10 μg) of GST-TH1 fusion protein and rotated for 3-5 h at 4 °C. The beads were then washed three times with lysis buffer. The beads were resuspended in SDS-PAGE sample buffer, boiled for 8 min, electrophoresed on a 15% SDS-polyacrylamide gel, and analyzed by Western blot. An in vitro binding assay was also performed as follow. Namely, purified GST-TH1 and His6-PAK1 proteins were incubated in IP buffer at 4 °C, and then GST pulldown was performed. Kinase Activity Assay-To evaluate PAK1 activity, 293T cells were transfected with various gene plasmids or with vector. After 24 h of incubation in DMEM containing 10% FBS, the cells were serum-starved for 8-20 h and then stimulated with or without 20% FBS for 15 min. The cells were washed three times with ice-cold PBS and lysed at 4 °C in IP lysis buffer. Equal total protein was immunoprecipitated with 2 μg of anti-GFP antibody or anti-PAK1 antibody at 4 °C overnight. The beads were washed twice in lysis buffer and twice in kinase buffer (50 mm HEPES, pH 7.5, 10 mm MgCl2, and 2 mm MnCl2). Kinase assays were performed in 30-μl reactions with 25 mm ATP, 10 μCi of [γ-32P]ATP, and 20 μg of myelin basic protein. After incubation at 30 °C for 30 min, the reactions were stopped by the addition of 8 μl of 5 × sample buffer, boiled, separated by 15% SDS-PAGE, and analyzed using autoradiography. Similar assays were used to measure the activity in immunoprecipitates of MEK1 and ERK from 293T cells. Confocal Microscopy-COS-1 or HeLa cells grown to 50% confluence on coverslips were transiently transfected with GFP or GFP-TH1 along with RFP-PAK1 or RFP-TH1. After 24 h of transfection, cells were washed with PBS, fixed in 4% formaldehyde, permeabilized in 0.2% Triton X-100/PBS, and blocked in 1% bovine serum albumin for 1 h at room temperature. The coverslips were stained with anti-paxillin antibody for 2 h at room temperature or overnight at 4 °C followed by incubation with Cy5-conjugated goat anti-mouse secondary antibody for 1 h at room temperature. Cells were washed three times with PBS, stained for F-actin and DNA with fluorescein isothiocyanate-phalloidin (5 μg/ml) and Hoechst 33258 (50 μg/ml) solution, respectively, in a dark chamber. The coverslips were washed as described above, inverted, mounted on slides, and examined in a Zeiss or Leica TCS SP5 confocal microscope. RNA Interference-RNA interference was undertaken using the pSilencer2.0 vector (Ambion Inc.). RNA interference target sequences were selected from the human TH1L sequence (GenBank™ accession number NM_198976). Target oligonucleotides were synthesized (5′-GATCCGCAGAATTGAGCACACTTTATCGAAATAAAGTGTGCTCAATTCTGCTTTTTTGGAAA-3′ and 3′-GCGTCTTAACTCGTGTGAAATAGCTTTATTTCACACGAGTTAAGACGAAAAAACCTTTTCGA-5′), annealed, and cloned into pSilencer vector between the BamHI and HindIII sites. For shRNA expression, HeLa cells were transfected with non-targeting shRNA vector or vector encoding TH1 shRNA, and 24 h later the cells were treated. Wound Healing Assay and Boyden Chamber Assay-An in vitro wound healing assay and Boyden chamber assay were performed as described previously (34Shan D. Chen L. Njardarson J.T. Gaul C. Ma X. Danishefsky S.J. Huang X.Y. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3772-3776Crossref PubMed Scopus (149) Google Scholar, 35Yang S. Huang X.Y. J. Biol. Chem. 2005; 280: 27130-27137Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Briefly, HeLa cells in DMEM containing 10% FBS were seeded into wells of 6-well plates. After the cells grew to confluency, wounds were made with sterile pipette tips. Wells were rinsed three times with DMEM without serum, and media containing 10% FBS and 100 ng/ml EGF were added. After overnight incubation at 37 °C, cells were fixed with 4% formaldehyde, stained with 0.1% crystal violet, and photographed. Cell migration assay was performed using Boyden chambers (tissue culture-treated, 6.5-mm diameter, 10-μm thickness, 8-μm pores; Transwell®, Costar Corp., Cambridge, MA) containing polycarbonate membranes. Serum-starved cells were trypsinized and counted. Then 100 μl of 1 ∼ 3 × 106 cells in serum-free medium was added to the upper chamber, and 600 μl of the appropriate medium with 10% FBS or 100 ng/ml EGF was added to the lower chamber. The Transwell was incubated for 12-18 h at 37 °C. Nonmigratory cells on the upper membrane surface were removed with a cotton swab, and the migratory cells on the undersurface of the membrane were fixed and stained with 0.1% crystal violet for 20 min at room temperature. Photographs of three random regions were taken, and the number of cells was counted to calculate the average number of cells that had transmigrated. TH1 Interacts with PAK1 in Cells-Previous studies on the biological function of TH1 and PAK1 in the RAF-MEK-MAPK pathway suggested a functional linkage between TH1 and PAK1 (25Slack-Davis J.K. Eblen S.T. Zecevic M. Boerner S.A. Tarcsafalvi A. Diaz H.B. Marshall M.S. Weber M.J. Parsons J.T. Catling A.D. J. Cell Biol. 2003; 162: 281-291Crossref PubMed Scopus (208) Google Scholar, 27Sundberg-Smith L.J. Doherty J.T. Mack C.P. Taylor J.M. J. Biol. Chem. 2005; 280: 2055-2064Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 31Liu W. Shen X. Yang Y. Yin X. Xie J. Yan J. Jiang J. Liu W. Wang H. Sun M. Zheng Y. Gu J. J. Biol. Chem. 2004; 279: 10167-10175Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). To determine whether the link was physical, the interaction of TH1 and PAK1 was examined. We expressed GFP-tagged PAK1 and HA-tagged TH1 in 293T cells. Although an anti-GFP antibody coimmunoprecipitated HA-TH1 in GFP-PAK1 and HA-TH1 cotransfected cells, this antibody did not coimmunoprecipitate HA-TH1 in control cells cotransfected with HA-TH1 and GFP vector or with HA vector and GFP-PAK1 (Fig. 1A). Conversely, anti-HA antibody also coimmunoprecipitated GFP-PAK1 in GFP-PAK1 and HA-TH1 cotransfected 293T cells (Fig. 1B). Supporting the importance of this interaction, TH1-PAK1 association also took place between endogenous proteins as well as during protein overexpression. Thus immunoprecipitation of endogenous TH1 from HeLa cells resulted in coimmunoprecipitation of endogenous PAK1 (Fig. 1C). To further confirm the above results, we examined subcellular localization of TH1 and PAK1 using confocal fluorescence microscopy analysis. We selected COS-1 cells to transfect with pEGFP-TH1 or pEGFP-N3 along with pDsRed-PAK1, respectively. At exactly 24 h after transfection, the cells were fixed and analyzed under confocal microscopy. As shown in Fig. 1D, in control cells, GFP was distributed in whole cellular compartments, especially in the nucleus, whereas red fluorescent protein-fused PAK1 (RFP-PAK1) was widely distributed in the cytoplasm, and there was no colocalization between GFP and RFP-PAK1. However, in cells coexpressing GFP-TH1 and RFP-PAK1, we observed that the double-transfected cells contained yellow granules, indicating colocalization of TH1 and PAK1 in both the cytoplasm and nucleus (Fig. 1D, Normal). Taken together, these data indicate that TH1 interacts with PAK1 in cultured cells. TH1 Interacts Directly with PAK1 in Vitro-To ascertain whether TH1 interacts directly with PAK1, recombinant proteins GST-TH1 and His6-tagged PAK1 were purified from E. coli (Fig. 2A), and an in vitro binding assay was conducted in a cell-free system. The purified recombinant protein His6-PAK1 was incubated with GST-TH1 or GST fusion protein. Glutathione-agarose beads were used to pull down GST or GST-TH1 from the formed complex. Western blots with anti-PAK1 antibody revealed that PAK1 interacted only with GST-TH1 but not GST (Fig. 2B). These results demonstrate that TH1 formed a complex with PAK1 in a cell-free system. TH1 Binding Site Is Located in the Carboxyl-terminal Kinase Domain of PAK1-The regulatory domain of PAK1 spans residues 1-248 of its amino terminus, whereas amino acids 248-545 comprise the kinase domain. The kinase domain of PAK1 is structurally composed of two lobes. The small lobe mainly involves binding to ATP, and the large lobe provides a binding site for peptide substrates (Fig. 2C). A phosphotransfer reaction was achieved by coordination between the two lobes of the kinase domain and their interaction with substrates. To determine where the TH1-binding domain resided on PAK1, we initially engineered constructs encoding aa 1-394, the amino-terminal regulatory region (aa 1-270), and the carboxyl-terminal kinase domain (aa 271-545) and expressed them in 293T cells. Then, the whole cell lysates were incubated with E. coli-expressed GST-TH1 or GST fusion protein to perform the in vitro binding assay. As shown in Fig. 2D, a GST pulldown assay showed that TH1 could bind directly with M1 (aa 1-394) and M2 (aa 271-545), whereas no binding with M3 (aa 1-270) was detected. Thus, the carboxyl-terminal 271-545 amino acids of PAK1 were necessary and sufficient for the interaction of TH1. Overexpression of TH1 Inhibits PAK1 Kinase Activity-Because TH1 preferentially associates with the carboxyl-terminal kinase domain of PAK1, we hypothesized that TH1 could influence PAK1 kinase activity. To verify the hypothesis, 293T cells were transiently transfected with GFP-PAK1 WT, GFP-PAK1-K299R (a kinase-dead version of the protein (36Alahari S.K. Reddig P.J. Juliano R.L. EMBO J. 2004; 23: 2777-2788Crossref PubMed Scopus (71) Google Scholar, 37Lei M. Robinson M.A. Harrison S.C. Structure (London). 2005; 13: 769-778Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar)), the active PAK1 mutant, GFP-PAK1-T423E (the threonine to glutamic acid substitution mimicked the phosphorylation of Thr-423 and partially activated the kinase (2Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (869) Google Scholar, 8Chong C. Tan L. Lim L. Manser E. J. Biol. Chem. 2001; 276: 17347-17353Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 37Lei M. Robinson M.A. Harrison S.C. Structure (London). 2005; 13: 769-778Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar)) along with HA-TH1 or vector. At 24 h after transfection, the cells were serum-starved for 16 h. The immunoprecipitated PAK1 proteins were tested for kinase activity using myelin basic protein as a substrate. As shown in Fig. 3A, the kinase activity of the active mutant PAK1-T423E was inhibited by TH1, whereas the kinase activity of PAK1 WT disappeared under serum-starved conditions. These data also suggest that TH1 directly affected PAK1 itself to lead to the inhibition of PAK1 kinase activity instead of the blockade of the signaling pathway leading to PAK1 activation. To further study whether the overexpression of TH1 also inhibited the endogenous PAK1 kinase activity in cells, we transfected 293T cells with the active mutant Rac1 (Q61L) and HA-TH1 or vector. Immunoprecipitated endogenous PAK1 proteins were subjected to in vitro kinase assays. As seen in Fig. 3B, serum stimulation strongly increased PAK1 kinase activity, whereas overexpression of TH1 blocked the increase in PAK1 activity. Because threonine 423 phosphorylation of the PAK1 is critical for the conformational change of the activation loop, assembly of the active configuration of catalytic domain, and activation of PAK1 (2Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (869) Google Scholar, 6Lei M. Lu W. Meng W. Parrini M.C. Eck M.J. Mayer B.J. Harrison S.C. Cell. 2000; 102: 387-397Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 7Parrini M.C. Lei M. Harrison S.C. Mayer B.J. Mol. Cell. 2002; 9: 73-83Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 38Guo D. Tan Y.C. Wang D. Madhusoodanan K.S. Zheng Y. Maack T. Zhang J.J. Huang X.Y. Cell. 2007; 128: 341-355Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), we analyzed the phosphorylation state of PAK1 at site 423 in 293T cells cotransfected with GFP-PAK1 and HA-TH1 or vector. As depicted in Fig. 3C, serum stimulation strongly increased the phosphorylation level of PAK1 at site 423, whereas coexpression of HA-TH1 blocked the increase. In summary, these data strongly suggest that TH1 inhibits the activation of PAK1 in cells. TH1 Binds with Active PAK1 Strongly and Interferes with MAPK Signaling-Because of the binding site of TH1 to the kinase domain of PAK1, we reasoned that their interaction might be related to the active state of the PAK1 kinase. To further explore this issue, HA-TH1 was coexpressed with GFP-PAK1-K299R, GFP-PAK1 WT, or GFP-PAK1-T423E. TH1 immunoprecipitated via its HA tag showed enhanced co-precipitation of PAK1-T423E as compared with the wild-type PAK1, whereas in serum-starved cells, co-precipitation of kinase-dead PAK1 was hardly found (Fig. 4A). Notably, coimmunoprecipitation of the wild-type PAK1 dramatically increased from cells cotransfected with the active mutant Cdc42 (Q61L) and stimulated with EGF as compared with that from serum-starved cells (Fig. 4A). Identical results were also obtained with d" @default.
• W2110546905 created "2016-06-24" @default.
• W2110546905 creator A5001764217 @default.
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• W2110546905 date "2009-03-01" @default.
• W2110546905 modified "2023-09-28" @default.
• W2110546905 title "Trihydrophobin 1 Interacts with PAK1 and Regulates ERK/MAPK Activation and Cell Migration" @default.
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• W2110546905 doi "https://doi.org/10.1074/jbc.m806144200" @default.
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