Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Regulation of Platelet-derived Growth Factor Receptor Activation by Afadin through SHP-2

2007; Elsevier BV; Volume: 282; Issue: 52 Linguagem: Inglês

10.1074/jbc.m707461200

ISSN

1083-351X

Autores

Shinsuke Nakata, Naoyuki Fujita, Y. Kitagawa, Ryoko Okamoto, Hisakazu Ogita, Yoshimi Takai,

Tópico(s)

Polysaccharides and Plant Cell Walls

Resumo

Upon binding of platelet-derived growth factor (PDGF), PDGF receptor is autophosphorylated at tyrosine residues in its cytoplasmic region, which induces the activation of diverse intracellular signaling pathways such those involving Ras-ERK, c-Src, and Rap1-Rac. Signaling through activated Ras-ERK promotes cell cycle and cell proliferation. The sequential activation of Rap1 and Rac affects cellular morphology and induces the formation of leading-edge structures, including lamellipodia, peripheral ruffles, and focal complexes, resulting in the enhancement of cell movement. In addition to the promotion of cell proliferation, the Ras-ERK signaling is involved in the regulation of cellular morphology. Here, we showed a novel role of afadin in the regulation of PDGF-induced intracellular signaling and cellular morphology in NIH3T3 cells. Afadin was originally identified as an actin filament-binding protein, which binds to a cell-cell adhesion molecule nectin and is involved in the formation of cell-cell junctions. When afadin was tyrosine-phosphorylated by c-Src activated in response to PDGF, afadin physically interacted with and increased the phosphatase activity of Src homology 2 domain-containing phosphatase-2 (SHP-2), a protein-tyrosine phosphatase that dephosphorylates PDGF receptor, leading to the prevention of hyperactivation of PDGF receptor and the Ras-ERK signaling. In contrast, knockdown of afadin or SHP-2 induced the hyperactivation of PDGF receptor and Ras-ERK signaling and consequently suppressed the formation of leading-edge structures. Thus, afadin plays a critical role in the proper regulation of the PDGF-induced activation of PDGF receptor and signaling by Ras-ERK. This effect, which is mediated by SHP-2, impacts cellular morphology. Upon binding of platelet-derived growth factor (PDGF), PDGF receptor is autophosphorylated at tyrosine residues in its cytoplasmic region, which induces the activation of diverse intracellular signaling pathways such those involving Ras-ERK, c-Src, and Rap1-Rac. Signaling through activated Ras-ERK promotes cell cycle and cell proliferation. The sequential activation of Rap1 and Rac affects cellular morphology and induces the formation of leading-edge structures, including lamellipodia, peripheral ruffles, and focal complexes, resulting in the enhancement of cell movement. In addition to the promotion of cell proliferation, the Ras-ERK signaling is involved in the regulation of cellular morphology. Here, we showed a novel role of afadin in the regulation of PDGF-induced intracellular signaling and cellular morphology in NIH3T3 cells. Afadin was originally identified as an actin filament-binding protein, which binds to a cell-cell adhesion molecule nectin and is involved in the formation of cell-cell junctions. When afadin was tyrosine-phosphorylated by c-Src activated in response to PDGF, afadin physically interacted with and increased the phosphatase activity of Src homology 2 domain-containing phosphatase-2 (SHP-2), a protein-tyrosine phosphatase that dephosphorylates PDGF receptor, leading to the prevention of hyperactivation of PDGF receptor and the Ras-ERK signaling. In contrast, knockdown of afadin or SHP-2 induced the hyperactivation of PDGF receptor and Ras-ERK signaling and consequently suppressed the formation of leading-edge structures. Thus, afadin plays a critical role in the proper regulation of the PDGF-induced activation of PDGF receptor and signaling by Ras-ERK. This effect, which is mediated by SHP-2, impacts cellular morphology. Afadin is a nectin- and actin filament (F-actin) 2The abbreviations used are: F-actinactin filamentAJadherens junctionPDGFplatelet-derived growth factorERKextracellular signal-regulated kinasePTPprotein-tyrosine phosphataseSH2Src homology 2SHP-2SH2 domain-containing phosphatase-2DMEMDulbecco's modified Eagle's mediumAbantibodypAbpolyclonal AbmAbmonoclonal AbBSAbovine serum albuminGSTglutathione S-transferaseMBPmaltose-binding proteinBrdUrdbromodeoxyuridineMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseCAconstitutively activeGFPgreen fluorescent proteinWTwild-typesiRNAsmall interfering RNALMWlow molecular weight 2The abbreviations used are: F-actinactin filamentAJadherens junctionPDGFplatelet-derived growth factorERKextracellular signal-regulated kinasePTPprotein-tyrosine phosphataseSH2Src homology 2SHP-2SH2 domain-containing phosphatase-2DMEMDulbecco's modified Eagle's mediumAbantibodypAbpolyclonal AbmAbmonoclonal AbBSAbovine serum albuminGSTglutathione S-transferaseMBPmaltose-binding proteinBrdUrdbromodeoxyuridineMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseCAconstitutively activeGFPgreen fluorescent proteinWTwild-typesiRNAsmall interfering RNALMWlow molecular weight-binding protein that connects nectin to the actin cytoskeleton (1Mandai K. Nakanishi H. Satoh A. Obaishi H. Wada M. Nishioka H. Itoh M. Mizoguchi A. Aoki T. Fujimoto T. Matsuda Y. Tsukita S. Takai Y. J. Cell Biol. 1997; 139: 517-528Crossref PubMed Scopus (391) Google Scholar). Nectin is aCa2+-independent immunoglobulin-like molecule that first forms cell-cell adhesion and then assembles cadherin at the nectin-based cell-cell adhesion sites, resulting in the formation of adherens junctions (AJs) in epithelial cells and fibroblasts (2Takai Y. Irie K. Shimizu K. Sakisaka T. Ikeda W. Cancer Sci. 2003; 94: 655-667Crossref PubMed Scopus (277) Google Scholar, 3Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (468) Google Scholar). The nectin family of proteins comprises four members, nectin-1, -2, -3, and -4. A series of our studies revealed the roles and modes of action of nectin and afadin for the formation of AJs (2Takai Y. Irie K. Shimizu K. Sakisaka T. Ikeda W. Cancer Sci. 2003; 94: 655-667Crossref PubMed Scopus (277) Google Scholar, 3Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (468) Google Scholar, 4Fukuhara T. Shimizu K. Kawakatsu T. Fukuyama T. Minami Y. Honda T. Hoshino T. Yamada T. Ogita H. Okada M. Takai Y. J. Cell Biol. 2004; 166: 393-405Crossref PubMed Scopus (91) Google Scholar, 5Fukuyama T. Ogita H. Kawakatsu T. Fukuhara T. Yamada T. Sato T. Shimizu K. Nakamura T. Matsuda M. Takai Y. J. Biol. Chem. 2005; 280: 815-825Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 6Hoshino T. Sakisaka T. Baba T. Yamada T. Kimura T. Takai Y. J. Biol. Chem. 2005; 280: 24095-24103Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 7Izumi G. Sakisaka T. Baba T. Tanaka S. Morimoto K. Takai Y. J. Cell Biol. 2004; 166: 237-248Crossref PubMed Scopus (151) Google Scholar, 8Kawakatsu T. Ogita H. Fukuhara T. Fukuyama T. Minami Y. Shimizu K. Takai Y. J. Biol. Chem. 2005; 280: 4940-4947Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 9Kawakatsu T. Shimizu K. Honda T. Fukuhara T. Hoshino T. Takai Y. J. Biol. Chem. 2002; 277: 50749-50755Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Sato T. Fujita N. Yamada A. Ooshio T. Okamoto R. Irie K. Takai Y. J. Biol. Chem. 2006; 281: 5288-5299Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Nectin initiates the formation of cell-cell adhesion and then induces the activation of Rap1, Cdc42, and Rac small G proteins through the activation of c-Src. Subsequently, Cdc42 and Rac bind to IQGAP1 and induce the reorganization of the actin cytoskeleton, thereby causing the accumulation of non-trans-interacting E-cadherin at the nectin-based cell-cell adhesion sites. On the other hand, afadin, which binds to Rap1 activated by the action of nectin, associates with p120ctn and inhibits the endocytosis of accumulated non-trans-interacting E-cadherin to facilitate the trans-interaction of E-cadherin. In parallel with these processes, Cdc42 increases the number of filopodia and cell-cell contact sites, whereas Rac induces the formation of lamellipodia, which efficiently zip the cell-cell adhesion between the filopodia, acting like a "zipper." We recently found that nectin also interacts with integrin αvβ3 and that the activation of integrin αvβ3 is necessary for the nectin-induced signaling and formation of AJs (11Sakamoto Y. Ogita H. Hirota T. Kawakatsu T. Fukuyama T. Yasumi M. Kanzaki N. Ozaki M. Takai Y. J. Biol. Chem. 2006; 281: 19631-19644Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). actin filament adherens junction platelet-derived growth factor extracellular signal-regulated kinase protein-tyrosine phosphatase Src homology 2 SH2 domain-containing phosphatase-2 Dulbecco's modified Eagle's medium antibody polyclonal Ab monoclonal Ab bovine serum albumin glutathione S-transferase maltose-binding protein bromodeoxyuridine mitogen-activated protein kinase/extracellular signal-regulated kinase kinase constitutively active green fluorescent protein wild-type small interfering RNA low molecular weight actin filament adherens junction platelet-derived growth factor extracellular signal-regulated kinase protein-tyrosine phosphatase Src homology 2 SH2 domain-containing phosphatase-2 Dulbecco's modified Eagle's medium antibody polyclonal Ab monoclonal Ab bovine serum albumin glutathione S-transferase maltose-binding protein bromodeoxyuridine mitogen-activated protein kinase/extracellular signal-regulated kinase kinase constitutively active green fluorescent protein wild-type small interfering RNA low molecular weight Afadin furthermore plays an essential role in the recruitment of claudins to the apical side of the nectin-based cell-cell adhesion sites to form tight junctions (10Sato T. Fujita N. Yamada A. Ooshio T. Okamoto R. Irie K. Takai Y. J. Biol. Chem. 2006; 281: 5288-5299Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). We recently showed that cell-cell adhesion based on the nectin-afadin complex is indispensable for the formation of tight junctions but that the cadherin-based cell-cell adhesion is not always essential for it under certain conditions (12Yamada A. Fujita N. Sato T. Okamoto R. Ooshio T. Hirota T. Morimoto K. Irie K. Takai Y. Oncogene. 2006; 25: 5085-5102Crossref PubMed Scopus (47) Google Scholar), although it had been believed from several lines of circumstantial evidence that cadherin-based cell-cell adhesion is required for the formation of tight junctions (13Gonzalez-Mariscal L. Chavez de Ramirez B. Cereijido M. J. Membr. Biol. 1985; 86: 113-125Crossref PubMed Scopus (224) Google Scholar, 14Gumbiner B. Stevenson B. Grimaldi A. J. Cell Biol. 1988; 107: 1575-1587Crossref PubMed Scopus (653) Google Scholar, 15Watabe M. Nagafuchi A. Tsukita S. Takeichi M. J. Cell Biol. 1994; 127: 247-256Crossref PubMed Scopus (363) Google Scholar, 16Yap A.S. Brieher W.M. Gumbiner B.M. Annu. Rev. Cell Dev. Biol. 1997; 13: 119-146Crossref PubMed Scopus (681) Google Scholar). Therefore, afadin critically functions in the formation of both AJs and tight junctions. Platelet-derived growth factor (PDGF) is a family of growth factors consisting of three isoforms, PDGF-AA, -AB, and -BB (17Heldin C.H. Westermark B. Physiol. Rev. 1999; 79: 1283-1316Crossref PubMed Scopus (1932) Google Scholar). Binding of PDGF to its receptor causes the autophosphorylation of specific tyrosine residues in the intracellular parts of the receptor, leading to the activation of diverse intracellular signaling pathways such as Ras-ERK, c-Src, and Rap1-Rac signalings. It is well known that the Ras-ERK signaling enhances cell cycle progression and facilitates cell proliferation (18Heldin C.H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1421) Google Scholar, 19McCormick F. Curr. Opin. Genet. Dev. 1994; 4: 71-76Crossref PubMed Scopus (208) Google Scholar, 20Satoh T. Fantl W.J. Escobedo J.A. Williams L.T. Kaziro Y. Mol. Cell. Biol. 1993; 13: 3706-3713Crossref PubMed Scopus (87) Google Scholar). c-Src activated in response to PDGF tyrosine-phosphorylates various proteins to control the PDGF receptor-mediated signal transduction (21DeMali K.A. Godwin S.L. Soltoff S.P. Kazlauskas A. Exp. Cell Res. 1999; 253: 271-279Crossref PubMed Scopus (50) Google Scholar). Moreover, we recently found that the sequential activation of Rap1 and Rac locally at the leading edge, where PDGF receptor, integrin αvβ3, and nectin-like molecule-5 are accumulated and clustered (22Ikeda W. Kakunaga S. Takekuni K. Shingai T. Satoh K. Morimoto K. Takeuchi M. Imai T. Takai Y. J. Biol. Chem. 2004; 279: 18015-18025Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 23Minami Y. Ikeda W. Kajita M. Fujito T. Amano H. Tamaru Y. Kuramitsu K. Sakamoto Y. Monden M. Takai Y. J. Biol. Chem. 2007; 282: 18481-18496Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), 3H. Amano, W. Ikeda, S. Kawano, M. Kajita, Y. Tamaru, N. Inoue, Y. Minami, A. Yamada, and Y. Takai, unpublished observation. 3H. Amano, W. Ikeda, S. Kawano, M. Kajita, Y. Tamaru, N. Inoue, Y. Minami, A. Yamada, and Y. Takai, unpublished observation. influences the formation of leading-edge structures including lamellipodia, peripheral ruffles, focal complexes, and focal adhesions, resulting in the enhancement of cell movement. 4M. Takahashi, Y. Rikitake, Y. Nagamatsu, T. Hara, W. Ikeda, K. Hirata, and Y. Takai, unpublished observation. 4M. Takahashi, Y. Rikitake, Y. Nagamatsu, T. Hara, W. Ikeda, K. Hirata, and Y. Takai, unpublished observation. In addition, it has been reported that the Ras-ERK signaling plays roles in cellular morphology as well as cell proliferation (24Huang C. Jacobson K. Schaller M.D. J. Cell Sci. 2004; 117: 4619-4628Crossref PubMed Scopus (872) Google Scholar, 25Zhong C. Kinch M.S. Burridge K. Mol. Biol. Cell. 1997; 8: 2329-2344Crossref PubMed Scopus (139) Google Scholar). Tyrosine-phosphorylated PDGF receptor is in turn dephosphorylated and inactivated by protein-tyrosine phosphatases (PTPs) such as Src homology 2 (SH2) domain-containing phosphatase-2 (SHP-2) (26Chiarugi P. Cirri P. Raugei G. Manao G. Taddei L. Ramponi G. Biochem. Biophys. Res. Commun. 1996; 219: 21-25Crossref PubMed Scopus (31) Google Scholar, 27Markova B. Herrlich P. Ronnstrand L. Bohmer F.D. Biochemistry. 2003; 42: 2691-2699Crossref PubMed Scopus (44) Google Scholar). SHP-2 is a widely expressed cytosolic non-transmembrane PTP containing two SH2 domains at the N terminus and a single central phosphatase domain. It is reported that when PDGF receptor is autophosphorylated by PDGF, SHP-2 binds to PDGF receptor, and its phosphatase activity is up-regulated to dephosphorylate PDGF receptor (28Lechleider R.J. Sugimoto S. Bennett A.M. Kashishian A.S. Cooper J.A. Shoelson S.E. Walsh C.T. Neel B.G. J. Biol. Chem. 1993; 268: 21478-21481Abstract Full Text PDF PubMed Google Scholar, 29Pluskey S. Wandless T.J. Walsh C.T. Shoelson S.E. J. Biol. Chem. 1995; 270: 2897-2900Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). We show here a novel role of afadin in the regulation of the activation of PDGF receptor and Ras-ERK signaling. When afadin is tyrosine-phosphorylated by c-Src activated in response to PDGF, it binds to SHP-2 and stimulates the phosphatase activity of SHP-2, resulting in dephosphorylation of PDGF receptor. This contributes to the fine tuning of the activation of PDGF receptor and Ras-ERK signaling. We also found that the afadin- and SHP-2-mediated regulation of PDGF receptor and Ras-ERK signaling are important for the formation of leading-edge structures necessary for directional cell movement. Cell Culture, Expression Vectors, Transfection, and Knockdown Experiment—NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. Full-length (amino acids 1-1829), ΔRA (amino acids 351-1829), ΔDIL (amino acids 1-646 and 893-1829), ΔFAB (amino acids 1-1663), ΔPDZ (amino acids 1-1015 and 1122-1829), Δ1 (amino acids 1-1131), Δ2 (amino acids 982-1829), Δ3 (amino acids 1329-1829), and Δ4 (amino acids 1509-1829) of rat afadin cDNAs were subcloned into pEGFP-C1 (Clontech) and pCMV-FLAG (a kind gift from Dr. K. Matsumoto, Nagoya University, Nagoya, Japan). RNA interference-resistant mutants were constructed as described (10Sato T. Fujita N. Yamada A. Ooshio T. Okamoto R. Irie K. Takai Y. J. Biol. Chem. 2006; 281: 5288-5299Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The point mutants of full-length afadin at Y1237F and afadin-Δ2 at Y1139F, Y1158F, Y1196F, Y1226F, Y1233F, Y1237F, Y1259F, and Y1292F were generated by mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene). Full-length (amino acids 1-593), Δ1 (amino acids 1-234), Δ2 (amino acids 1-110), Δ3 (amino acids 111-234), and Δ4 (amino acids 241-597) of mouse SHP-2 cDNAs were subcloned into pCIneo-Myc (Promega). The full-length cDNA of Myc-tagged rat LMW-PTP and Myc-tagged constitutively active mutant of MEK1 (pSRα-Myc-MEK1-CA) were kindly supplied by Dr. T. Kondo (Nagasaki University, Nagasaki, Japan) and Dr. E. Nishida (Kyoto University, Kyoto, Japan), respectively. Expression vectors for constitutively active (pUSE-c-Src-CA) and kinase inactive (pUSE-Src (K297R)) mutants of c-Src was purchased from Upstate Biotechnology. Afadin-knockdown NIH3T3 cells, in which afadin is stably knocked down, were generated as follows; the fragment containing the H1-RNA promoter and short hairpin RNA sequence against afadin was excised from the pBS-H1-afadin vector, which was previously created to transiently knock down afadin as described (10Sato T. Fujita N. Yamada A. Ooshio T. Okamoto R. Irie K. Takai Y. J. Biol. Chem. 2006; 281: 5288-5299Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), and ligated into the EB virus-based vector, pEB, which was generated by deletion of the CAG promoter and DsRed gene from pRUBY-M2 kindly supplied by Dr. Y. Miwa (University of Tsukuba, Tsukuba, Japan) to construct the pEB-H1-afadin vector. NIH3T3 cells stably expressing the short hairpin RNA specific for afadin were generated by the transfection of pEB-H1-afadin into NIH3T3 cells followed by selection with 500 μg/ml G418 (Nacalai Tesque). Control NIH3T3 cells for afadin short hairpin RNA (shRNA) were similarly produced using scrambled shRNA sequence (5′-CCATCTCAATTCTTGGACG-3′). To knock down SHP-2, double-stranded 25-nucleotide RNA duplex (Stealth™ RNA interference; Invitrogen) for SHP-2 (5′-CCACUUUGGCUGAACUGGUUCAGUA-3′) was transfected into NIH3T3 cells with HiPerFect transfection reagent (Qiagen). As a control for SHP-2 small interfering RNA (siRNA), the scrambled RNA duplex (5′-CCAGGUUAGUCGUCACUUGAUCGUA-3′) was also purchased from Invitrogen and transfected into NIH3T3 cells. For DNA transfection, the Lipofectamine 2000 or the Lipofectamine LTX reagent (Invitrogen) was used. Antibodies and Reagents—A rabbit anti-afadin polyclonal Ab (pAb) and a mouse anti-afadin monoclonal Ab (mAb) were prepared as described (30Sakisaka T. Nakanishi H. Takahashi K. Mandai K. Miyahara M. Satoh A. Takaishi K. Takai Y. Oncogene. 1999; 18: 1609-1617Crossref PubMed Scopus (78) Google Scholar). Abs listed below were purchased from commercial sources; a rabbit anti-GFP pAb was from MBL, a mouse anti-FLAG M2 mAb was from Sigma, a rabbit anti-phospho-PDGF receptor (Tyr716) pAb was from Upstate Biotechnology, a rabbit anti-phospho-PDGF receptor (Tyr1009) pAb was from BIOSOURCE, a rabbit anti-PDGF receptor pAb was from Santa Cruz, a mouse anti-Ras mAb was from Upstate Biotechnology, a mouse anti-SHP-2 mAb was from BD Transduction Laboratories, a mouse anti-phosphotyrosine (PY 20) mAb was from BD Transduction Laboratories, a mouse anti-phospho-ERK1/2 mAb was from Cell Signaling Technology, a rabbit anti-ERK1/2 pAb was from Cell Signaling Technology, a rabbit anti-c-Src pAb was from Cell Signaling Technology, a rabbit anti-N-cadherin pAb was from Takara, and a rabbit anti-glyceraldehyde-3-phosphate dehydrogenase mAb was from Cell Signaling Technology. Hybridoma cells expressing a mouse anti-Myc mAb were purchased from the American Type Collection. Rhodamine phalloidin was purchased from Molecular Probes. Horseradish peroxidase-conjugated and fluorophore-conjugated secondary Abs were purchased from Amersham Biosciences and Chemicon, respectively. PDGF-BB and fatty acid-free bovine serum albumin (BSA) were purchased from PEPROTECH and Sigma, respectively. Vitronectin was purified from human plasma (Kohjinbio) as described (31Yatohgo T. Izumi M. Kashiwagi H. Hayashi M. Cell Struct. Funct. 1988; 13: 281-292Crossref PubMed Scopus (437) Google Scholar). Phosphorylation of PDGF Receptor and ERK and Activation of Ras—To examine the level of the phosphorylation of PDGF receptor and ERK in each kind of NIH3T3 cells after the PDGF stimulation, cells were serum-starved for 16 h and treated with 15 ng/ml PDGF at 25 °C for the indicated periods of time. After being washed with ice-cold PBS, cells were harvested using prewarmed Laemmli buffer (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar) containing 1 mm Na3VO4, 10 mm NaF, and a phosphatase inhibitor cocktail I (Sigma), boiled for 5 min, and sonicated 3 times for 10 s with 20-s cooling periods. Protein concentrations of the samples were determined using an RC DC protein assay kit (Bio-Rad) with BSA as a reference protein. The samples were subjected to SDS-PAGE followed by Western blotting using the indicated phospho-specific Abs. To assess the activation of Ras, the pulldown assay was performed as described previously (33Kakunaga S. Ikeda W. Shingai T. Fujito T. Yamada A. Minami Y. Imai T. Takai Y. J. Biol. Chem. 2004; 279: 36419-36425Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Briefly, after the treatment with PDGF, cells were lysed with Buffer A (50 mm Tris-HCl at pH 7.5, 200 mm NaCl, 5 mm MgCl2, 1% Nonidet P-40, 10% glycerol, 10 μm p-aminophenylmethanesulfonyl fluoride, 2 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mm Na3VO4) containing 10 μg of glutathione S-transferase (GST)-Raf-RBD+CRD (the Ras binding and cysteine-rich domains of Raf-1 fused to GST) and incubated at 2 °C for 30 min. The cell extract was obtained by centrifugation at 20,000 × g at 0 °C for 5 min and incubated with 50 μl of glutathione-agarose beads (Amersham Biosciences) at 2 °C for 1 h. After the beads were washed with Buffer A, proteins bound to the beads were eluted with Laemmli buffer and subjected to SDS-PAGE followed by Western blotting using the anti-Ras mAb. Band intensity was assessed using ImageJ software (National Institutes of Health) and paired Student's t tests were performed for statistical analysis. Immunoprecipitation Assay—HEK293 cells expressing in various combinations of the indicated molecules were lysed with Buffer B (20 mm Tris-HCl at pH 7.5, 150 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 1% Nonidet P-40, 10 mm NaF, 1 mm Na3VO4, 10 μg/ml leupeptin, 2 μg/ml aprotinin, and 10 μm p-amidinophenylmethanesulfonyl fluoride). The cell lysates were rotated for 30 min and subjected to centrifugation at 12,000 × g for 20 min. The supernatant was then incubated with the anti-FLAG mAb or the anti-Myc mAb at 4 °C for 2 h followed by incubation with the protein G-Sepharose beads at 4 °C for 2 h. After the beads were extensively washed with Buffer B, the bound proteins were eluted from the beads by boiling with the SDS sample buffer for 5 min and subjected to SDS-PAGE followed by Western blotting using the indicated Abs. To investigate the interaction of endogenous SHP-2 with afadin in a c-Src-dependent manner, NIH3T3 cells were treated with or without 10 μm PP2 (Calbiochem-Novabiochem) dissolved in 0.2% Me2SO, 10 μm PP3 (Calbiochem-Novabiochem) dissolved in 0.2% Me2SO, 10 μm SU6656 (Sigma) dissolved in 0.2% Me2SO, or 0.2% Me2SO as a negative control in DMEM for 4 h and lysed with Buffer B, and the cell lysates were incubated with the anti-SHP-2 mAb or control mouse IgG by incubation with protein G-Sepharose beads. The immunoprecipitated samples were then analyzed by Western blotting. Biotinylation Assay for Internalization of PDGF Receptor upon PDGF Stimulation—After stimulation with 15 ng/ml PDGF for the indicated periods of time, cells were incubated with 0.2 mg/ml sulfosuccinimidyl 2-(biotinamido) ethyldithioproprionate (sulfo-NHS-SS-biotin; Pierce) at 4 °C for 30 min. The cells were then lysed in radioimmune precipitation assay buffer (20 mm Tris-HCl at pH 7.4, 150 mm NaCl, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mm EDTA, 10 μg/ml leupeptin, 100 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin). The cell lysates were centrifuged, and the supernatants were incubated with streptavidin beads (Sigma) to collect bound biotinylated proteins. The samples were then subjected to SDS-PAGE followed by Western blotting. Direct Binding of SHP-2 to Afadin—Recombinant GST-fused full-length SHP-2 (GST-SHP-2) was prepared as described (34Takekuni K. Ikeda W. Fujito T. Morimoto K. Takeuchi M. Monden M. Takai Y. J. Biol. Chem. 2003; 278: 5497-5500Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Recombinant maltose-binding protein (MBP)-fused full-length afadin (MBP-afadin) was prepared as described (6Hoshino T. Sakisaka T. Baba T. Yamada T. Kimura T. Takai Y. J. Biol. Chem. 2005; 280: 24095-24103Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Briefly, High Five insect cells were infected with the baculovirus bearing the afadin cDNA, cultured for 64 h, treated with or without 0.1 mm pervanadate for 8 h, and then purified by the use of TALON metal affinity beads (Clontech). Pervanadate, which strongly inhibits tyrosine dephosphorylation and, thus, retains tyrosine phosphorylation, was prepared as described (35Huyer G. Liu S. Kelly J. Moffat J. Payette P. Kennedy B. Tsaprailis G. Gresser M.J. Ramachandran C. J. Biol. Chem. 1997; 272: 843-851Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar). The tyrosine phosphorylation of purified MBP-afadin was confirmed by Western blotting. To examine the affinities of SHP-2 for afadin, GST-SHP-2 or GST was incubated with MBP-afadin (20 pmol) immobilized on 20 μl of amylose resin beads in 400 μl of Buffer C (20 mm Tris-HCl at pH 8.0, 27.5 mm NaCl, 25 mm KCl, and 0.1% Triton X-100) at 4 °C for 2 h. After the beads were extensively washed with Buffer C, the bound proteins were eluted by boiling in the SDS sample buffer. The samples were then subjected to SDS-PAGE followed by staining with Coomassie Brilliant Blue. The amount of bound GST-SHP-2 was determined by comparing the band intensity of various amounts of BSA using a densitometer Fluorchem™ (Alpha Innotech Corp.). The Kd value was calculated by Scatchard analysis. Phosphatase Assay for SHP-2—Control NIH3T3 or afadin-knockdown NIH3T3 cells were seeded at density of 2.5 × 103 cells/cm2, cultured for 18 h, starved of serum with DMEM containing 0.5% BSA for 16 h, and then stimulated with DMEM containing 0.5% BSA and 15 ng/ml PDGF at 25 °C for 0 or 2 min. After stimulation, the cells were lysed with Buffer D (25 mm HEPES at pH 7.4, 150 mm NaCl, 2 mm EDTA, 0.5% Triton X-100). The cell lysates were rotated for 30 min and subjected to centrifugation at 12,000 × g for 20 min. The supernatant was then incubated with the anti-SHP-2 mAb at 4 °C for 2 h followed by incubation with the protein G-Sepharose beads at 4 °C for 2 h. Then the beads were extensively washed twice with Buffer D and twice with PTP buffer (20 mm HEPES at pH 7.5, 1 mm EDTA, 5% glycerol, 1 mm dithiothreitol). Beads were re-suspended in 50 μl of PTP buffer supplemented with 250 μm phosphopeptide, TSTEPQpYQPGENL (pY is phosphotyrosine, Upstate Biotechnology) and incubated at 37 °C for 30 min. To assess the amount of free phosphate from reactions, aliquots of supernatants were added in 96-well plates with 100 μl of malachite green solution (Upstate Biotechnology) and incubated at room temperature for 15 min. Absorbances were read at 620 nm with a microplate reader. The absorbance was compared with a phosphate standard curve to determine the release of phosphate in picomoles. For the quantities of inorganic phosphate released, the absorbance values were in the middle of the optical density range where the absorbance curve is linear. To control the expression levels of SHP-2 in the immune complex in the assays, aliquots of the immune complexes were Western blotted using the anti-SHP-2 mAb. Data are expressed as the means ± S.E. of three independent experiments. Paired Student's t test was performed for statistical analysis. Cell Proliferation and Directional Stimulation by PDGF—Cell proliferation assessed by BrdUrd incorporation was performed as described previously (36Fujito T. Ikeda W. Kakunaga S. Minami Y. Kajita M. Sakamoto Y. Monden M. Takai Y. J. Cell Biol. 2005; 171: 165-173Crossref PubMed Scopus (80) Google Scholar). Briefly, control and afadin-knockdown and SHP-2-knockdown NIH3T3 cells were starved of serum with DMEM containing 0.5% BSA for 24 h and then stimulated by 30 ng/ml PDGF and 10 μg/ml insulin for the indicated periods of time. Cells were incubated with BrdUrd for 2 h before the end of the stimulation. For directional stimulation of cells with PDGF, the μ-Slide VI flow (Ibidi), which has six parallel channels and was coated with 5 μg/ml vitronectin, was used to generate a concentration gradient of PDGF as described previously (23Minami Y. Ikeda W. Kajita M. Fujito T. Amano H. Tamaru Y. Kuramitsu K. Sakamoto Y. Monden M. Takai Y. J. Biol. Chem. 2007; 282: 18481-18496Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The concentration gradient of PDGF was made using DMEM containing 30 ng/ml PDGF and 0.5% BSA according to the manufacturer's protocol. Cells were seeded at a density of 5 × 103 cells/cm2, cultured for 18 h, and starved of serum with DMEM containing 0.5% BSA for 1 h in the presence or absence of 10 μm U0126 (Calbiochem-Novabiochem). After 30 min of directio

Referência(s)