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• W1998051038 abstract "c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) (also termed JNK-interacting protein 3; JIP3) is a member of a family of scaffold factors for the mitogen-activated protein kinase (MAPK) cascades, and it also forms a complex with focal adhesion kinase (FAK). Here we demonstrate that JSAP1 serves as a cooperative scaffold for activation of JNK and regulation of cell migration in response to fibronectin (FN) stimulation. JSAP1 mediated an association between FAK and JNK, which was induced by either co-expression of Src or attachment of cells to FN. Complex formation of FAK with JSAP1 and p130 Crk-associated substrate (p130Cas) resulted in augmentation of FAK activity and phosphorylation of both JSAP1 and p130Cas, which required p130Cas hyperphosphorylation and was abolished by inhibition of Src. JNK activation by FN was enhanced by JSAP1, which was suppressed by disrupting the FAK/p130Cas pathway by expression of a dominant-negative form of p130Cas or by inhibiting Src. We also documented the co-localization of JSAP1 with JNK and phosphorylated FAK at the leading edge and stimulation of cell migration by JSAP1 expression, which depended on its JNK binding domain and was suppressed by inhibition of JNK. The level of JSAP1 mRNA correlated with advanced malignancy in brain tumors, unlike other JIPs. We propose that the JSAP1·FAK complex functions cooperatively as a scaffold for the JNK signaling pathway and regulator of cell migration on FN, and we suggest that JSAP1 is also associated with malignancy in brain tumors. c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) (also termed JNK-interacting protein 3; JIP3) is a member of a family of scaffold factors for the mitogen-activated protein kinase (MAPK) cascades, and it also forms a complex with focal adhesion kinase (FAK). Here we demonstrate that JSAP1 serves as a cooperative scaffold for activation of JNK and regulation of cell migration in response to fibronectin (FN) stimulation. JSAP1 mediated an association between FAK and JNK, which was induced by either co-expression of Src or attachment of cells to FN. Complex formation of FAK with JSAP1 and p130 Crk-associated substrate (p130Cas) resulted in augmentation of FAK activity and phosphorylation of both JSAP1 and p130Cas, which required p130Cas hyperphosphorylation and was abolished by inhibition of Src. JNK activation by FN was enhanced by JSAP1, which was suppressed by disrupting the FAK/p130Cas pathway by expression of a dominant-negative form of p130Cas or by inhibiting Src. We also documented the co-localization of JSAP1 with JNK and phosphorylated FAK at the leading edge and stimulation of cell migration by JSAP1 expression, which depended on its JNK binding domain and was suppressed by inhibition of JNK. The level of JSAP1 mRNA correlated with advanced malignancy in brain tumors, unlike other JIPs. We propose that the JSAP1·FAK complex functions cooperatively as a scaffold for the JNK signaling pathway and regulator of cell migration on FN, and we suggest that JSAP1 is also associated with malignancy in brain tumors. Cell adhesion to the extracellular matrix regulates many cellular functions, including cell differentiation, proliferation, apoptosis, and migration (1Geiger B. Bershadsky A. Pankov R. Yamada K.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 793-805Crossref PubMed Scopus (1857) Google Scholar). Integrin stimulation by extracellular matrix proteins such as FN 2The abbreviations used are: FNfibronectinERKextracellular signal-regulated kinaseSembryonic stemFAKfocal adhesion kinaseGFPgreen fluorescent proteinGSTglutathione S-transferaseJIPJNK interacting proteinJNKc-Jun N-terminal kinaseJSAP1JNK/stress-activated protein kinase-associated protein 1MAPKmitogen-activated protein kinaseMKKMAPK kinaseMEKKMAPK kinase kinaseCrk-associate substrateCasSHSrc homologyWTwild type leads to activation of MAPKs, including JNK and extracellular signal-regulated kinase (ERK) in a variety of cell types. Among the types of MAPK signaling transmitted by integrins, JNK activation is believed to correlate particularly with increased cell migration and invasion (2Huang C. Jacobson K. Schaller M.D. J. Cell Sci. 2004; 117: 4619-4628Crossref PubMed Scopus (895) Google Scholar, 3Xia Y. Karin M. Trends Cell Biol. 2004; 14: 94-101Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). FN stimulation of cells is known to activate JNK through an FAK/p130Cas/CrkII/DOCK180/ELMO/Rac1 pathway (2Huang C. Jacobson K. Schaller M.D. J. Cell Sci. 2004; 117: 4619-4628Crossref PubMed Scopus (895) Google Scholar, 4Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 5Dolfi F. Garcia-Guzman M. Ojaniemi M. Nakamura H. Matsuda M. Vuori K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15394-15399Crossref PubMed Scopus (158) Google Scholar, 6Larsen M. Tremblay M.L. Yamada K.M. Nat. Rev. Mol. Cell. Biol. 2003; 4: 700-711Crossref PubMed Scopus (111) Google Scholar, 7Takino T. Tamura M. Miyamori H. Araki M. Matsumoto K. Sato H. Yamada K.M. J. Cell Sci. 2003; 116: 3145-3155Crossref PubMed Scopus (54) Google Scholar, 8Takino T. Nakada M. Miyamori H. Yamashita J. Yamada K.M. Sato H. Cancer Res. 2003; 63: 2335-2337PubMed Google Scholar, 9Tamura M. Gu J. Takino T. Yamada K.M. Cancer Res. 1999; 59: 442-449PubMed Google Scholar). This signaling is highly conserved across species and seems to be important for promoting cell migration. Mutations in CrkII, DOCK180, ELMO, Rac1, and JNK homologues in Drosophila all result in phenotypes with defects in cell migration (3Xia Y. Karin M. Trends Cell Biol. 2004; 14: 94-101Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 4Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 10Martin-Blanco E. Pastor-Pareja J.C. Garcia-Bellido A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7888-7893Crossref PubMed Scopus (109) Google Scholar). Embryonic fibroblasts from JNK-deficient and MAPK kinase kinase1 (MEKK1)-deficient mice are impaired in serum-induced JNK activation and cell migration (3Xia Y. Karin M. Trends Cell Biol. 2004; 14: 94-101Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 11Yujiri T. Nawata R. Takahashi T. Sato Y. Tanizawa Y. Kitamura T. Oka Y. J. Biol. Chem. 2003; 278: 3846-3851Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 12Yujiri T. Ware M. Widmann C. Oyer R. Russell D. Chan E. Zaitsu Y. Clarke P. Tyler K. Oka Y. Fanger G.R. Henson P. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7272-7277Crossref PubMed Scopus (208) Google Scholar, 13Javelaud D. Laboureau J. Gabison E. Verrecchia F. Mauviel A. J. Biol. Chem. 2003; 278: 24624-24628Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Recent studies have demonstrated that JNK regulates cell migration by phosphorylating paxillin and regulating microtubule assembly by phosphorylating microtubule-associated protein 2 (14Wittmann T. Waterman-Storer C.M. J. Cell Sci. 2001; 114: 3795-3803Crossref PubMed Google Scholar, 15Huang C. Rajfur Z. Borchers C. Schaller M.D. Jacobson K. Nature. 2003; 424: 219-223Crossref PubMed Scopus (417) Google Scholar, 16Chang L. Jones Y. Ellisman M.H. Goldstein L.S. Karin M. Dev. Cell. 2003; 4: 521-533Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). However, the mechanism by which JNK is recruited and activated in association with focal adhesions is largely unknown. fibronectin extracellular signal-regulated kinase embryonic stem focal adhesion kinase green fluorescent protein glutathione S-transferase JNK interacting protein c-Jun N-terminal kinase JNK/stress-activated protein kinase-associated protein 1 mitogen-activated protein kinase MAPK kinase MAPK kinase kinase Cas Src homology wild type JIP family members (JIP1, -2, and -3) were originally identified as JNK-binding proteins and are thought to serve as scaffold factors for MAPK signaling cascades (17Ito M. Yoshioka K. Akechi M. Yamashita S. Takamatsu N. Sugiyama K. Hibi M. Nakabeppu Y. Shiba T. Yamamoto K.I. Mol. Cell. Biol. 1999; 19: 7539-7548Crossref PubMed Scopus (228) Google Scholar, 18Verhey K.J. Meyer D. Deehan R. Blenis J. Schnapp B.J. Rapoport T.A. Margolis B. J. Cell Biol. 2001; 152: 959-970Crossref PubMed Scopus (499) Google Scholar, 19Verhey K.J. Rapoport T.A. Trends Biochem. Sci. 2001; 26: 545-550Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). JSAP1 binds not only to MAPK pathway constituents, including all JNK isoforms, MAPK kinases (MKK) 1, 4, and 7, MEKK1, mixed-lineage protein kinase 3, and c-Raf-1, but also to the motor protein kinesin (17Ito M. Yoshioka K. Akechi M. Yamashita S. Takamatsu N. Sugiyama K. Hibi M. Nakabeppu Y. Shiba T. Yamamoto K.I. Mol. Cell. Biol. 1999; 19: 7539-7548Crossref PubMed Scopus (228) Google Scholar, 18Verhey K.J. Meyer D. Deehan R. Blenis J. Schnapp B.J. Rapoport T.A. Margolis B. J. Cell Biol. 2001; 152: 959-970Crossref PubMed Scopus (499) Google Scholar, 19Verhey K.J. Rapoport T.A. Trends Biochem. Sci. 2001; 26: 545-550Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). We found previously that JSAP1 interacts with FAK (20Takino T. Yoshioka K. Miyamori H. Yamada K.M. Sato H. Oncogene. 2002; 21: 6488-6497Crossref PubMed Scopus (30) Google Scholar). This association was enhanced by c-Src, and it promoted cell spreading on FN. FAK is known to be essential for cell migration and for transmitting integrin-stimulated signals to MAPK (1Geiger B. Bershadsky A. Pankov R. Yamada K.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 793-805Crossref PubMed Scopus (1857) Google Scholar, 6Larsen M. Tremblay M.L. Yamada K.M. Nat. Rev. Mol. Cell. Biol. 2003; 4: 700-711Crossref PubMed Scopus (111) Google Scholar). FAK activated by cell adhesion to extracellular matrix undergoes autophosphorylation at Tyr-397 and thereby associates with Src family kinases, leading to enhancement of its tyrosine phosphorylation and kinase activity. Binding of FAK to c-Src also induces the formation of a multimolecular signaling complex in which FAK functions as a scaffold (21Westhoff M.A. Serrels B. Fincham V.J. Frame M.C. Carragher N.O. Mol. Cell. Biol. 2004; 24: 8113-8133Crossref PubMed Scopus (193) Google Scholar). Notably, JNK activation and FAK autophosphorylation at Tyr-397 are both attenuated in JSAP1 knock-out mice (22Ha H.Y. Cho I.H. Lee K.W. Song J.Y. Kim K.S. Yu Y.M. Lee J.K. Song J.S. Yang S.D. Shin H.S. Han P.L. Dev. Biol. 2005; 277: 184-199Crossref PubMed Scopus (56) Google Scholar), suggesting that JSAP1 might be involved in FAK-mediated JNK activation. Although many studies have suggested critical roles for JNK signaling in tumor cell migration and invasion, the role of JNK scaffold proteins in tumor malignancy is not well examined. Here, we provide evidence that the JSAP1·FAK complex functions as an effective scaffold for the JNK pathway and particularly for stimulating cell migration, and that JSAP1 mRNA is elevated in brain tumors. These results suggest that JSAP1 may also participate in the acquisition of malignancy in brain tumors. Cell Culture and Reagents—The human astrocytoma cell line U87MG (American Type Culture Collection) was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. Transient transfections were performed by a calcium phosphate method or by using Trans IT (PanVera). Reagents used were FN (Asahi Techno Glass), SP600125 (Alexis Biochemicals), poly-l-lysine (Sigma), and rhodamine-phalloidin (Molecular Probes). Immunological reagents used were anti-myc, anti-phosphotyrosine, anti-paxillin, anti-FAK, and anti-p130Cas antibodies (BD Biosciences); anti-FAK[pY397] antibody (BIOSOURCE); anti-myc antibody (Cell Signaling); anti-active JNK antibody (Promega); anti-FAK, anti-His, anti-glutathione S-transferase (GST), and anti-JNK1 antibodies (Santa Cruz Biotechnology); anti-α-tubulin, anti-VSV, anti-FLAG M2 antibodies (Sigma); an anti-Src antibody (Upstate); horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG antibodies (Amersham Biosciences); and AlexaFluor 488, 546, and 633 anti-mouse and anti-rabbit IgG antibodies (Molecular Probes); and an anti-JSAP1 rabbit antiserum (from Dr. M. Ito, Kitasato University, Japan). Expression Plasmids—pcDNA-myc-JSAP1, pcDNA-FLAG-JSAP1, pcDNA-His-S-JSAP1, pcDNA-His-S-JSAP1-ΔJBD (deletion mutant of amino acid residues 210-226 of JSAP1), pcDNA-His-S-JSAP1-Δ2 (double deletion mutant of amino acid residues 1-342 and 1054-1305 of JSAP1), pcDNA-FLAG-JNK1, pRK-green fluorescent protein (GFP), pRK-FLAG-FAK, pRK-VSV-FAK, and pSG-c-Src were constructed as described previously (20Takino T. Yoshioka K. Miyamori H. Yamada K.M. Sato H. Oncogene. 2002; 21: 6488-6497Crossref PubMed Scopus (30) Google Scholar, 23Kuboki Y. Ito M. Takamatsu N. Yamamoto K.I. Shiba T. Yoshioka K. J. Biol. Chem. 2000; 275: 39815-39818Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 24Sato S. Ito M. Ito T. Yoshioka K. Gene. 2004; 329: 51-60Crossref PubMed Scopus (24) Google Scholar). A cDNA encoding JIP1 (GenBank™ accession number NM_005456) was obtained by reverse transcription-PCR with human placenta cDNA and was cloned into the pEAK-FLAG expression vector (pEAK-FLAG-JIP1). The expression plasmids for p130Cas (pSSRα-p130Cas) and its deletion mutants (deleted SH3 domain, ΔSH3; substrate domain, ΔSD; Src binding domain, ΔSB) (25Nakamoto T. Sakai R. Honda H. Ogawa S. Ueno H. Suzuki T. Aizawa S. Yazaki Y. Hirai H. Mol. Cell. Biol. 1997; 17: 3884-3897Crossref PubMed Scopus (136) Google Scholar) were kind gifts from Dr. H. Hirai (Tokyo University, Japan), and the FLAG-epitope was inserted at their N terminus by site-directed mutagenesis. The pHA246pur puromycin-resistance plasmid was kindly provided by Hein te Riele (Netherlands Cancer Institute, Amsterdam, The Netherlands). Immunoprecipitation Analyses—At 36 h after transfection, cells were washed twice with ice-cold phosphate-buffered saline and lysed in a buffer containing 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 2 mm Na3VO4, 2 mm NaF, and 1% Nonidet P-40. In experiments to analyze the association between JNK and FAK, the lysis buffer consisted of 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 2 mm Na3VO4, 2 mm NaF, 1% Triton X-100, and 10% glycerol. Lysates were used for precipitation with the indicated antibodies or S-protein-agarose (Novagen) for 2 h at 4 °C,and antibody complexes were collected with protein G-Sepharose (Amersham Biosciences) in a further 1-h incubation at 4 °C. The precipitates were analyzed by immunoblotting. Measurement of FAK Activity—As described previously (20Takino T. Yoshioka K. Miyamori H. Yamada K.M. Sato H. Oncogene. 2002; 21: 6488-6497Crossref PubMed Scopus (30) Google Scholar), immunoprecipitated FAK was washed twice in kinase buffer (20 mm HEPES (pH 7.4), 150 mm NaCl, 10 mm MgCl2) and was incubated with 25 μCi of [γ-32P]ATP for 30 min at 37 °C. The reaction mixtures were subjected to SDS-PAGE, and the 32P-labeled materials were detected by a Typhoon 9200 Image Analyzer (Amersham Biosciences). Immunofluorescence Staining—Glass coverslips were incubated with 10 μg/ml FN in phosphate-buffered saline overnight at 4 °C and then blocked with 10 mg/ml bovine serum albumin. Cells were allowed to spread onto the FN-coated coverslips for the indicated periods, fixed with 4% paraformaldehyde in phosphate-buffered saline for 15 min, and then permeabilized with 4% paraformaldehyde/0.5% Triton X-100 for 5 min. The cells were then stained with the indicated antibodies and examined by confocal laser microscopy (Carl Zeiss). Measurement of Cell Motility—U87MG cells were co-transfected with 0.5 μg of pHA262pur, pRK-GFP, FLAG-JNK1, and 1 μg of His-S-JSAP1-WT, -ΔJBD, or control plasmids. Transfectants were selected with 1.25 μg/ml puromycin for 48 h. This selection for transient transfectants routinely resulted in 90% positive cells expressing GFP. Puromycin-selected cells were replated on 35-mm glass-bottom dishes coated with 10 μg/ml FN; the cells were cultured overnight in complete medium. Cell movements were monitored using Olympus inverted microscopes. Video images were collected at 10-min intervals for 4 h. Image stacks were converted to QuickTime movies, and the positions of nuclei were tracked to quantify cell motility using Move-tr/2D software (Library, Tokyo, Japan). Measurement of JNK Activity—JNK activity was measured using the SAPK/JNK assay kit from Cell Signaling. Briefly, the cell lysates were incubated with GST-c-Jun (amino acid residues 1-89), and precipitates were incubated in the presence of ATP for 30 min at 30 °C. Phosphorylation of c-Jun on Ser-63 by JNK bound to GST-c-Jun was detected by immunoblotting using anti-phospho-c-Jun (Ser 63) antibody. Embryonic Stem Cell Culture—The murine ES cell line E14K (gift of Dr. Hiroshi Nishina, Tokyo Medical and Dental University) was maintained as described previously (26Nishina H. Fischer K.D. Radvanyi L. Shahinian A. Hakem R. Rubie E.A. Bernstein A. Mak T.W. Woodgett J.R. Penninger J.M. Nature. 1997; 385: 350-353Crossref PubMed Scopus (309) Google Scholar) with the minor modification that KnockOut Serum Replacement (Invitrogen) was used instead of fetal calf serum. The generation of Jsap1(-/-) ES cell lines will be described elsewhere. Clinical Samples and Histology—Under an institutional review board-approved protocol, fresh human brain tumor tissues were obtained from 26 patients with astrocytic tumors who underwent therapeutic removal of brain tumors. Normal brain tissues were obtained from three patients undergoing temporal lobectomy for epilepsy. Histological diagnosis was made by standard light-microscopic evaluation. The classification of human brain tumors used in this study was based on the revised WHO criteria for tumors of the central nervous system (27Kleihues P. Burger P.C. Scheithauer B.W. Histological Typing of Tumours of Central Nervous System.Springer-Verlag, Berlin. 1993; Google Scholar). The 26 astrocytic tumors consisted of 7 low-grade astrocytomas, 8 anaplastic astrocytomas, and 11 glioblastomas. All of the tumor tissues were obtained at primary resection, and none of the patients had been subjected to chemotherapy or radiation therapy before resection. Quantitative reverse transcription-PCR—Real-time quantitative PCR was performed using a LightCycler (Roche Diagnostics) with SYBR green fluorescence signal detection after each cycle of amplification as described previously (28Mariani L. McDonough W.S. Hoelzinger D.B. Beaudry C. Kaczmarek E. Coons S.W. Giese A. Moghaddam M. Seiler R.W. Berens M.E. Cancer Res. 2001; 61: 4190-4196PubMed Google Scholar). Briefly, total RNA was isolated from human brain or brain tumor tissues. PCR was performed using the following primers: JIP1, sense (5′-TTTCCTGCCTATTACGCCATC-3′) and antisense (5′-CACCCCGCACGCTGAT-3′) (amplicon size, 238 bp); JIP2, sense (5′-CCCCACGGAGGACATCTACC-3′) and antisense (5′-GGGGACCGAGGACGAGAGTTC-3′) (amplicon size, 188 bp); JSAP1, sense (5′-TGAGAGACCACGGGAACCCAC-3′) and antisense (5′-GACTAATGCGGAATGCGAAAC-3′) (amplicon size, 245 bp); and histone H3.3, sense (5′-CCACTGAACTTCTGATTCGC-3′) and antisense (5′-GCGTGCTAGCTGGATGTCTT-3′) (amplicon size, 215 bp) (GenBank™ accession numbers AF074091, AF136382, NM_033392, and NM_002107 for JIP1, JIP2, JIP3, and histone H3.3, respectively). The PCR data were analyzed with LightCycler analysis software as previously described (28Mariani L. McDonough W.S. Hoelzinger D.B. Beaudry C. Kaczmarek E. Coons S.W. Giese A. Moghaddam M. Seiler R.W. Berens M.E. Cancer Res. 2001; 61: 4190-4196PubMed Google Scholar). Quantification was based on the number of cycles necessary to produce a detectable amount of product above background. The difference in the cycle number (d) was normalized to the housekeeping gene histone H3.3 and then used to calculate the -fold difference in copy number according to the formula f = 2(d), where f = -fold difference in specific gene expression and d = cycle number difference between compared sources of mRNA (corrected for differences in histone H3.3). JSAP1 Forms a Scaffold for JNK with FAK—JSAP1 has been identified as a scaffold factor for JNK and we previously reported that JSAP1 also associates with FAK (17Ito M. Yoshioka K. Akechi M. Yamashita S. Takamatsu N. Sugiyama K. Hibi M. Nakabeppu Y. Shiba T. Yamamoto K.I. Mol. Cell. Biol. 1999; 19: 7539-7548Crossref PubMed Scopus (228) Google Scholar, 20Takino T. Yoshioka K. Miyamori H. Yamada K.M. Sato H. Oncogene. 2002; 21: 6488-6497Crossref PubMed Scopus (30) Google Scholar). To examine whether formation of the JSAP1 and FAK complex affects JNK signaling, e.g. as a scaffold factor, JSAP1-WT or a mutant lacking the JNK-binding domain of JSAP1 (ΔJBD) was co-expressed with FAK, c-Src, and JNK1 in 293T cells, and their interaction was analyzed by immunoprecipitation. Co-expression of FAK/c-Src significantly stimulated complex formation between JSAP1 and JNK1, and the JNK1 in the complex was phosphorylated (Fig. 1B). Although both JSAP1-WT and JSAP1-ΔJBD were tyrosine-phosphorylated and co-precipitated with FAK, only JSAP1-WT but not JSAP1-ΔJBD was co-precipitated with JNK1. To confirm that both FAK and JNK1 reside on the same JSAP1 scaffold, immunoprecipitation with an antibody against FAK was performed. JNK1 was co-precipitated with FAK only in cells co-expressing JSAP1-WT but not JSAP1-ΔJBD, whereas both JSAP1-WT and JSAP1-ΔJBD were co-precipitated with FAK (Fig. 1C). FAK was co-precipitated with JNK1 only in the cells co-expressing JSAP1-WT but not JSAP1-ΔJBD. The tyrosine phosphorylation of JSAP1-WT and JSAP1-ΔJBD was equally augmented by adhesion of cells to FN in place of FAK·Src expression, and only JSAP1-WT but not JSAP1-ΔJBD associated with JNK1 (Fig. 1D). Moreover, immunoprecipitation analysis using lysates from mouse brain tissue demonstrated the existence of endogenous JSAP1·FAK·JNK complex (Fig. 1E). These results suggest that JSAP1 forms a scaffold for the JNK pathway with FAK following formation of a FAK·Src complex, and thus the association of JSAP1 with JNK is facilitated under conditions that induce FAK·Src complex formation such as FN stimulation. JSAP1 Elevates FAK Activity and p130Cas Phosphorylation—FAK is a critical regulator of FN-induced JNK activation and cell migration. FAK activity is negatively regulated by its N-terminal FERM domain (29Schlaepfer D.D. Jones K.C. Hunter T. Mol. Cell. Biol. 1998; 18: 2571-2585Crossref PubMed Scopus (358) Google Scholar, 30Dunty J.M. Gabarra-Niecko V. King M.L. Ceccarelli D.F. Eck M.J. Schaller M.D. Mol. Cell. Biol. 2004; 24: 5353-5368Crossref PubMed Scopus (96) Google Scholar), which binds to JSAP1. We next investigated whether JSAP1 affects FAK activity. In vitro kinase assays showed that expression of JSAP1-WT or JSAP1-ΔJBD increased FAK activity by 3-fold (Fig. 2A). In contrast, JSAP1-Δ2, which fails to bind to FAK, had no effect. FAK activity was elevated 10-fold by co-expression of c-Src under the same conditions, indicating that activation by JSAP1 is substantial but not maximal. p130Cas is phosphorylated by a FAK·Src complex and mediates FAK-induced JNK activation by forming a complex with CrkII (5Dolfi F. Garcia-Guzman M. Ojaniemi M. Nakamura H. Matsuda M. Vuori K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15394-15399Crossref PubMed Scopus (158) Google Scholar). The effects of JSAP1 expression on p130Cas phosphorylation and FAK activity were studied using deletion mutants of p130Cas (Fig. 2, B and C). p130Cas expressed by transfection of the gene was not tyrosine-phosphorylated, and co-expression of FAK slightly induced its phosphorylation. Although expression of JSAP1 alone did not affect p130Cas phosphorylation, co-expression of JSAP1 with FAK strongly stimulated it. This JSAP1·FAK-enhanced phosphorylation of p130Cas was not observed with the mutants p130Cas-ΔSH3 or -ΔSB, but p130Cas-ΔSD was slightly phosphorylated under the same conditions. Because FAK is known to interact with the SH3 domain of p130Cas through its C-terminal proline-rich region and to phosphorylate the Src-binding domain of p130Cas, these results suggest that JSAP1 stimulates phosphorylation of p130Cas by Src at the substrate domain followed by phosphorylation of its Src-binding domain by FAK. In vitro kinase assays showed that FAK activity was slightly elevated by p130Cas expression, which was stimulated by co-expression of JSAP1. In contrast, p130Cas mutants had no effect. JSAP1 phosphorylation was also induced by co-expression of FAK with p130Cas. This is supported by the results presented in Fig. 2D showing that phosphorylation of both p130Cas and JSAP1 induced by co-expression with FAK was reduced by Src inhibitor PP2 treatment. These results suggest that complex formation of FAK with JSAP1 and p130Cas promotes p130Cas phosphorylation and FAK activation through Src. JSAP1 but Not JIP1 Enhances JNK Activation—Although JSAP1 is a member of the JIP family, the primary structure of JSAP1 is different from the others (18Verhey K.J. Meyer D. Deehan R. Blenis J. Schnapp B.J. Rapoport T.A. Margolis B. J. Cell Biol. 2001; 152: 959-970Crossref PubMed Scopus (499) Google Scholar). To compare JSAP1 with JIP1 in terms of association with FAK, JSAP1 or JIP1 tagged with FLAG epitope was co-expressed with FAK and c-Src, and immunoprecipitation analysis was carried out. As shown in Fig. 3A, only JSAP1 but not JIP1 co-precipitated FAK. JNK activation was faintly induced by cultivation of cells on FN or by JSAP1 expression when cultured on PLL (Fig. 3B). In contrast, consistent with its binding to FAK, the expression of JSAP1 but not JIP1 significantly enhanced the JNK activating signal from FN (Fig. 3B). To confirm the role of JSAP1 in JNK activation, U87MG cells co-transfected with either JSAP1-WT or JSAP1-ΔJBD and JNK1 were cultured on FN, and JNK phosphorylation was examined (Fig. 3C). JSAP1-WT expression augmented JNK phosphorylation in a dose-dependent manner, whereas JSAP1-ΔJBD expression did not affect it. As shown in Fig. 3D, the JNK phosphorylation enhanced by JSAP1 was suppressed by the Src inhibitor and a dominant negative form of p130Cas (ΔSD). These results suggest that JSAP1 expression promotes FN-induced JNK activation by facilitating the FAK/p130Cas pathway. Because FAK, p130Cas, CrkII, and Rac1 are known to accumulate at the leading edge in migrating cells (31Hsia D.A. Mitra S.K. Hauck C.R. Streblow D.N. Nelson J.A. Ilic D. Huang S. Li E. Nemerow G.R. Leng J. Spencer K.S. Cheresh D.A. Schlaepfer D.D. J. Cell Biol. 2003; 160: 753-767Crossref PubMed Scopus (461) Google Scholar, 32Webb 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 (1081) Google Scholar), we next examined the localization of JSAP1 in U87MG cells cultured on FN. Immunofluorescence staining demonstrated the extension of tubulin-containing microtubules toward the cell periphery (Fig. 3E) and the well organized distribution of paxillin (Fig. 3F) and autophosphorylated FAK (Fig. 3G) at the leading front of migrating U87MG cells. It has been reported that JSAP1 is transported to the tips of neurites through its interaction with the tetratricopeptide repeat domain of kinesin light chains (24Sato S. Ito M. Ito T. Yoshioka K. Gene. 2004; 329: 51-60Crossref PubMed Scopus (24) Google Scholar, 33Kelkar N. Delmotte M.H. Weston C.R. Barrett T. Sheppard B.J. Flavell R.A. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9843-9848Crossref PubMed Scopus (66) Google Scholar). We observed that JSAP1 is distributed diffusely in the cytoplasm but is also concentrated at the cell periphery where paxillin is well organized and microtubules have elongated. These results indicate that JSAP1 functions together with FAK scaffold at the leading edge. JSAP1-deficient ES Cells Are Impaired in Lamellipodial Protrusion— ES cells provide a highly informative in vitro cell system to study the phenotypic effects of gene disruption at the cellular level. Jsap1-/- murine ES cell lines have been described elsewhere (Fig. 4A). Attachment of wild-type Jsap1+/+ ES cells to FN induced lamellipodial protrusion and membrane ruffling (Fig. 4, B and C). In contrast, Jsap1-null (Jsap1-/-) ES cells displayed a substantial decrease of the rate of lamellipodial protrusion and membrane ruffling (Fig. 4, B and D). Rac1 and p130Cas/Crk has been shown to induce lamellipodial protrusion and membrane ruffle formation associated with cell migration (5Dolfi F. Garcia-Guzman M. Ojaniemi M. Nakamura H. Matsuda M. Vuori K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15394-15399Crossref PubMed Scopus (158) Google Scholar, 38Palazzo A.F. Eng C.H. Schlaepfer D.D. Marcantonio E.E. Gundersen G.G. Science. 2004; 303: 836-839Crossref PubMed Scopus (366) Google Scholar). These observations suggest that JSAP1 is required for FN-induced lamellipodial protrusion and membrane ruffling through the p130Cas/Crk/Rac1 pathway, which led us to test the hypothesis that JSAP1 contributes to cell motility. JSAP1 Expression Promotes Cell Migration—To explore whether JSAP1 is involved in cell migration, the distribution of JSAP1 and JNK1 in wound-edge cells was monitored. As shown in Fig. 5A, JNK1 was barely localized" @default.
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• W1998051038 title "JSAP1/JIP3 Cooperates with Focal Adhesion Kinase to Regulate c-Jun N-terminal Kinase and Cell Migration" @default.
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