A proinflammatory cytokine IFN-gamma stimulates microglia in the injured brain; however, signaling pathways for IFN-gamma-mediated microglia activation are not well characterized. In the present study, a protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) acts in concert with IFN-gamma to enhance nitric oxide (NO) production in murine microglial BV2 cells by synergistically increasing expression of inducible NO synthase (iNOS). The synergistic NO production by PMA was in part decreased by a PKC inhibitor Gö6976. PMA alone induced activation of nuclear factor-kappa B (NF-kappaB) and extracellular signal-regulated kinase (ERK) of mitogen-activated protein kinases (MAPKs) subtypes, whereas IFN-gamma alone had little effect. PMA and IFN-gamma synergistically enhanced activity of NF-kappaB, but not ERK. The inhibitors of NF-kappaB (pyrrolidine dithiocarbamate, PDTC) and ERK (1,4-diamino-2,3-dicyano-1,4 bis[2-aminophenylthio]butadiene; U0126) markedly decreased synergistic NO production in BV2 cells treated with IFN-gamma and PMA in combination. We found further that co-treatment with IFN-gamma and PMA synergistically induced interferon regulatory factor-1 (IRF-1), which is the major transcription factor for IFN-gamma-mediated iNOS expression. The present results demonstrate the cooperative interaction of multiple signaling pathways in the induction of NO production in activated microglial cells, and suggest that the functional interplay of these pathways may be important for the onset of microglia-mediated inflammatory responses in brain.
The aim of the current study was to investigate the effects of glucosamine (GlcN) on septic lethality and sepsis-induced inflammation using animal models of mice and zebrafish. GlcN pretreatment improved survival in the cecal ligation and puncture (CLP)-induced sepsis mouse model and attenuated lipopolysaccharide (LPS)-induced septic lung injury and systemic inflammation. GlcN suppressed LPS-induced M1-specific but not M2-specific gene expression. Furthermore, increased expressions of inflammatory genes in visceral tissue of LPS-injected zebrafish were suppressed by GlcN. GlcN suppressed LPS-induced activation of mitogen-activated protein kinase (MAPK) and NF-κB in lung tissue. LPS triggered a reduction in O-GlcNAc levels in nucleocytoplasmic proteins of lung, liver, and spleen after 1 day, which returned to normal levels at day 3. GlcN inhibited LPS-induced O-GlcNAc down-regulation in mouse lung and visceral tissue of zebrafish. Furthermore, the O-GlcNAcase (OGA) level was increased by LPS, which were suppressed by GlcN in mouse and zebrafish. OGA inhibitors suppressed LPS-induced expression of inflammatory genes in RAW264.7 cells and the visceral tissue of zebrafish. Stable knockdown of Oga via short hairpin RNA led to increased inducible nitric oxide synthase (iNOS) expression in response to LPS with or without GlcN in RAW264.7 cells. Overall, our results demonstrate a protective effect of GlcN on sepsis potentially through modulation of O-GlcNAcylation of nucleocytoplasmic proteins.
Impaired brain glucose metabolism is considered a hallmark of brain dysfunction and neurodegeneration. Disruption of the hexosamine biosynthetic pathway (HBP) and subsequent O-linked N-acetylglucosamine ( O-GlcNAc) cycling has been identified as an emerging link between altered glucose metabolism and defects in the brain. Myriads of cytosolic and nuclear proteins in the nervous system are modified at serine or threonine residues with a single N-acetylglucosamine ( O-GlcNAc) molecule by O-GlcNAc transferase (OGT), which can be removed by β- N-acetylglucosaminidase ( O-GlcNAcase, OGA). Homeostatic regulation of O-GlcNAc cycling is important for the maintenance of normal brain activity. Although significant evidence linking dysregulated HBP metabolism and aberrant O-GlcNAc cycling to induction or progression of neuronal diseases has been obtained, the issue of whether altered O-GlcNAcylation is causal in brain pathogenesis remains uncertain. Elucidation of the specific functions and regulatory mechanisms of individual O-GlcNAcylated neuronal proteins in both normal and diseased states may facilitate the identification of novel therapeutic targets for various neuronal disorders. The information presented in this review highlights the importance of HBP/ O-GlcNAcylation in the neuronal system and summarizes the roles and potential mechanisms of O-GlcNAcylated neuronal proteins in maintaining normal brain function and initiation and progression of neurological diseases.
Although elevated expression and increased tyrosine phosphorylation of focal adhesion kinase (FAK) are crucial for tumor progression, the mechanism by which FAK promotes oncogenic transformation is unclear. We have therefore determined the role of FAK phosphorylation at tyrosine 861 in the oncogenic transformation of NIH3T3 fibroblasts. FAK phosphorylation at tyrosine 861 was increased in both constitutively H-Ras-transformed and H-Ras-inducible NIH3T3 cells, in parallel with cell transformation. However, H-Ras-inducible cells transfected with the nonphosphorylatable mutant FAK Y861F showed decreased migration/invasion, focus forming activity and anchorage-independent growth, compared with either wild-type or kinase-defective FAK. In contrast to unaltered FAK/Src activity, the association of FAK and p130CAS was decreased in FAK Y861F-transfected cells, and FAK phosphorylation at tyrosine 861 enhanced this association in vitro. Consistently, FAK Y861F-transfected cells were defective in activation of c-Jun NH2-terminal kinase and in expression of matrix metalloproteinase-9 during transformation. Taken together, these results strongly suggest that FAK phosphorylation at tyrosine 861 is crucial for H-Ras-induced transformation through regulation of the association of FAK with p130CAS. Although elevated expression and increased tyrosine phosphorylation of focal adhesion kinase (FAK) are crucial for tumor progression, the mechanism by which FAK promotes oncogenic transformation is unclear. We have therefore determined the role of FAK phosphorylation at tyrosine 861 in the oncogenic transformation of NIH3T3 fibroblasts. FAK phosphorylation at tyrosine 861 was increased in both constitutively H-Ras-transformed and H-Ras-inducible NIH3T3 cells, in parallel with cell transformation. However, H-Ras-inducible cells transfected with the nonphosphorylatable mutant FAK Y861F showed decreased migration/invasion, focus forming activity and anchorage-independent growth, compared with either wild-type or kinase-defective FAK. In contrast to unaltered FAK/Src activity, the association of FAK and p130CAS was decreased in FAK Y861F-transfected cells, and FAK phosphorylation at tyrosine 861 enhanced this association in vitro. Consistently, FAK Y861F-transfected cells were defective in activation of c-Jun NH2-terminal kinase and in expression of matrix metalloproteinase-9 during transformation. Taken together, these results strongly suggest that FAK phosphorylation at tyrosine 861 is crucial for H-Ras-induced transformation through regulation of the association of FAK with p130CAS. Focal adhesion kinase (FAK) 1The abbreviations used are: FAK, focal adhesion kinase; SH2, Src homology 2; mAb, monoclonal antibody; GST, glutathione S-transferase; JNK, c-Jun NH2-terminal kinase; FBS, fetal bovine serum; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium.1The abbreviations used are: FAK, focal adhesion kinase; SH2, Src homology 2; mAb, monoclonal antibody; GST, glutathione S-transferase; JNK, c-Jun NH2-terminal kinase; FBS, fetal bovine serum; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium. is a non-receptor cytoplasmic tyrosine kinase that modulates multiple cell functions, including migration, proliferation, and survival (1Wang H.B. Dembo M. Hanks S.K. Wang Y.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11295-11300Crossref PubMed Scopus (396) Google Scholar, 2Frisch S.M. Vuori K. Ruoslahti E. Chan-Hui P.Y. J. Cell Biol. 1996; 134: 793-799Crossref PubMed Scopus (995) Google Scholar). Elevated expression and increased tyrosine phosphorylation of FAK have been reported in several types of malignant tumors, suggesting that FAK may play a role in tumor progression (3Wang D. Grammer J.R. Cobbs C.S. Stewart J.E. Lui Z. Rhoden R. Hecker T.P. Ding Q. Gladson C.L. J. Cell Sci. 2000; 113: 4221-4230Crossref PubMed Google Scholar, 4Hecker T.P. Grammer J.R. Gillespie G.Y. Stewart J. Gladson C.L. Cancer Res. 2002; 62: 2699-2707PubMed Google Scholar), especially because the deregulation of processes normally regulated by FAK, namely adhesion-dependent cell growth, survival, and motility, are critical aspects of tumor progression (5Richardson A. Parsons T. Nature. 1996; 280: 538-540Crossref Scopus (452) Google Scholar, 6Xu L.H. Yang X. Craven R.J. Cance W.G. Cell Growth & Differ. 1998; 9: 999-1005PubMed Google Scholar). The ability of FAK to transduce downstream signals depends on its phosphorylation at tyrosine residues and its ability to interact with several intracellular signaling molecules, including the Src family kinases (7Cobb B.S. Schaller M.D. Leu T.-H. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (483) Google Scholar), p130CAS (8Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (387) Google Scholar), Grb2 (9Schlaepfer D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (400) Google Scholar), and phosphatidylinositol 3-kinase (10Chen H.-C. Guan J.-L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10148-10152Crossref PubMed Scopus (476) Google Scholar, 11Chen H.-C. Appeddu P.A. Isoda H. Guan J.-L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). FAK phosphorylation at tyrosine 397 results in its direct interaction with Src, which contributes to the transformation of fibroblasts (12Schaller M.D. J. Endocrinol. 1996; 150: 1-7Crossref PubMed Scopus (74) Google Scholar, 13Parsons J.T. J. Cell Sci. 2003; 116: 1409-1416Crossref PubMed Scopus (1139) Google Scholar). Subsequent recruitment to the complex of proteins containing Src homology 2 (SH2) domains, including Grb2 and c-Crk, is likely to trigger adhesion-induced cellular responses, including changes to the actin cytoskeleton and activation of the Ras-mitogen-activated protein kinase pathway (14Schlaepfer D.D. Jones K.C. Hunter T. Mol. Cell. Biol. 1998; 18: 2571-2585Crossref PubMed Scopus (357) Google Scholar, 15Dolfi 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 (157) Google Scholar). p130CAS docking protein was initially identified as a major phosphotyrosine-containing protein in cells transformed by the v-src and v-crk oncogenes (16Kanner S.B. Reynolds A.B. Vines R.R. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3328-3332Crossref PubMed Scopus (397) Google Scholar). In addition, p130CAS may be a substrate of v-Src kinase and a binding target for the SH2 domain of v-Crk (17Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (593) Google Scholar) during retroviral transformation. Indeed, transfection of antisense p130CAS mRNA into ras- and v-src-transformed cells led to their reversion (18Auvinen M. Paasinen-Sohns A. Hirai H. Andersson L.C. Holtta E. Mol. Cell. Biol. 1995; 15: 6513-6525Crossref PubMed Scopus (73) Google Scholar), and mouse embryonic fibroblasts lacking p130CAS were resistant to Src-induced transformation (19Honda H. Oda H. Nakamoto T. Honda Z. Sakai R. Suzuki T. Saito T. Nakamura K. Nakao K. Ishikawa T. Katsuki M. Yazaki Y. Hirai H. Nat. Genet. 1998; 19: 361-365Crossref PubMed Scopus (307) Google Scholar). p130CAS-deficient mouse embryonic fibroblasts also showed impaired actin bundling and cell migration, and these properties were restored after re-expression of p130CAS (19Honda H. Oda H. Nakamoto T. Honda Z. Sakai R. Suzuki T. Saito T. Nakamura K. Nakao K. Ishikawa T. Katsuki M. Yazaki Y. Hirai H. Nat. Genet. 1998; 19: 361-365Crossref PubMed Scopus (307) Google Scholar), further indicating that this protein is essential in signal transduction during cell migration and transformation. Phosphorylation of FAK at tyrosine 861 (FAK Tyr861 phosphorylation) is especially interesting, because it is known to regulate migration of prostate carcinoma cells with increasing metastatic potential (20Slack J.K. Adams R.B. Rovin J.D. Bissonette E.A. Stoker C.E. Parsons J.T. Oncogene. 2001; 20: 1152-1163Crossref PubMed Scopus (189) Google Scholar), as well as the migration and survival of vascular endothelial cells (21Abu-Ghazaleh R. Kabir J. Jia H. Lobo M. Zachary I. Biochem. J. 2001; 15: 255-264Crossref Google Scholar). In addition, FAK Tyr861 phosphorylation is increased in metastatic breast cancer cells (22Vadlamudi R.K. Sahin A.A. Adam L. Wang R.A. Kumar R. FEBS Lett. 2003; 22: 76-80Crossref Scopus (75) Google Scholar) and ras-transformed fibroblasts (23Lim Y. Han I. Kwon H.J. Oh E.S. J. Biol. Chem. 2002; 277: 12735-12740Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) and decreased in detransformed cells by trichostatin A (23Lim Y. Han I. Kwon H.J. Oh E.S. J. Biol. Chem. 2002; 277: 12735-12740Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). We therefore hypothesized that FAK Tyr861 phosphorylation may regulate transforming activity in transformed/cancer cells. Here we report that FAK Tyr861 phosphorylation is crucial for H-Ras-induced transformation by regulating the association between FAK and p130CAS. Reagents and Antibodies—Doxycyclin and puromycin were purchased from Sigma, and fibronectin was purchased from Invitrogen. Monoclonal antibodies (mAbs) to phosphotyrosine (4G10) and p130CAS (8G4-E8) were purchased from UBI (Hauppauge, NY), mAb to HA (12CA5) was purchased from Roche Applied Science, mAbs to H-Ras (F235), GST (B-14), phospho-specific ERK (E-4), and ERK2 (K-23) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and mAbs to JNK and phospho-specific JNK (pT183/pY185) were purchased from Cell Signaling Technology (Beverly, MA). Mouse mAb to FAK and rabbit polyclonal antibodies to phosphorylation site-specific FAK[PY397] and FAK[PY861] were purchased from BioSource Quality Controlled Biochemicals, Inc. (Morgan Hill, CA). Establishment of a Doxycyclin-regulated H-Ras NIH3T3 Cell Line— Mouse wild-type H-Ras cDNA in pcDNA3.1 (Invitrogen) was a generous gift from Dr. Zea Young Ryoo of the Catholic University of Korea. The full-length H-Ras (G12R) cDNA was enzymatically excised and subcloned, using the GeneTailer site-directed mutagenesis system (Invitrogen), into the BamHI site of the tetracycline-inducible vector, pTRE-IRES-EGFP, a generous gift from Dr Hong Jian Zhu of the Ludwig Institute for Cancer Research (Melbourne, Australia). To obtain NIH3T3 cells with doxycyclin-induced (a tetracycline derivative) H-Ras (G12R) expression, pTRE-H-Ras-IRES-EGFP and pEFpurop-Tet-on (the generous gift of Dr. Hong Jian Zhu) were cotransfected into NIH3T3 cells with FuGENE6 reagent (Roche Applied Science), and the cells were selected with puromycin. Positive clones were those that expressed H-Ras in the presence of doxycyclin, as shown by Western analysis using anti H-Ras antibody (F235 clone; Santa Cruz). These H-Ras NIH3T3 cells were grown in a 5% CO2 atmosphere at 37 °C in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (FBS) (Invitrogen), 60 mg/ml penicillin, and 100 mg/ml streptomycin. Puromycin (2 μg/ml) was added to the medium for H-Ras expression clones. Construction of Mutant FAK Mammalian Expression Vectors—Site-directed mutagenesis of full-length cDNA encoding FAK in the pRC/CMV vector was performed using the Transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA). The synthetic oligonucleotide, CCA ACA CAT CTT TCA GCC TGT GGG G (Tyr861 → Phe), was used to change tyrosine to phenylalanine. cDNAs encoding FAK and its mutant Y861F were inserted into pRC/CMV at the NotI/XbaI cloning sites, which generated in-frame fusions of a sequence encoding three HA epitopes (YPYDVPDYA) at the 3′ end of the FAK coding sequences. Expression vectors encoding epitope-tagged WT FAK (pKH3-FAK) and kinase-defective FAK (pKH3-kdFAK) were kindly provided by Dr. Jun-Lin Guan of Cornell University (Ithaca, NY). Transfections—Transient transfections were carried out using LipofectAMINE reagent (Invitrogen), as described by the manufacturer. In brief, NIH3T3 and H-Ras-inducible NIH3T3 cells were plated in 100-mm dishes and grown to ∼80% confluency. To each culture was added 5 ml of a mixture of 15 μl of LipofectAMINE and 4 μg of plasmid DNA, and the cells were incubated for 6 h at 37 °Cina5%CO2, 95% air incubator. To each was added 5 ml of DMEM containing 20% FBS, and the cells were further incubated for 24 h. The medium was then aspirated and replaced with 5 ml of DMEM containing 10% FBS. Immunoprecipitation and Immunoblotting—The cultures were washed twice with phosphate-buffered saline, and the cells were lysed in RIPA buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 10 mm NaF, 2 mm Na3VO4) containing a protease inhibitor mixture (1 μg/ml aprotinin, 1 μg/ml antipain, 5 μg/ml leupeptin, 1 μg/ml pepstatin A, and 20 μg/ml phenylmethylsulfonyl fluoride). The lysates were clarified by centrifugation at 10,000 × g for 15 min at 4 °C, denatured with SDS sample buffer, boiled, and analyzed by SDS-PAGE. For immunoprecipitations, each sample, containing 200–1,000 μg of total protein, was incubated with the relevant antibody for 2 h at 4 °C, followed by incubation with protein G-Sepharose beads for 1 h. Immune complexes were collected by centrifugation. The proteins were transferred onto polyvinylidene difluoride membranes (Amersham Biosciences), which were incubated with the appropriate primary antibodies, followed by species-specific horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences). The signals were detected by ECL (Amersham Biosciences). Focus Forming and Soft Agar Growth Assays—In the focus forming assay, 5 × 104 H-Ras-transformed NIH3T3 cells were plated and incubated for 10–14 days, fixed in 99% methanol, and stained with Wright-Giemsa stain. For analysis of colony formation in soft agar, 1 × 105 cells in 2 ml of DMEM containing 10% FBS, 2 μg/ml puromycin, and 0.3% agarose were seeded in 6-mm plates containing a 0.6% agarose underlay. The cultures were fed every 4 days, and the formation of colonies was scored after 3 weeks. Tumor Cell Migration and Invasion Assay—Fibronectin (10 μg/μl in phosphate-buffered saline) was added to each well of a Transwell plate (Costar; 8-μm pore size), and the membranes were allowed to dry for 1 h at 25 °C. The Transwell plates were assembled in a 24-well plate, and the lower chambers were filled with DMEM containing 10% FBS and 0.1% bovine serum albumin. Cells (5 × 104) were added to each upper chamber, and the plate was incubated at 37 °C in 5% CO2 for 3 h. The cells that had migrated to the lower surface of the filters were stained with 0.6% hematoxylin and 0.5% eosin and counted. For invasion assays, 24-well Transwell plates (Costar; 8-μm pore size) were coated with fibronectin (10 μg/μl) on the lower side of the membrane and with Matrigel (30 μg/μl) on the upper side. In Vitro FAK/Src Kinase Assays—FAK immunoprecipitates were washed twice with 1× RIPA buffer and once with 10 mm Tris buffer. The pellets were dissolved in 20 μl of kinase buffer (10 mm Tris, pH 7.4, 10 mm MnCl2, 2 mm MgCl2, 0.02% Triton X-100), and the reactions were started by adding 10 μCi of [γ-32P]ATP, 1 μm cold ATP, and GST-paxillin and incubated at 25 °C for 5 min. For the Src kinase assay, Src immunoprecipitates were dissolved in 20 μl of kinase buffer (10 mm Tris, pH 7.4, 5 mm MnCl2) and preincubated for 5 min at 25 °C. To each sample was added 2 μg of acid-denatured enolase as exogenous substrate, and the samples were incubated at 25 °C for 5 min. Cell Adhesion and Spreading Assays—Cell adhesion and spreading assays were performed on fibronectin coated tissue plates essentially as described (24Woods A. McCarthy J.B. Furcht L.T. Couchman J.R. Mol. Biol. Cell. 1993; 4: 605-613Crossref PubMed Scopus (182) Google Scholar). Briefly, fibronectin was diluted in serum-free medium, added to tissue culture plates (2 μg/cm2) and incubated at 25 °C for at least 1 h to allow its adsorption. After washing with phosphate-buffered saline, the plates were blocked by incubating them with 0.2% heat-inactivated bovine serum albumin for 1 h and then washed with serum-free medium (2 × 10 min). The cells were detached with 0.05% trypsin, 0.53 mm EDTA, suspended in serum-free medium containing 0.25 mg/ml of soybean trypsin inhibitor, harvested, resuspended in serum-free medium, plated onto fibronectin-coated plates, and incubated for various periods of time at 37 °C. GST Pull-down Assays—Three cDNA constructs encoding proline-rich domains of FAK, PR1PR2 (amino acids 711–877), PR1PR2F (amino acids 711–877, Tyr861 → Phe), and FAK PR2 (amino acids 811–877), were generated by PCR amplification. The PCR products were cloned into the BamHI/EcoRI site of the pGEX-4T-1 expression vector (Amersham Biosciences). The recombinant proteins were purified on glutathione-Sepharose 4B columns, phosphorylated in vitro with purified Src (23Lim Y. Han I. Kwon H.J. Oh E.S. J. Biol. Chem. 2002; 277: 12735-12740Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 25Eliceiri B.P. Puente X.S. Hood J.D. Stupack D.G. Schlaepfer D.D. Huang X.Z. Sheppard D. Cheresh D.A. J. Cell Biol. 2002; 157: 149-160Crossref PubMed Scopus (300) Google Scholar), and mixed with H-Ras-inducible NIH3T3 cell lysates. After incubation at 4 °C on a rotator for 2 h, the precipitated complex was eluted with SDS-PAGE sample buffer and resolved by SDS-PAGE. RNA Extraction and Reverse Transcription Polymerase Chain Reaction—Total RNA was extracted from cultured cells and used as templates for reverse transcriptase. Aliquots of cDNA were amplified using primers for MMP-2 (5′-AATACCTGAA-3′ (forward) and 5′-AAGGGGAACTTGCAGT-3′ (reverse)); MMP-9 (5′-GCTTTGCTGCCCC-3′ (forward) and 5′-GGAAAGGCGTGTGCCAG-3′ (reverse)); and rat β-actin (5′-TGGAATCCTGTGGCATCCATGAAAC-3′ (forward) and 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′ (reverse)). The amplification protocol consisted of an initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C (MMP-9 and β-actin) or 55 °C (MMP-2) for 30 s, and extension at 72 °C for 60 s. The PCR products (545 bp for MMP-2, 544 bp for MMP-9, and 349 bp for β-actin) were cloned and sequenced to confirm their identity. Gelatinase Activity—Conditioned culture media from 5 × 106 cells were subjected to nonreduced SDS-PAGE containing 1 mg/ml of gelatin. The gels were washed three times with 2.5% (v/v) Triton X-100 for 30 min each at 25 °C and three times with water for 10 min each, incubated at 37 °C overnight in 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.02% NaN3, 10 mm CaCl2, stained with Coomassie Brilliant Blue, and destained. Protease activity was visualized as a clear zone against a blue background. FAK Phosphorylation at Tyrosine 861 Is Increased in H-Ras-transformed Cells—To investigate the potential role of FAK phosphorylation in Ras-induced transformation, we assayed FAK Tyr861 phosphorylation in two different ras-transformed cells. We found that, in contrast to FAK Tyr397 phosphorylation, FAK Tyr861 phosphorylation was dramatically higher in H-Ras transformed NIH3T3 and K-ras transformed rat2 cells than in their respective untransformed cells (Fig. 1A). To further examine the correlation of FAK Tyr861 phosphorylation with transformation, NIH3T3 cells were transfected with a tetracycline-inducible H-Ras expression vector. In response to 2 μg/ml doxycyclin, these cells gradually expressed H-Ras (Fig. 1C) and exhibited a spindle-like morphology with small round cell bodies (Fig. 1B). In addition, FAK Tyr861 phosphorylation was increased in a time-dependent manner over 96 h (Fig. 1C). In contrast, when these transformed cells were removed from doxycyclin containing medium and cultured in the absence of doxycyclin, H-Ras expression was decreased, cell morphology reverted to a normal phenotype (Fig. 1, B and D), and FAK Tyr861 phosphorylation was decreased (Fig. 1D). Taken together, these data strongly suggest that increased FAK Tyr861 phosphorylation is correlated with the transformation of NIH3T3 cells. FAK Tyrosine Phosphorylation at 861 Is Required for H-Ras Transformation of NIH3T3 Cells—To investigate the role of FAK Tyr861 phosphorylation, we performed site-directed mutagenesis to replace tyrosine 861 with nonphosphorylatable phenylalanine residues (Y861F) and transfected HA-tagged Y861F into NIH3T3 cells (Fig. 2A). Compared with control cells, proliferations of wild-type FAK (wtFAK), kinase-defective FAK (kdFAK), and Y861F-transfected cells were not much altered, but cell migration was markedly increased in wtFAK-transfected cells and decreased in kdFAK- and Y861F-transfected cells (Fig. 2, B and C), implying that FAK Tyr861 phosphorylation contributes to cell transformation through the regulation of migration rather than proliferation. Consistently, H-Ras-inducible NIH3T3 cells transfected with the Y861F mutant showed decreased migration, invasion, focus forming activity and anchorage-independent growth in soft agar (Fig. 3), indicating that FAK Tyr861 phosphorylation is critical for H-Ras transformation of NIH3T3 cells.Fig. 3FAK phosphorylation at tyrosine 861 is essential for transforming activities in H-Ras-transformed NIH3T3 cells. H-Ras-inducible NIH3T3 cells transfected with vector, wtFAK, kdFAK, or Y861F mutant of FAK cDNA were treated with 2 g/ml of doxycyclin for the indicated periods of time. A, cells (5 × 104) were trypsinized and allowed to migrate on fibronectin-coated (10 μg/ml) Transwell plates for 2 h as described in the legend to Fig. 2. The percentages of cells migrating, relative to that of vector, are shown. B, cells (5 × 104) were loaded onto the upper compartments of Matrigel-coated Transwell plates and incubated for 13 h, and the number of invasive cells was counted. The percentages of invasive cells, relative to that of vector, are shown. C, cells were replated and incubated for 14 days, and transformed foci were visualized by Wright-Giemsa staining and counted. This experiment was repeated three times with nearly identical results. D, cells (1 × 105/dish) were seeded in soft agar and allowed to grow 21 days, and the number of viable colonies was counted. All of the results represent the averages of at least three independent experiments.View Large Image Figure ViewerDownload (PPT) FAK Phosphorylation at Tyrosine 861 of FAK Regulates Its Interaction with p130CAS—Because FAK-mediated signaling involves interactions with the Src family kinases, FAK Tyr861 phosphorylation may affect the activity of Src kinase. The in vitro kinase assay, however, showed that transfection of FAK Y861F mutant had no effect on the activity of Src (Fig. 4A). Similarly, FAK Tyr861 phosphorylation had no effect on the activity of FAK (Fig. 4B), making it unlikely that FAK Tyr861 phosphorylation regulates H-Ras transformation via the regulation of enzymatic activity. p130CAS has been shown to play a central role in transformation mediated by the v-src and H-Ras oncogenes (26Auvinen M. Paasinen-Sohns A. Hirai H. Andersson L. Holtta E. Mol. Cell. Biol. 1995; 15: 6513-6652Crossref PubMed Scopus (75) Google Scholar), as well as being essential for FAK-mediated (27Wei L. Yang Y. Zhang X. Yu Q. J. Cell. Biochem. 2002; 87: 439-449Crossref PubMed Scopus (53) Google Scholar) cancer cell survival and migration (28Hauck C.R. Sieg D.J. Hsia D.A. Loftus J.C. Gaarde W.A. Monia B.P. Schlaepfer D.D. Cancer Res. 2001; 61: 7079-7090PubMed Google Scholar). We found that overexpression of Y861F, but neither wtFAK nor kdFAK, caused a decreased interaction between p130CAS and FAK in NIH3T3 cells (Fig. 5A). The interaction of p130CAS with FAK was increased in H-Ras-transformed cells but decreased in cells overexpressing Y861F (Fig. 5B), supporting the importance of the interaction of p130CAS with FAK in transformation. Interestingly, the total phosphorylation of FAK was decreased in Y861F-transfected cells, but there was no detectable change in p130CAS phosphorylation (Fig. 5B).Fig. 5Mutation of tyrosine 861 to phenylalanine leads to reduced interaction with p130CAS. A, NIH3T3 cell extracts were immunoprecipitated with antibody to FAK (left panel) or p130CAS (right panel), and the amount of protein in each immunoprecipitate was determined by immunoblotting with antibodies to FAK, p130CAS, HA, and phosphotyrosine (PY). B, H-Ras-inducible NIH3T3 cell lysates, FAK immunoprecipitates, and p130CAS immunoprecipitates were immunoblotted with the indicated antibodies. The levels of FAK and p130CAS protein in each were assayed by immunoblotting with specific antibodies. C, cells were plated on fibronectin-coated plates for the indicated periods of time. FAK and p130CAS immunoprecipitates were immunoblotted with the indicated antibodies. The level of protein in each immunoprecipitate was determined by immunoblotting with antibodies to FAK and p130CAS.View Large Image Figure ViewerDownload (PPT) Because the integrin pathways are involved in the phosphorylation of FAK, the recruitment of adaptor proteins, and the subsequent activation of downstream effecter molecules, we assayed the interaction between p130CAS and FAK after plating the cells on fibronectin. Again, both the interaction of p130CAS with FAK and the phosphorylation of FAK were decreased in Y861F-transfected cells during spreading (Fig. 5C). The proline-rich domain1 (PR1) of FAK is required for its interaction with the SH3 domain of p130CAS (8Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (387) Google Scholar). Because FAK Tyr861 is located between PR1 and PR2, FAK Tyr861 phosphorylation may affect the interaction of this protein with p130CAS. We therefore performed a GST pull-down assay using three GST-FAK recombinants (PR1PR2, PR1PR2F, and PR2) and H-Ras-inducible NIH3T3 whole cell lysate. We found that in vitro phosphorylation of GST-PR1PR2 (23Lim Y. Han I. Kwon H.J. Oh E.S. J. Biol. Chem. 2002; 277: 12735-12740Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 25Eliceiri B.P. Puente X.S. Hood J.D. Stupack D.G. Schlaepfer D.D. Huang X.Z. Sheppard D. Cheresh D.A. J. Cell Biol. 2002; 157: 149-160Crossref PubMed Scopus (300) Google Scholar) caused a dramatic increase in the amount of p130CAS bound to GST-PR1PR2 (Fig. 6, compare the second and third lanes with the fifth and sixth lanes), whereas we were unable to detect p130CAS bound to in vitro phosphorylated GST-PR2. These results strongly suggest that FAK Tyr861 phosphorylation regulates the interaction of FAK with p130CAS. A Mutation in FAK Tyrosine 861 Results in Decreased JNK Activation and Matrix Metalloproteinase-9 Expression—The FAK/p130CAS complex has been observed to regulate cell transformation through JNK-mediated transcriptional regulation (29Shibata K. Kikkawa F. Nawa A. Thant A.A. Naruse K. Mizutani S. Hamaguchi M. Cancer Res. 1998; 58: 900-903PubMed Google Scholar). We found that the activation of JNK was consistently defective in H-Ras-inducible NIH3T3 transfected with the Y861F mutant (Fig. 7A). We also observed consistent decreases in the expression of MMP-9 mRNA (Fig. 7B), and gelatin zymography of cell conditioned media showed decreased expression of MMP-9 (Fig. 7C). These results confirmed that FAK Tyr861 phosphorylation regulates the interaction of FAK with p130CAS, resulting in the activation of JNK and the secretion of MMP-9. Although the relationship between FAK Tyr861 phosphorylation and transformation and/or tumorigenesis has been noted (20Slack J.K. Adams R.B. Rovin J.D. Bissonette E.A. Stoker C.E. Parsons J.T. Oncogene. 2001; 20: 1152-1163Crossref PubMed Scopus (189) Google Scholar, 21Abu-Ghazaleh R. Kabir J. Jia H. Lobo M. Zachary I. Biochem. J. 2001; 15: 255-264Crossref Google Scholar, 22Vadlamudi R.K. Sahin A.A. Adam L. Wang R.A. Kumar R. FEBS Lett. 2003; 22: 76-80Crossref Scopus (75) Google Scholar, 30Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1444) Google Scholar), the mechanism by which this phosphorylation affects cell phenotype has not been directly addressed. The results shown here indicate that FAK Tyr861 phosphorylation regulates H-Ras-induced transformation of fibroblasts by regulating the interaction between FAK and p130CAS. We observed a clear correlation between FAK Tyr861 phosphorylation and the transformed morphology and activity in these cells. The ability of FAK to integrate signals requires the integrity of tyrosine 397, a major autophosphorylation site that mediates the SH2-dependent binding of Src family kinases, which are crucial regulators of cell transformation. We therefore thought it likely that FAK Tyr861 phosphorylation might regulate cell transformation by regulating FAK phosphorylation at tyrosine 397. We found, however, that FAK Tyr861 phosphorylation did not affect FAK Tyr397 phosphorylation or the phosphorylation of the endogenous FAK substrate, paxillin. Elevated Src activity has been shown to correlate with increased FAK phosphorylation in tumor cells, and a stable complex between FAK and activated Src has been described in Src-transformed cells (31Hauck C.R. Hsia D.A. Puente X.S. Cheresh D.A. Schlaepfer D.D. EMBO J. 2002; 21: 6289-6302Crossref PubMed Scopus (158) Google Scholar), suggesting that increased Src activity/interaction could potentially contribute to transformation (4Hecker T.P. Grammer J.R. Gillespie G.Y. Stewart J. Gladson C.L. Cancer Res. 2002; 62: 2699-2707PubMed Google Scholar, 33Guan J.-L. Shalloway D. Nature. 1992; 358: 690-692Crossref PubMed Scopus (724) Google Scholar). We found, however, that Src activity was not much different in cells transfected with FAK Y861F mutant compared with those transfected with vector and that the interaction of FAK with Src was not affected by this mutation (data not shown). Taken together, these findings indicate that it is unlikely that FAK Tyr861 phosphorylation regulates cell transformation through FAK/Src complex activity. p130CAS has been considered a putative substrate of the v-Src kinase and a binding target for the SH2 domain of v-Crk during retroviral transformation (17Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (593) Google Scholar). Recently, p130CAS phosphorylation and the interaction of p130CAS with FAK have been reported as important in cell transformation (8Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (387) Google Scholar, 35Burnham M.R. Harte M.T. Richardson A. Parsons J.T. Bouton A.H. Oncogene. 1996; 12: 2467-2472PubMed Google Scholar). Our data demonstrated that the association of FAK with p130CAS was decreased in FAK Y861F-transfected cells and that, in vitro, FAK Tyr861 phosphorylation enhanced this association. In addition, the interaction of p130CAS with FAK was decreased during cell transformation in H-Ras-inducible NIH3T3 cells expressing the Y861F mutant. These results strongly suggest that FAK Tyr861 phosphorylation regulates cell transformation through regulation of the interaction between FAK and p130CAS. Interestingly, although total FAK phosphorylation was decreased in Y861F-transfected cells, there was no detectable change of p130CAS phosphorylation, which is an important event leading to cell transformation and consistent with the unchanged Src and FAK activity observed in these cells. The signal downstream of the FAK/p130CAS complex may affect ras transformation of fibroblasts in several ways. The activation of p130CAS creates binding sites for adaptor proteins such as Crk, which is critical for cell transformation (32Nievers M.G. Birge R.B. Greulich H. Verkleij A.J. Hanafusa H. van Bergen en Henegouwen P.M. J. Cell Sci. 1997; 110: 389-399PubMed Google Scholar). Rac-mediated JNK activation downstream of the p130CAS-Crk complex is essential for cell invasion (34Hsia 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 (459) Google Scholar) and acts to alter the transcriptional regulation of cell invasion-associated gene targets, including MMP-9 (29Shibata K. Kikkawa F. Nawa A. Thant A.A. Naruse K. Mizutani S. Hamaguchi M. Cancer Res. 1998; 58: 900-903PubMed Google Scholar). In addition, FAK promotes cell motility and focal contact remodeling events, in part through the regulation of MMP-9 secretion (28Hauck C.R. Sieg D.J. Hsia D.A. Loftus J.C. Gaarde W.A. Monia B.P. Schlaepfer D.D. Cancer Res. 2001; 61: 7079-7090PubMed Google Scholar). Our data demonstrated that both the activation of JNK and the secretion of MMP-9 were defective in the FAK Y861F mutant. These findings strongly support the notion that FAK Tyr861 phosphorylation is crucial for FAK/p130CAS-mediated cell transformation. Because in vitro phosphorylated tyrosine 861 clearly enhanced the interaction of FAK with p130CAS, it is likely that FAK Tyr861 phosphorylation regulates this interaction. This result was somewhat unexpected, because this interaction is through the SH3 domain of p130CAS and the PR1 domain of FAK, in which tyrosine 861 is not present. It is possible, however, that FAK Tyr861 phosphorylation serves to stabilize pre-existing interactions by creating an additional SH2-binding site. We are currently attempting to identify another adaptor molecule that contains an SH2 domain and binds to FAK in a Tyr861 phosphorylation-dependent manner.
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