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• W2053887013 abstract "Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCα, PKCδ, or PKCε was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc. Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCα, PKCδ, or PKCε was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc. mitogen-activated protein kinase protein kinase C 12-0-tetradecanoyl-phorbol-13-acetate extracellular regulated kinase 2 mitogen-activated kinase kinase focal adhesion kinase phosphatidylinositol 3-kinase phospholipase C epidermal growth factor phosphotyrosine phosphatase phosphate-buffered saline Dulbecco's modified Eagle's medium Ras-binding domain hemagglutinin immunoprecipitation(s) GTPase-activating protein Adhesion of cells to extracellular matrices through integrin transmembrane receptors initiates the assembly of an actin cytoskeletal complex at the inner surface of the membrane that is required for filopodia, lamellipodia, focal adhesion, and stress fiber formation. Multiple intracellular signaling molecules are stimulated following integrin-dependent adhesion, some of which require assembly of these actin complexes for activation. Integrin targeted signaling molecules include members of the mitogen-activated protein kinase (MAPK)1 signaling pathways, Rho family GTPases, nonreceptor tyrosine kinases such as focal adhesion kinase (FAK) and Src, and members of the lipid signaling pathways such as phosphatidylinositol 3-kinase (PI 3-K), and protein kinase C (PKC) (reviewed in Refs. 1Schlaepfer D.D. Hunter T. Trends Cell Biol. 1998; 8: 151-157Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar and 2Aplin A.E. Howe A. Alahari S.K. Juliano R.L. Pharmacol Rev. 1998; 50: 197-263PubMed Google Scholar). How ligand binding to integrins activates these signaling events and how activation of the different molecules mediates integrin functions is still poorly understood. The protein kinase C family of serine/threonine kinases can be classified into three major subgroups (3Blobe G.C. Stribling S. Obeid L.M. Hannun Y.A. Cancer Surveys. 1996; 27: 213-248PubMed Google Scholar). The classical PKCs consist of PKCα, βI, βII, and γ, which are Ca2+/lipid-dependent kinases. The novel PKCs, PKCδ, ε, η, and θ are Ca2+-independent but require lipid for activation. The third class, atypical PKCs, consists of PKCζ and ι/λ, which are neither Ca2+- nor lipid-dependent. Finally, PKCμ, although similar to novel PKCs, contains a membrane-spanning domain and is often placed in a separate class. PKCs are activated in cells following stimulation with a wide variety of agonists, including growth factors, antigens, cytokines, and neurotransmitters. In addition, phorbol esters such as 12-0-tetradecanoylphorbol 13-acetate (TPA) are direct stimulators of classical and novel PKCs. Acute treatment with TPA causes translocation of classical and novel PKCs from the cytosol to the membrane, an event that is necessary for activation of at least some PKC members. However, prolonged treatment with TPA results in degradation and loss of expression of some TPA-responsive PKCs. PKC has been shown to play an important role in cell-regulated events such as secretion, differentiation, tumorigenesis, mitogenesis, signal transduction (3Blobe G.C. Stribling S. Obeid L.M. Hannun Y.A. Cancer Surveys. 1996; 27: 213-248PubMed Google Scholar), intracellular transport (4Ktistakis N.T. Bioessays. 1998; 20: 495-504Crossref PubMed Scopus (26) Google Scholar), gene expression (5Smithgall T.E. Pharmacol. Rev. 1998; 50: 1-19PubMed Google Scholar), and cytoskeletal regulation (6Keenan C. Kelleher D. Cell Signal. 1998; 10: 225-232Crossref PubMed Scopus (165) Google Scholar). Several in vivo PKC targets have been identified; the best characterized being the 80-kDa phosphoprotein MARCKS (7Blackshear P.J. J. Biol. Chem. 1993; 268: 1501-1504Abstract Full Text PDF PubMed Google Scholar). In addition to MARCKS, other cytoskeletal proteins such as pleckstrin, talin, vinculin, annexins, α- and γ-adducin, Src suppressed protein kinase C substrate, and paxillin are potential PKC substrates in vivo (6Keenan C. Kelleher D. Cell Signal. 1998; 10: 225-232Crossref PubMed Scopus (165) Google Scholar,8Hyatt S.L. Liao L. Chapline C. Jaken S. Biochemistry. 1994; 33: 1223-1228Crossref PubMed Scopus (80) Google Scholar, 9De Nichilo M.O. Yamada K.M. J. Biol. Chem. 1996; 271: 11016-11022Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 10Matsuoka Y. Li X. Bennett V. J. Cell Biol. 1998; 142: 485-497Crossref PubMed Scopus (173) Google Scholar, 11Lin X. Tombler E. Nelson P.J. Ross M. Gelman I.H. J. Biol. Chem. 1996; 271: 28430-28438Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 12Chapline C. Cottom J. Tobin H. Hulmes J. Crabb J. Jaken S. J. Biol. Chem. 1998; 273: 19482-19489Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Possible in vivo PKC targets involved in receptor-induced signaling events include the serine/threonine kinase Raf-1 (13Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1159) Google Scholar, 14Cai H. Smola U. Wixler V. Eisenmann-Tappe I. Diaz-Meco M.T. Moscat J. Rapp U. Cooper G.M. Mol. Cell. Biol. 1997; 17: 732-741Crossref PubMed Scopus (263) Google Scholar), tyrosine phosphatase SHP1 (15Brumell J.H. Chan C.K. Butler J. Borregaard N. Siminovitch K.A. Grinstein S. Downey G.P. J. Biol. Chem. 1997; 272: 875-882Crossref PubMed Scopus (59) Google Scholar), Gγ12subunit (16Asano T. Morishita R. Ueda H. Asano M. Kato K. Eur. J. Biochem. 1998; 251: 314-319Crossref PubMed Scopus (13) Google Scholar), and the EGF and insulin receptors (17Chen P. Xie H. Wells A. Mol. Biol. Cell. 1996; 7: 871-881Crossref PubMed Scopus (100) Google Scholar, 18Berti L. Mosthaf L. Kroder G. Kellerer M. Tippmer S. Mushack J. Seffer E. Seedorf K. Haring H. J. Biol. Chem. 1994; 269: 3381-3386Abstract Full Text PDF PubMed Google Scholar). Several lines of evidence indicate that PKC may be important in integrin-mediated adhesion and signaling events. First, treatment of many different cell types with TPA causes increased adhesion, spreading, and migration of cells on extracellular matrices (19Huang X. Wu J. Spong S. Sheppard D. J. Cell Sci. 1998; 111: 2189-2195Crossref PubMed Google Scholar, 20Defilippi P. Venturino M. Gulino D. Duperray A. Boquet P. Fiorentini C. Volpe G. Palmieri M. Silengo L. Tarone G. J. Biol. Chem. 1997; 272: 21726-21734Crossref PubMed Scopus (84) Google Scholar). Conversely, inhibition of PKC with pharmacological agents blocks cell adhesion and cell spreading (21Chun J. Jacobson B.S. Mol. Biol. Cell. 1993; 4: 271-281Crossref PubMed Scopus (67) Google Scholar, 22Lewis J.M. Cheresh D.A. Schwartz M.A. J. Cell Biol. 1996; 134: 1323-1332Crossref PubMed Scopus (177) Google Scholar) and have been reported to inhibit cell migration (23Liao L. Jaken S. Cell Growth Differ. 1993; 4: 309-316PubMed Google Scholar), FAK phosphorylation (24Haimovich B. Kaneshiki N. Ji P. Blood. 1996; 87: 152-161Crossref PubMed Google Scholar, 25Vuori K. Ruoslahti E. J. Biol. Chem. 1993; 268: 21459-21462Abstract Full Text PDF PubMed Google Scholar), and focal adhesion formation (26Woods A. Couchman J.R. J. Cell Sci. 1992; 101: 277-290Crossref PubMed Google Scholar). Second, several PKC isoforms have been implicated in adhesion-dependent events. PKCα and PKCδ are associated with focal adhesions (27Jaken S. Leach K. Klauck T. J. Cell Biol. 1989; 109: 697-704Crossref PubMed Scopus (253) Google Scholar, 28Barry S.T. Critchley D.R. J. Cell Sci. 1994; 107: 2033-2045Crossref PubMed Google Scholar), and PKCα and PKCε translocate to the membrane following integrin activation (29Haller H. Lindschau C. Maasch C. Olthoff H. Kurscheid D. Luft F.C. Circ. Res. 1998; 82: 157-165Crossref PubMed Scopus (76) Google Scholar, 30Chun J. Ha M. Jacobson B.S. J. Biol. Chem. 1996; 271: 13008-13012Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Third, integrin engagement leads to increased phospholipase C (PLC) activity, increased diacylglycerol levels, and arachidonic acid production, pathways involved in PKC activation (21Chun J. Jacobson B.S. Mol. Biol. Cell. 1993; 4: 271-281Crossref PubMed Scopus (67) Google Scholar, 29Haller H. Lindschau C. Maasch C. Olthoff H. Kurscheid D. Luft F.C. Circ. Res. 1998; 82: 157-165Crossref PubMed Scopus (76) Google Scholar, 31Banno Y. Nakashima S. Ohzawa M. Nozawa Y. J. Biol. Chem. 1996; 271: 14989-14994Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 32Cybulsky A.V. Carbonetto S. Cyr M. McTavish A.J. Huang Q. Am. J. Physiol. 1993; 264: C323-C332Crossref PubMed Google Scholar). Although the specific PKC isoforms involved in integrin-mediated events are beginning to be defined, how PKCs regulate integrin-induced signaling events and what their targets are have not been fully explored. To characterize the role of PKC in integrin-dependent signaling, we monitored the effects of inhibiting PKC on fibronectin-induced signaling events in Cos7 cells using pharmacological and molecular genetic approaches. Down-regulating classical and novel PKC isoforms with TPA or over-expression of dominant-negative mutants of PKC in Cos7 cells did not block integrin-induced FAK or paxillin tyrosine phosphorylation; however, fibronectin-induced Erk2 activation was significantly reduced. Inhibition of PKC also greatly reduced fibronectin-induced MEK1, Raf-1, and Ras activation as well as Shc tyrosine phosphorylation and Grb2 association. These results indicate that fibronectin-induced PKC activation plays a role in modulating the MAPK pathway by regulating early events upstream of Shc. Cos7 cells were maintained in DMEM supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 50 units penicillin, 50 μg/ml streptomycin, and 2 mm glutamine. For adhesion assays, tissue culture plates were coated with 5 μg/ml fibronectin (Collaborative Biomedical) in PBS overnight at 4 °C and blocked with 1% bovine serum albumin prior to use. Near confluent plates of cells were serum starved overnight in DMEM containing 0.1% fetal bovine serum. Cells were washed with PBS and trypsinized in 0.01% trypsin containing 5 mm EDTA for 5 min at 37 °C. Trypsinization was terminated with 1 mg/ml soybean trypsin inhibitor (Life Technologies, Inc.) in PBS containing 5 mm EDTA. Cells were collected, washed in PBS/5 mm EDTA, resuspended in serum free DMEM, and held in suspension at 37 °C for at least 30 min. Cells were either left in suspension or placed on fibronectin-coated plates at a density of 3–4 × 106 cells/10-cm plate for various times. TPA (Calbiochem) at 25–100 ng/ml was added to plates 18–24 h before plating on fibronectin. Suspension cells were lysed in 2× lysis buffer as defined below and diluted with 1× buffer when necessary. Adherent cells were washed once with DMEM prior to lysis to remove nonadherent cells and lysed in 1× lysis buffer on the plate. Monoclonal antibodies for immunoblotting PKC isoforms were purchased from Transduction Laboratories, except PKCγ, PKCβI, and PKCβII isoform-specific polyclonal antibodies, which were from Santa Cruz. Polyclonal antibodies against Grb2, FAK, PKCδ, MAPK, MEK1, Raf-1, and Ras used for immunoprecipitation assays were purchased from Santa Cruz. Polyclonal Shc and Ras antibodies and monoclonal MAPK, FAK, and paxillin antibodies were purchased from Transduction Laboratories. Monoclonal PLCγ-1 antibody was purchased from Upstate Biochemical. HA tag antibody was generated in cell culture from 12CA5 hybridoma cells (33Wadzinski B.E. Eisfelder B.J. Peruski L.F. Mumby M.C. Johnson G.L. J. Biol. Chem. 1992; 267: 16883-16888Abstract Full Text PDF PubMed Google Scholar), which were kindly provided by Dr. Jeff Settleman (Massachusetts General Hospital, Charlestown, MA). Anti-phosphotyrosine 4G10 antibody was provided by Dr. Tom Roberts (Dana Farber Cancer Institute, Boston, MA). The pcDNA3-Flag-Raf-1 construct was generated by subcloning as described previously (34King W.G. Mattaliano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (387) Google Scholar). pSVL-HA-Erk2 was a gift from Mike Weber (University of Charlottesville, VA). pcDNA3-HA-FAK was provided by Tony Hunter (Scripps, San Diego, CA). pSRD plasmids containing wild type PKCε and kinase dead PKCε (K−) have been described previously (35Ueda Y. Hirai S. Osada S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). The regulatory domain truncation mutant of PKCδ (pSRD-PKCδ RD) encodes the N-terminal region of mouse PKCδ from amino acid residues 1–298 connected to C-terminal amino acids 655–674. The regulatory domain truncation mutant PKCε (pSRDHis-PKCε RD) encodes the N-terminal region of rabbit PKCε from amino acid residues 1–385 and is tagged with His6/T710 tag at the N terminus. The kinase domain truncation mutant of mouse PKCδ (pSRD-PKCδ KD) encodes amino acid residues 348–674 and is preceded by Met. The kinase domain truncation mutant of rabbit PKCε (pSRDHis-PKCε KD) encodes amino acid residues 386–736 preceded by the His6/T710 tag. The kinase domain truncation mutant of rabbit PKCα (pSRD-PKCα KD) encodes amino acid residues 298–672. Cos7 cells were transfected at 2 × 106 cells/10-cm plate with 8–10 μg of total DNA using the LipofectAMINE procedure as described by the manufacturer (Life Technologies, Inc.). 48 h after transfection, cells were used in adhesion assays as described above. Erk2, MEK1, and Raf-1 kinase assays were performed as described previously (36Reuter C.W. Catling A.D. Weber M.J. Methods Enzymol. 1995; 255: 245-256Crossref PubMed Scopus (47) Google Scholar, 37Alessi D.R. Cohen P. Ashworth A. Cowley S. Leevers S.J. Marshall C.J. Methods Enzymol. 1995; 255: 279-290Crossref PubMed Scopus (155) Google Scholar). Extracts were clarified by centrifugation at 13,000 × g for 10 min, and protein concentrations were determined using Coomassie Blue Reagent (Bio-Rad). Erk2, MEK1, and Raf-1 substrates were 0.25 mg/ml myelin basic protein (Life Technologies, Inc.), 30 μg/ml kinase dead GST-MAPK (Upstate Biochemical), and 12.5 μg/ml kinase dead GST-MEK1 (Upstate Biochemical), respectively. Reactions were incubated at 30 °C for 15–30 min and terminated with 40 μl of 2× SDS Sample buffer by heating to 95 °C for 5 min and analyzed by SDS-polyacrylamide gel electrophoresis. Gels were transferred to polyvinylidene difluoride (Bio-Rad), and after autoradiography and quantitation by phosphoimaging (Fuji) blots were subjected to immunoblotting for the respective kinases. Cells adherent to fibronectin or left in suspension were lysed in RIPA buffer (10 mm Tris, pH 7.2, 158 mm NaCl, 1 mmEDTA, 0.1% SDS, 1% sodium deoxycholate, 1% Triton-X, 1 mm Na3VO4, 1 mmphenylmethylsulfonyl fluoride, 100 units/ml aprotinin, and 10 μg/ml leupeptin), passed through a 25ga needle, and clarified by centrifugation at 13,000 × g for 10 min. Protein concentrations were determined using the BCA assay (Pierce). Immunoprecipitations (IP) were incubated for 2–4 h at 4 °C with protein A-conjugated agarose beads (Pierce) to capture the complexes. All IP were washed three times with RIPA, resuspended in 2× SDS sample buffer, boiled, and analyzed by SDS-polyacrylamide gel electrophoresis. Gels were transferred to a polyvinylidene difluoride membrane and probed by immunoblotting. After blocking in 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 and incubating with primary antibody, blots were incubated with horseradish peroxidase-conjugated secondary antibody and visualized with chemiluminescence reagent (NEN Life Science Products). Blots were stripped in 2% SDS at 65 °C for 30 min, rinsed extensively, and reprobed as indicated in the figure legends. Cells were lysed in hypotonic cytosolic buffer (10 mm Tris, pH 7.4, 0.5 mm EDTA, 1 mm Na3VO4, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 100 units/ml aprotinin) for 20 min on ice after washing with PBS. Cells were collected and broken open by Dounce homogenization. The soluble cytosolic fraction (S30) was collected after centrifugation at 30,000 × g for 30 min. The pellets (P30) were further fractionated into membrane and Triton X-100-insoluble fractions by solubilizing in cytosolic buffer containing 1% Triton X-100. The soluble membrane fraction (P30Mem) was recovered after centrifugation at 30,000 × g for 30 min. The Triton X-100-insoluble pellets were resuspended in RIPA, and supernatants (P30Ins) were collected after centrifugation at 14,000 × g. The ratio of total cytosolic protein:membrane protein:Triton-insoluble was calculated, and that ratio was maintained when extracts were loaded onto SDS gels. Both soluble and insoluble membrane extracts were pooled for immunoprecipitation of PKCδ from membrane fractions. The amount of GTP bound to Ras was measured according to the protocol of Vaillancourt et al.(38Vaillancourt R.R. Harwood A.E. Winitz S. Methods Enzymol. 1994; 238: 255-258Crossref PubMed Scopus (15) Google Scholar), which was modified as described by Zheng et al. (39Zheng L. Sjolander A. Eckerdal J. Andersson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8431-8436Crossref PubMed Scopus (68) Google Scholar) and Clark and Hynes (40Clark E.A. Hynes R.O. J. Biol. Chem. 1996; 271: 14814-14818Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Briefly, confluent, serum starved cells were washed with phosphate-free DMEM and incubated with 0.25 mCi of32PO4/plate for 4–5 h. Cells were washed, trypsinized, and placed in suspension as described above, except that TBS was substituted for PBS. Suspended cells were incubated in phosphate-free DMEM and 1 mCi/ml 32PO4 for 1 h at 37 °C. Cells were plated onto fibronectin coated plates for 20–30 min, until cells were adherent and beginning to spread. Plated cells were washed with PBS, lysed, processed, and immunoprecipitated as described (39Zheng L. Sjolander A. Eckerdal J. Andersson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8431-8436Crossref PubMed Scopus (68) Google Scholar). GTP/GDP was eluted from the Ras immunoprecipitates with 25 μl 0.75 mKH2PO4 (pH 3.4) at 68 °C for 10 min. TLC was carried out on polyethyleneimine-cellulose plates. GTP and GDP unlabeled standards were run in parallel and visualized under UV light. The percentage of GTP bound relative to the ratio of GTP/GDP was quantitated on a Fuji phosphoimaging system. The ability of GTP-Ras to bind the effector region in Raf-1 was monitored as described previously (41Taylor S.J. Shalloway D. Curr. Biol. 1996; 6: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). As a first step toward defining the role of PKC in fibronectin-induced signaling in Cos7 cells, we examined which PKC isoforms are expressed in Cos7 cells. Immunoblot analysis of Cos7 cell extracts with antibodies specific for eleven different PKC isoforms revealed that Cos7 cells express eight PKC isoforms (Fig. 1 A): classical PKCα, PKCβI, and PKCβII, novel PKCδ and PKCε, atypical PKCζ and PKCλ as well as PKCμ. Prolonged treatment of cells with TPA results in degradation and loss of expression of TPA-responsive PKCs, effectively resulting in a cell that is null for those PKCs. Of the PKC isoforms in Cos7 cells, only PKCα, β, δ, and ε should be affected by prolonged TPA exposure. Treatment of Cos7 cells for 24 h with 100 ng/ml TPA eliminated PKCα, βI, and δ but only reduced PKCε by 70% as monitored by immunoblotting of whole cell lysates (Fig. 1 B). Higher doses or longer TPA treatment did not further reduce PKCε levels (data not shown). As expected, atypical PKC isoforms and PKCμ were unaffected by long term TPA treatment. Plating cells on fibronectin did not change the level of PKC expression either before or after TPA-mediated down-regulation (Fig. 1 B). The best characterized mechanism leading to in vivo activation of classical and novel PKCs following receptor stimulation involves an increase in intracellular diacylglycerol levels, which is mediated by PLC isozymes (42Nakamura S. Nishizuka Y. J. Biochem. (Tokyo). 1994; 115: 1029-1034Crossref PubMed Scopus (138) Google Scholar). The observations that PLCs translocate to integrin complexes and that adhesion of epithelial cells to collagen induces PLCγ activation through β1 integrin indicate that activation of PLC may be important for integrin signaling events (31Banno Y. Nakashima S. Ohzawa M. Nozawa Y. J. Biol. Chem. 1996; 271: 14989-14994Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 32Cybulsky A.V. Carbonetto S. Cyr M. McTavish A.J. Huang Q. Am. J. Physiol. 1993; 264: C323-C332Crossref PubMed Google Scholar, 43Plopper G.E. McNamee H.P. Dike L.E. Bojanowski K. Ingber D.E. Mol. Biol. Cell. 1995; 6: 1349-1365Crossref PubMed Scopus (472) Google Scholar). To determine whether PLCγ can be activated by fibronectin in Cos7 cells, PLCγ1 was immunoprecipitated from cell extracts following adhesion of Cos7 cells to fibronectin, and the levels of tyrosine phosphorylation were monitored by immunoblotting with anti-phosphotyrosine antibody, because tyrosine phosphorylation of PLCγ1 activates it. Plating cells on fibronectin resulted in a rapid increase in tyrosine phosphorylation of PLCγ1 (Fig. 2 A). No tyrosine phosphorylation of PLCγ1 was detected in suspension cells. Thus adhesion of Cos7 cells to fibronectin activates PLCγ1, which could lead to activation of PKC through diacylglycerol production. TPA-responsive PKCs translocate from the cytosolic fraction into detergent-soluble membrane fractions during activation. To determine whether adhesion of Cos7 cells to fibronectin causes an increase in membrane-associated PKCs, cells were subjected to biochemical fractionation as outlined under “Experimental Procedures.” Cells were fractionated into cytosolic (S30) and membrane fractions (P30). The membrane fraction was further fractionated into Triton X-100-soluble (P30Mem) and Triton X-100-insoluble (P30Ins) fractions, and each was analyzed by immunoblotting with different PKC isoform antibodies. Adhesion to fibronectin resulted in translocation of a small fraction of PKCα and PKCβI to the soluble membrane fraction, both of which were predominately cytosolic in suspension cells (Fig.2 B). A corresponding increase in soluble membrane-associated PKC kinase activity was also observed (data not shown). Unlike PKCβI, PKCβII was found to be primarily in the P30 pellet and fractionated equally between the soluble membrane and detergent-insoluble fraction in suspension cells. This distribution did not change following adhesion to fibronectin (Fig. 2 B). Approximately a third of total PKCε was associated with the P30 pellet in suspension cells. Following adhesion to fibronectin a small but reproducible increase in the amount of PKCε in the soluble membrane fraction (P30Mem) occurred concurrently with a reduction in mobility (Fig. 2 B). Like PKCε, approximately a third of the total PKCδ was found in the P30 pellet, but PKCδ distribution did not change following adhesion. Growth factors and TPA have been shown to induce tyrosine phosphorylation of PKCδ (44Li W. Mischak H. Yu J. Wang L. Mushinski J.F. Heidaran M.A. Pierce J.H. J. Biol. Chem. 1994; 269: 2349-2352Abstract Full Text PDF PubMed Google Scholar, 45Li W. Yu J. Michieli P. Beeler J.F. Ellmore N. Heidaran M.A. Pierce J.H. Mol. Cell. Biol. 1994; 14: 6727-6735Crossref PubMed Google Scholar). Adhesion of Cos7 cells to fibronectin also resulted in inducible tyrosine phosphorylation of membrane-associated PKCδ, as seen by immunoprecipitation of PKCδ and immunoblotting with anti-phosphotyrosine antibody (Fig.2 C). Thus adhesion of Cos7 cells to fibronectin induces activation of PLCγ1, membrane association of PKCα, βI, and ε, and tyrosine phosphorylation of membrane-associated PKCδ. TPA-induced PKC down-regulation did not adversely affect the ability of Cos7 cells to adhere to fibronectin (Fig.3 A). However, prolonged TPA treatment did result in delayed spreading on fibronectin (Fig.3 B). Spreading in untreated cells could be seen as early as 10 min after plating on fibronectin (60%), but TPA-treated cells were poorly spread at 10 min (5%). By 45 min TPA-treated cells had reached the same level of spreading as untreated cells at 10 min (58%). At 60 min after plating only 64% of the TPA-treated cells had spread compared with 90% for untreated cells. Thus inhibition of PKC function in Cos7 cells by long term TPA treatment affects cell spreading but not cell adhesion. We next investigated which integrin-induced downstream signaling events are affected by loss of novel or classical PKC isoform function. Adhesion to extracellular matrices induces tyrosine phosphorylation of two focal adhesion-associated proteins, FAK and paxillin (46Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1182) Google Scholar). To determine whether inhibition of PKC affects fibronectin-induced FAK or paxillin phosphorylation, FAK and paxillin were immunoprecipitated from cells plated on fibronectin for various times and probed by immunoblotting with anti-phosphotyrosine antibody. Adhesion to fibronectin induced robust tyrosine phosphorylation of both FAK and paxillin (Fig.4 A, P-tyr blot). TPA-induced PKC down-regulation did not block fibronectin-induced FAK or paxillin phosphorylation, although it did reduce tyrosine phosphorylation at very early (10 min) stages of cell adhesion. Previous studies demonstrated that inhibition of PKC blocked integrin-induced FAK phosphorylation (22Lewis J.M. Cheresh D.A. Schwartz M.A. J. Cell Biol. 1996; 134: 1323-1332Crossref PubMed Scopus (177) Google Scholar, 25Vuori K. Ruoslahti E. J. Biol. Chem. 1993; 268: 21459-21462Abstract Full Text PDF PubMed Google Scholar). Because TPA-induced PKC down-regulation did not completely inhibit PKCε expression, we examined the effect of inhibiting PKCε on integrin-induced FAK phosphorylation. Either of the two dominant-negative mutants of PKCε (PKCε K− or PKCε RD) was co-expressed with HA-tagged FAK, and the ability of fibronectin to induce FAK tyrosine phosphorylation was monitored. The PKCε mutants were expressed at levels greater than 10-fold over endogenous levels (Fig. 4 B,PKC blot) as determined by immunoblotting of whole cell extracts with PKCε antibody. The kinase inactive PKCε mutant (PKCε K−) and the regulatory domain of PKCε (PKCε RD) have previously been shown to act as a dominant-negative mutants (14Cai H. Smola U. Wixler V. Eisenmann-Tappe I. Diaz-Meco M.T. Moscat J. Rapp U. Cooper G.M. Mo" @default.
• W2053887013 created "2016-06-24" @default.
• W2053887013 creator A5039815523 @default.
• W2053887013 creator A5043845770 @default.
• W2053887013 creator A5067673954 @default.
• W2053887013 date "1999-04-01" @default.
• W2053887013 modified "2023-09-30" @default.
• W2053887013 title "Protein Kinase C Regulates Integrin-induced Activation of the Extracellular Regulated Kinase Pathway Upstream of Shc" @default.
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