Bombesin, Lysophosphatidic Acid, and Epidermal Growth Factor Rapidly Stimulate Focal Adhesion Kinase Phosphorylation at Ser-910
2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês
10.1074/jbc.m210876200
ISSN1083-351X
AutoresIsabel Hunger‐Glaser, Eduardo Pérez Salazar, James Sinnett‐Smith, Enrique Rozengurt,
Tópico(s)Cellular Mechanics and Interactions
ResumoA rapid increase in the tyrosine phosphorylation of focal adhesion kinase (FAK) has been extensively documented in cells stimulated by multiple signaling molecules, but virtually nothing is known about the regulation of FAK phosphorylation at serine residues. Stimulation of Swiss 3T3 cells with bombesin promoted a striking increase (∼13-fold) in the phosphorylation of FAK at Ser-910, as revealed by site-specific antibodies that recognized the phosphorylated state of this residue. Lysophosphatidic acid and epidermal growth factor (EGF) also stimulated FAK phosphorylation at Ser-910. Direct activation of protein kinase C isoforms with phorbol-12,13-dibutyrate (PDB) also promoted striking phosphorylation of FAK at Ser-910. Treatment with the protein kinase C inhibitor GF I or Ro 31-8220 or chronic exposure to PDB prevented the increase in FAK phosphorylation at Ser-910 induced by bombesin or PDB but not by EGF. Treatment with the ERK inhibitors U0126 and PD98059 prevented FAK phosphorylation at Ser-910 in response to all of the stimuli tested. Furthermore, incubation of activated ERK2 with FAK immunocomplexes leads to FAK phosphorylation at Ser-910 in vitro. Our results demonstrate, for the first time, that stimulation with bombesin, lysophosphatidic acid, PDB, or EGF induces phosphorylation of endogenous FAK at Ser-910 via an ERK-dependent pathway in Swiss 3T3 cells. A rapid increase in the tyrosine phosphorylation of focal adhesion kinase (FAK) has been extensively documented in cells stimulated by multiple signaling molecules, but virtually nothing is known about the regulation of FAK phosphorylation at serine residues. Stimulation of Swiss 3T3 cells with bombesin promoted a striking increase (∼13-fold) in the phosphorylation of FAK at Ser-910, as revealed by site-specific antibodies that recognized the phosphorylated state of this residue. Lysophosphatidic acid and epidermal growth factor (EGF) also stimulated FAK phosphorylation at Ser-910. Direct activation of protein kinase C isoforms with phorbol-12,13-dibutyrate (PDB) also promoted striking phosphorylation of FAK at Ser-910. Treatment with the protein kinase C inhibitor GF I or Ro 31-8220 or chronic exposure to PDB prevented the increase in FAK phosphorylation at Ser-910 induced by bombesin or PDB but not by EGF. Treatment with the ERK inhibitors U0126 and PD98059 prevented FAK phosphorylation at Ser-910 in response to all of the stimuli tested. Furthermore, incubation of activated ERK2 with FAK immunocomplexes leads to FAK phosphorylation at Ser-910 in vitro. Our results demonstrate, for the first time, that stimulation with bombesin, lysophosphatidic acid, PDB, or EGF induces phosphorylation of endogenous FAK at Ser-910 via an ERK-dependent pathway in Swiss 3T3 cells. A rapid increase in the tyrosine phosphorylation of the nonreceptor tyrosine kinase FAK 1The abbreviations used are: FAK, focal adhesion kinase; GPCR, G protein-coupled receptor; DMEM, Dulbecco's modified Eagle's medium; LPA, lysophosphatidic acid; Ab, antibody; PDB, phorbol 12,13-dibutyrate; EGF, epidermal growth factor; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PTx, pertussis toxin; FAT, focal adhesion targeting; RIPA, radioimmune precipitation; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; FRNK, FAK-related nonkinase; GST, glutathione S-transferase. (1Schaller M.D. Borgman C.A. Cobb B.S. Vines R.R. Reynolds A.B. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5192-5196Crossref PubMed Scopus (1295) Google Scholar, 2Hanks S.K. Calalb M.B. Harper M.C. Patel S.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8487-8491Crossref PubMed Scopus (731) Google Scholar), which localizes to focal adhesion plaques (3Parsons J.T. J. Cell Sci. 2003; 116: 1409-1416Crossref PubMed Scopus (1150) Google Scholar), has been identified as a prominent early event in cells stimulated by diverse signaling molecules that regulate cell proliferation, migration, and apoptosis (4Hanks S.K. Polte T.R. Bioessays. 1997; 19: 137-145Crossref PubMed Scopus (440) Google Scholar, 5Rozengurt E. Am. J. Physiol. 1998; 275: G177-G182PubMed Google Scholar). Specifically, FAK is phosphorylated at multiple sites, including tyrosines 397, 576, 577, 861, and 925, in cells stimulated by mitogenic agonists that act via heptahelical GPCRs including bombesin (6Zachary I. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1992; 267: 19031-19034Abstract Full Text PDF PubMed Google Scholar, 7Sinnett-Smith J. Zachary I. Valverde A.M. Rozengurt E. J. Biol. 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Fry D.W. Hanks S.K. Mol. Cell. Biol. 1999; 19: 4806-4818Crossref PubMed Scopus (343) Google Scholar, 28Ruest P.J. Roy S. Shi E.G. Mernaugh R.L. Hanks S.K. Cell Growth Differ. 2000; 11: 41-48PubMed Google Scholar); bacterial toxins (29Lacerda H.M. Lax A.J. Rozengurt E. J. Biol. Chem. 1996; 271: 439-445Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 30Lacerda H.M. Pullinger G.D. Lax A.J. Rozengurt E. J. Biol. Chem. 1997; 272: 9587-9596Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar); and activated variants of pp60src (31Guan J.L. Shalloway D. Nature. 1992; 358: 690-692Crossref PubMed Scopus (726) Google Scholar, 32Parsons J.T. Parsons S.J. Curr. Opin. Cell Biol. 1997; 9: 187-192Crossref PubMed Scopus (355) Google Scholar). It is increasingly recognized that FAK functions as a point of convergence and integration in the action of multiple signals (33Zachary I. Rozengurt E. Cell. 1992; 71: 891-894Abstract Full Text PDF PubMed Scopus (387) Google Scholar, 34Rozengurt E. Cancer Surv. 1995; 24: 81-96PubMed Google Scholar). FAK promotes the transmission of downstream signaling by binding and recruiting signaling and adaptor proteins (3Parsons J.T. J. Cell Sci. 2003; 116: 1409-1416Crossref PubMed Scopus (1150) Google Scholar). Autophosphorylation of FAK at Tyr-397, located N-terminal to the catalytic domain, creates a binding site for the tyrosine kinase Src (35Schaller M.D. Hildebrand J.D. Parsons J.T. Mol. Biol. Cell. 1999; 10: 3489-3505Crossref PubMed Scopus (183) Google Scholar) and other downstream signaling effectors. Subsequent Src-mediated phosphorylation of FAK at Tyr-576 and Tyr-577 is important for the maximal activation of FAK and down-stream signaling events (14Salazar E.P. Rozengurt E. J. Biol. Chem. 2001; 276: 17788-17795Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 27Owen J.D. Ruest P.J. Fry D.W. Hanks S.K. Mol. Cell. Biol. 1999; 19: 4806-4818Crossref PubMed Scopus (343) Google Scholar). Phosphorylation at Tyr-925 within the focal adhesion targeting (FAT) domain creates a binding site for the Src homology 2 domain of the adapter protein Grb2-SOS (Ras exchange factor) complex and provides a possible mechanism of activation of the Ras/Raf/MEK/ERK pathway (36Schlaepfer D.D. Hauck C.R. Sieg D.J. Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1036) Google Scholar). The FAT domain binds paxillin and talin, which are responsible for targeting FAK to the focal adhesions and for promoting downstream signaling (36Schlaepfer D.D. Hauck C.R. Sieg D.J. Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1036) Google Scholar, 37Tachibana K. Sato T. D'Avirro N. Morimoto C. J. Exp. Med. 1995; 182: 1089-1099Crossref PubMed Scopus (239) Google Scholar, 38Chen H.-C. Appeddu P.A. Parsons J.T. Hildebrand J.D. Schaller M.D. Guan J.-L. J. Biol. 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In addition to being phosphorylated at multiple tyrosines in response to external stimuli, FAK is also phosphorylated at serine residues (48Yamakita Y. Totsukawa G. Yamashiro S. Fry D. Zhang X. Hanks S.K. Matsumura F. J. Cell Biol. 1999; 144: 315-324Crossref PubMed Scopus (106) Google Scholar, 49Ma A. Richardson A. Schaefer E.M. Parsons J.T. Mol. Biol. Cell. 2001; 12: 1-12Crossref PubMed Scopus (77) Google Scholar). Recently, Ser-722 and Ser-910, in the COOH-terminal, noncatalytic region of FAK (termed FAK-related nonkinase (FRNK)), have been identified as prominent phosphorylation sites (49Ma A. Richardson A. Schaefer E.M. Parsons J.T. Mol. Biol. Cell. 2001; 12: 1-12Crossref PubMed Scopus (77) Google Scholar). Despite the importance of FAK in signal transduction, virtually nothing is known about the regulation of these phosphorylation events. In particular, none of the previous studies demonstrated that the phosphorylation of any of these serine residues of FAK can be regulated in response to cell stimulation by either GPCR agonists or ligands of tyrosine kinase receptors. In the present study, we demonstrate that stimulation of Swiss 3T3 cells with bombesin, LPA, PDB, or EGF induces a rapid and dramatic increase in Ser-910 phosphorylation of endogenous FAK. Treatment with the PKC inhibitor GF I or Ro 31-8220 or chronic exposure to PDB to down-regulate PKCs prevented the increase in FAK phosphorylation at Ser-910 induced by bombesin or PDB. Since bombesin or PDB induces ERK activation through a PKC-dependent pathway in Swiss 3T3 cells and because the amino acids surrounding Ser-910 conform to a consensus site for mitogen-activated protein kinases, we also examined whether the ERKs mediate FAK phosphorylation at Ser-910 in response to extracellular stimuli. Our results show that ERK inhibitors prevented FAK phosphorylation at Ser-910 in response to either bombesin or PDB. Furthermore, LPA, another GPCR agonist that induces ERK via pertussis toxin (PTx)-sensitive Gi rather than Gq and ligands of tyrosine kinase receptors that induce potent ERK pathway activation through PKC-independent pathways, including EGF, also stimulated a dramatic increase in FAK phosphorylation at Ser-910. In addition, activated ERK2 can directly phosphorylate FAK (or recombinant FRNK) at Ser-910 in vitro. Thus, our results show that stimulation with bombesin, LPA, PDB, or EGF stimulates phosphorylation of endogenous FAK at Ser-910 via an ERK-dependent pathway in Swiss 3T3 cells. Cell Culture—Stock cultures of Swiss 3T3 cells were maintained in DMEM, supplemented with 10% fetal bovine serum in a humidified atmosphere containing 10% CO2 and 90% air at 37 °C. For experimental purposes, Swiss 3T3 cells were plated in 100-mm dishes at 6 × 105 cells/dish in DMEM containing 10% fetal bovine serum and used after 6–8 days when the cells were confluent and quiescent. For assays using suspended cells, confluent and quiescent Swiss 3T3 cells in 100-mm dishes were washed three times with DMEM and subsequently suspended in 10 ml of DMEM by gentle scraping. The cell suspension was then incubated for 30 min at 37 °C before stimulation with the respective agonists. Cell Stimulation with Bombesin and Other Agonists—Confluent and quiescent Swiss 3T3 cells were washed twice with DMEM, equilibrated in the same medium at 37 °C for at least 30 min, and then treated with bombesin or other factors for the times indicated. We used 2 × 106 cells grown in 100-mm dishes containing 10 ml of DMEM for each experimental condition. The stimulation was terminated by aspirating the medium and solubilizing the cells in 1 ml of ice-cold RIPA buffer containing 50 mm HEPES, pH 7.4, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 10% glycerol, 1.5 mm MgCl2, 1 mm EGTA, 1 mm sodium orthovanadate, 10 mm sodium pyrophosphate, 100 mm NaF, and 1 mm phenylmethylsulfonyl fluoride. Immunoprecipitation—Lysates were clarified by centrifugation at 15,000 rpm for 10 min. Supernatants were transferred to fresh tubes, and proteins were immunoprecipitated at 4 °C for 4 h with protein A-agarose linked to polyclonal anti-FAK (C-20) antibody, as previously described. Immunoprecipitates were washed three times with RIPA buffer and extracted in 2× SDS-PAGE sample buffer (200 mm Tris-HCl, pH 6.8, 1 mm EDTA, 6% SDS, 4% 2-mercaptoethanol, 10% glycerol) by boiling for 10 min and resolved by SDS-PAGE. SDS-Polyacrylamide Gel Electrophoresis—Gel electrophoresis was performed with 8% acrylamide in the separating gel, 4% in the staking gel, and 0.1% SDS. Immunoprecipitates with paxillin were resolved with 8 and 12.5% acrylamide in the separating gel. Western Blotting—After SDS-PAGE, proteins were transferred to Immobilon membranes. After transfer, membranes were blocked using 5% nonfat dried milk in PBS, pH 7.2, and incubated overnight at 4 °C with the anti-Tyr(P) Ab (1 μg/ml), anti-FAK-Tyr-397 (0.1 μg/ml), anti-phospho-p44/42 MAPK (0.1 μg/ml), anti-FAK-Ser(P)-722 Ab (0.1 μg/ml), anti-FAK-Ser(P)-910 Ab (0.1 μg/ml), anti-ERK2 (0.1 μg/ml), or anti-paxillin (0.1 μg/ml) as indicated. The membranes were washed three times with PBS-0.1% Tween 20 and then incubated with secondary antibodies (horseradish peroxidase-conjugated donkey antibody to rabbit (NA 934V) or mouse (NA931V)) (1:5000) for 1 h at 22 °C. After washing three times with PBS plus 0.1% Tween 20, the immunoreactive bands were visualized using enhanced chemiluminescence (ECL) detection reagents. Autoradiograms were scanned using the GS-710 Calibrated Imaging Densitometer (Bio-Rad), and the labeled bands were quantified using the Quantity One software program (Bio-Rad). Generation and Overexpression of Mutant FAK—pFLAG-CMV™-2 expression vector (Sigma) containing FAK wild type was used to perform the mutation of Ser-910 to Ala with the QuikChange site-directed mutagenesis kit (Stratagene). COS-7 cells were cultured to 40–60% confluence on 100-mm plates, and then 6 μg of DNA were transfected with Lipofectin according to the instructions from Invitrogen. Cells were then incubated for 18 h at 37 °C with 5% CO2. Transfection medium was aspirated and replaced with 10 ml of culture medium containing 10% fetal bovine serum prior to further treatment. In Vitro Kinase Assay—Confluent Swiss 3T3 100 mm plates were lysed in RIPA buffer, and unstimulated FAK was immunoprecipitated. The immunocomplexes were washed twice with RIPA buffer and three times with kinase buffer (30 mm Tris-HCl, pH 7.4, 10 mm MgCl2, 1 mm dithiothreitol). The immunocomplexes in 70 μl of kinase buffer or GST-FRNK or GST-FRNKS910A in 100 μl of kinase buffer were incubated with activated ERK2 (Calbiochem) and 10 μm cold ATP or [γ-32P]ATP (specific activity 490 cpm/pmol) at 30 °C. The reaction was terminated by the addition of 2× sample buffer and analyzed by SDS-PAGE. GST-FRNK Fusion Protein—pFLAG-CMV™-2 expression vector containing FAK wild type and FAK-S910A were used to create FRNK (noncatalytic domain of FAK (amino acids 693–1053)) by PCR amplification using specific oligonucleotide primers (forward primer, 5′-CGGGATCCATGGAATCCAGGCGACAAG-3′; reverse primer, 5′-AGTTAGCGGCCGCTTAGTGGGGCCTGGACTG-3′) containing restriction sites for BamHI and NotI, respectively (underlined). The resulting PCR products were subcloned as a BamHI-NotI fragment into the vector pGEX-4T3 (Amersham Biosciences) to generate the bacterial expression constructs pGEX-GST-FRNK and pGEX-GST-FRNKS910A. The 57-kDa GST-FRNK and GST-FRNKS910A were expressed in Escherichia coli (BL21) for 2 h, induced by isopropyl-1-thio-β-d-galactopyranoside. The GST alone and the two fusion proteins were purified with the B-PER GST fusion protein purification kit from Pierce, according to the manufacturer's instructions. The buffer exchange was performed with an Ultrafree-0.5 centrifugal filter device (Millipore Corp.) and used immediately in the in vitro kinase assay. Materials—Bombesin, endothelin, and LPA were obtained from Sigma. Horseradish peroxidase-conjugated donkey antibodies to rabbit (NA 934V) or mouse (NA931V), and ECL reagents were from Amersham Biosciences. FAK polyclonal Ab C-20 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The phosphospecific polyclonal Abs to Ser-722 and Ser-910 of FAK were obtained from BioSource International (Camarillo, CA). Anti-Tyr(P) monoclonal antibody 4G10 and anti-FAK 2A7 were from Upstate Biotechnology, Inc. (Lake Placid, NY). SYPRO RED protein stain was purchased from Molecular Probes, Inc. (Eugene, OR). All other reagents used were of the purest grade available. Bombesin Induces FAK Phosphorylation at Ser-910 —Phosphorylation state-specific antibodies provide a powerful strategy to analyze site-specific phosphorylations of a variety of proteins. To examine whether the phosphorylation of endogenous FAK at Ser residues is regulated by GPCR agonists, quiescent cultures of Swiss 3T3 cells were incubated with 10 nm bombesin for various times, and lysates of these agonist-treated cells were immunoprecipitated with anti-FAK antibody. The resulting immunoprecipitates were analyzed by Western blotting using a site-specific antibody that recognizes the phosphorylated state of FAK at Ser-910. The Western blot analysis illustrated in Fig. 1 (upper panel) shows that prior to stimulation, pS910 immunoreactivity was virtually absent, indicating that Ser-910 of FAK was not phosphorylated in quiescent cultures of Swiss 3T3 cells. Upon bombesin stimulation, pS910 immunoreactivity of a single protein band in SDS-PAGE migrating at the expected apparent molecular mass for FAK (120 kDa) increased dramatically in a time-dependent manner. An increase in FAK phosphorylation, as revealed by immunoblotting with the pS910 antibody, was detected 3 min after the addition of the agonist, reached a maximum within 5–10 min, and remained relatively constant for up to 60 min. The maximal increase of FAK phosphorylation at Ser-910 induced by bombesin was 13.4 ± 1.2-fold, as compared with the unstimulated level. Bombesin stimulation of Swiss 3T3 cells promoted FAK phosphorylation at Ser-910 in a concentration-dependent manner (Fig. 1, middle panel). Consistent with the results shown in Fig. 1 (upper panel), the pS910 antibody only very weakly recognized FAK isolated from unstimulated cells. The results presented in Fig. 1 show that bombesin stimulation of Swiss 3T3 cells induces a striking increase in FAK phosphorylation at Ser-910. We also examined whether bombesin regulates FAK phosphorylation at Ser-722 by Western blotting using a site-specific antibody that detects the phosphorylated state of this residue in FAK. As shown in Fig. 1 (lower panel), the pS722 antibody recognized FAK isolated from unstimulated cells, indicating that Ser-722 of FAK, in contrast to Ser-910, was phosphorylated in the basal state of Swiss 3T3 cells. Furthermore, the addition of bombesin to these cells caused only small changes in the phosphorylation of Ser-722 as compared with that of Ser-910 (Fig. 1, lower panel). These results indicate that bombesin induces a selective increase in the phosphorylation of endogenous FAK at Ser-910 in Swiss 3T3 cells. Cytochalasin D Dissociates Bombesin-induced FAK Tyrosine Phosphorylation from FAK Phosphorylation at Ser-910 —Previously, we demonstrated that tyrosine phosphorylation of FAK and complex formation between FAK and Src in response to GPCR agonists requires an intact actin cytoskeleton (7Sinnett-Smith J. Zachary I. Valverde A.M. Rozengurt E. J. Biol. Chem. 1993; 268: 14261-14268Abstract Full Text PDF PubMed Google Scholar, 16Seufferlein T. Rozengurt E. J. Biol. Chem. 1994; 269: 9345-9351Abstract Full Text PDF PubMed Google Scholar, 17Seufferlein T. Rozengurt E. J. Biol. Chem. 1995; 270: 24343-24351Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 18Rankin S. Rozengurt E. J. Biol. Chem. 1994; 269: 704-710Abstract Full Text PDF PubMed Google Scholar, 29Lacerda H.M. Lax A.J. Rozengurt E. J. Biol. Chem. 1996; 271: 439-445Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 30Lacerda H.M. Pullinger G.D. Lax A.J. Rozengurt E. J. Biol. Chem. 1997; 272: 9587-9596Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 50Rodrïguez-Fernández J.L. Rozengurt E. J. Biol. Chem. 1996; 271: 27895-27901Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Specifically, treatment of the cells with cytochalasin D, which caps the barbed end of actin filaments and promotes their depolymerization, inhibits the increase in FAK tyrosine phosphorylation in response to bombesin and other GPCR agonists. Here, we examined whether cytochalasin d-mediated disruption of the actin cytoskeleton interferes with the increase in the phosphorylation of FAK at Ser-910 induced by bombesin. Quiescent Swiss 3T3 cells were exposed for 2 h to increasing concentrations of cytochalasin D and then stimulated with bombesin for another 30 min. Cell lysates were then immunoprecipitated with an anti-FAK antibody, and the immunoprecipitates were analyzed by Western blotting with either a specific anti-Tyr(P) antibody or with the antibody that recognizes phosphorylated FAK at Ser-910. As shown in Fig. 2A, treatment with cytochalasin D completely blocked the tyrosine phosphorylation of FAK induced by bombesin in a concentration-dependent manner. Maximal inhibitory effect was achieved at 2.4 μm, a concentration that completely disrupts the actin cytoskeleton and the assembly of focal adhesions (7Sinnett-Smith J. Zachary I. Valverde A.M. Rozengurt E. J. Biol. Chem. 1993; 268: 14261-14268Abstract Full Text PDF PubMed Google Scholar). In striking contrast, a similar treatment with cytochalasin D did not induce any significant effect on FAK phosphorylation at Ser-910 in response to bombesin (Fig. 2B). Dissolution of actin filaments can also be induced by preventing integrin-mediated organization of the actin cytoskeleton (i.e. by suspending cells in serum-free medium). To substantiate the results obtained with cytochalasin D, we also examined FAK phosphorylation at Ser-910 in suspended cells. As seen in Fig. 2B (inset), bombesin induced a marked increase in FAK phosphorylation at Ser-910 in Swiss 3T3 cells kept in suspension. The findings presented in Fig. 2 indicate that, in striking contrast to the increase in tyrosine phosphorylation, bombesin induces a dramatic increase in the phosphorylation of FAK at Ser-910 through a pathway that does not require an intact actin organization and the localization of FAK to focal adhesions. Bombesin Induces FAK Phosphorylation at Ser-910 through a PKC-dependent Pathway—Agonist stimulation of the bombesin GPCR activates the α-subunit of Gq, which stimulates the β isoforms of phospholipase C, which catalyze the production of inositol 1,4,5-trisphosphate that triggers the release of Ca2+ from internal stores, and diacylglycerol that activates the classical and novel isoforms of PKC (reviewed in Ref. 51Exton J.H. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 481-509Crossref PubMed Scopus (300) Google Scholar). Our previous studies demonstrated that treatment with cytochalasin D, at concentrations that disrupt the organization of the actin cytoskeleton and completely prevent the increase in the tyrosine phosphorylation of FAK, does not interfere with agonist-induced phosphoinositide hydrolysis, mobilization of Ca2+ from internal stores, and activation of PKC. These findings prompted us to determine whether the increase in FAK Ser-910 phosphorylation in response to bombesin is mediated through a PKC-dependent pathway. To determine whether direct activation of PKC increases FAK phosphorylation at Ser-910, quiescent Swiss 3T3 cells were treated with 100 nm PDB for various times and lysed. The lysates were immunoprecipitated with anti-FAK antibody, and the resulting immunoprecipitates were analyzed by Western blotting using the site-specific antibody that recognizes the phosphorylated state of FAK at Ser-910. As shown in Fig. 3, treatment with PDB caused a dramatic, time-dependent increase in FAK phosphorylation at Ser-910. PDB stimulated FAK Ser-910 phosphorylation in a concentration-dependent manner; maximal effect was achieved at 100 nm. Western blotting with anti-FAK antibody showed that the recovery of FAK from cell lysates was not altered by treatment with PDB. In addition, exposure to the membrane-permeant diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol, an analog of the endogenous activator of PKC, also increased FAK phosphorylation at Ser-910 (Fig. 3B, inset). Collectively, the results presented in Fig. 3 suggested that PKC activation provides a potential mechanism leading to FAK phosphorylation at Ser-910. Consequently, the following experiments were designed to examine the role of PKC in bombesin-induced FAK Ser-910 phosphorylation. Quiescent cultures of Swiss 3T3 cells were treated with GF I (also known as GF 109203X or bisindolylmaleimide I) or Ro 31-8220, potent inhibitors of phorbol ester-sensitive isoforms of PKC (7Sinnett-Smith J. Zachary I. Valverde A.M. Rozengurt E. J. Biol. Chem. 1993; 268: 14261-14268Abstract Full Text PDF PubMed Google Scholar, 52Toullec D. Pianetti P. Coste H. Bellevergue P. Grandperret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 53Uberall F. Giselbrecht S. Hellbert K. Fresser F. Bauer B. Gschwendt M. Grunicke H.H. Baier G. J. Biol. Chem. 1997; 272: 4072-4078Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 54Yeo E.J. Exton J.H. J. Biol. Chem. 1995; 270: 3980-3988Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), before bombes
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