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• W2003879013 abstract "In studies to define mechanisms of ERK activation in Chinese hamster ovary cells, we have observed an inverse correlation between CRKII-C3G complex formation and ERK activity. That is, we were able to coprecipitate the guanine nucleotide exchange factor C3G with the adaptor protein CRKII in lysates from suspended cells that had low ERK activity, but we could not do so or could do so less efficiently in lysates of adherent cells with increased ERK activity. Consistent with the presence of a functional CRKII-C3G complex, we detected more GTP-loaded RAP1 in suspension than adherent lysates. Overexpression of cDNAs encoding B-RAF, CRKII W109L, and PTP1B C215S activated ERK in suspension cells, the latter two constructs also disrupting CRKII-C3G complex formation. Finally, we have also observed that certain integrin α subunit cytoplasmic splice variants differentially regulate ERK1/2 but also in a manner that correlated with levels of a CRKII-C3G complex. Thus, these data suggest the involvement of integrins in an ERK suppression pathway mediated by CRKII-C3G complex formation and downstream signaling from activated RAP1. In studies to define mechanisms of ERK activation in Chinese hamster ovary cells, we have observed an inverse correlation between CRKII-C3G complex formation and ERK activity. That is, we were able to coprecipitate the guanine nucleotide exchange factor C3G with the adaptor protein CRKII in lysates from suspended cells that had low ERK activity, but we could not do so or could do so less efficiently in lysates of adherent cells with increased ERK activity. Consistent with the presence of a functional CRKII-C3G complex, we detected more GTP-loaded RAP1 in suspension than adherent lysates. Overexpression of cDNAs encoding B-RAF, CRKII W109L, and PTP1B C215S activated ERK in suspension cells, the latter two constructs also disrupting CRKII-C3G complex formation. Finally, we have also observed that certain integrin α subunit cytoplasmic splice variants differentially regulate ERK1/2 but also in a manner that correlated with levels of a CRKII-C3G complex. Thus, these data suggest the involvement of integrins in an ERK suppression pathway mediated by CRKII-C3G complex formation and downstream signaling from activated RAP1. mitogen-activated protein extracellular signal-regulated kinase Chinese hamster ovary MAP kinase/ERK kinase fluorescein isothiocyanate Dulbecco's modified Eagle's medium bovine serum albumin polyacrylamide gel electrophoresis hemagglutinin phosphate-buffered saline fibronectin fibrinogen myelin basic protein RAS-binding domain of RAF-1 glutathione S-transferase Signal transduction pathways stimulated by growth factors and adhesion to matrix proteins influence cell behavior. Although the initial mechanics of these transduction pathways are distinct, several downstream effects and mediators are held in common. One of these common downstream mediators is the mitogen-activated protein kinase (the MAP1 kinases ERK1 and ERK2). Canonical pathways leading to ERK activation typically involve small GTPases. Principally, GTP loading or activation of RAS triggers a downstream kinase activation cascade from RAF1 to MEK and finally to ERK1 and ERK2. Integrin-mediated adhesion has been reported to impact this pathway at various and sometimes controversial points (1.Clark E.A. Hynes R.O. J. Biol. Chem. 1996; 271: 14814-14818Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 2.Lin T.H. Aplin A.E. Shen Y. Chen Q. Schaller M. Romer L. Aukhil I. Juliano R.L. J. Cell Biol. 1997; 136: 1385-1395Crossref PubMed Scopus (221) Google Scholar, 3.Renshaw M.W. Ren X.-D. Schwartz M.A. EMBO J. 1997; 16: 5592-5599Crossref PubMed Scopus (270) Google Scholar, 4.Wary K.K. Mariotti A. Zurzolo C. Giancotti F.G. Cell. 1998; 94: 625-634Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar). A second GTPase that may affect ERK activity is RAP1. By competing with RAS for common downstream effectors, activated RAP1 is sometimes thought to be antagonistic to RAS-based signaling (5.Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (334) Google Scholar), although specific RAP1 effects may be species- or cell type-specific (6.Ishimaru S. Williams R. Clark E. Hanafusa H. Gaul U. EMBO J. 1999; 18: 145-155Crossref PubMed Scopus (46) Google Scholar, 7.Zwartkruis F.J.T. Wolthuis R.M.F. Nabben N.M.J.M. Franke B. Bos J.L. EMBO J. 1998; 17: 5905-5912Crossref PubMed Scopus (191) Google Scholar). Crucial in the activation of the small GTPases is the mobilization of adaptor protein-exchange factor complexes. Thus, the GRB2-SOS complex is recruited to the cell membrane in GRB2 SH2 domain-dependent interactions with tyrosine-phosphorylated growth factor receptors or phosphorylated intermediates of integrin signaling pathways, thereby enabling efficient guanine nucleotide exchange or activation of RAS. Likewise, RAP1 is activated by the relocation of the adaptor protein, CRKII, and its associated exchange factor C3G. A potential docking site for CRKII is p130CAS, a protein heavily phosphorylated upon integrin ligation (8.Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar). In addition to relocation of these complexes, recent studies suggest their association itself may be regulated, thereby affecting downstream signaling. In what can be thought of as a classical negative feedback loop, a kinase activity downstream of MEK, possibly MEK itself, can serine/threonine-phosphorylate SOS (9.Zhao H. Okada S. Pessin J.E. Koretzky G.A. J. Biol. Chem. 1998; 273: 12061-12067Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Phosphorylated SOS can dissociate from GRB2 (10.Waters S.B. Holt K.H. Ross S.E. Syu L.J. Guan K.L. Saltiel A.R. Koretzky G.A. Pessin J.E. J. Biol. Chem. 1995; 270: 20883-20886Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 11.Langlois W.J. Sasaoka T. Saltiel A.R. Olefsky J.M. J. Biol. Chem. 1995; 270: 25320-25323Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), thereby limiting GTP loading of RAS and attenuating ERK activation. In a similar manner, as best exemplified with the insulin receptor, ligand occupancy can lead to tyrosine phosphorylation of the adaptor protein CRKII (12.Okada S. Matsuda M. Anafi M. Pawson T. Pessin J.E. EMBO J. 1998; 17: 2554-2565Crossref PubMed Scopus (82) Google Scholar). Once tyrosine-phosphorylated, CRKII can form an intramolecular SH2 domain loop (13.Rosen M.K. Yamazaki T. Gish G.D. Kay C.M. Pawson T. Kay L.E. Nature. 1995; 374: 477-479Crossref PubMed Scopus (118) Google Scholar), altering its conformation and dissociating from C3G (12.Okada S. Matsuda M. Anafi M. Pawson T. Pessin J.E. EMBO J. 1998; 17: 2554-2565Crossref PubMed Scopus (82) Google Scholar, 14.Okada S. Pessin J.E. J. Biol. Chem. 1997; 272: 28179-28182Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Certain integrins are expressed with alternatively spliced cytoplasmic domains that often exist in a cell- or tissue-specific manner and can be induced upon differentiation (15.Fornaro M. Languino L.R. Matrix Biol. 1997; 16: 185-193Crossref PubMed Scopus (60) Google Scholar). There are four β1and β4 cytoplasmic variants, three of α7, and two each of β3, α3, and α6. In some cases these isoforms confer phenotypic variation. For example, overexpression of the β1C and β1D variants slow cell cycle progression (16.Belkin A.M. Retta S.F. J. Biol. Chem. 1998; 273: 15234-15240Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 17.Fornaro M. Zheng D.Q. Languino L.R. J. Biol. Chem. 1995; 270: 24666-24669Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 18.Meredith J. Takada Y. Fornaro M. Languino L.R. Schwartz M.A. Science. 1995; 269: 1570-1572Crossref PubMed Scopus (117) Google Scholar), whereas β1B appears to have dominant negative effects on cell adhesion and motility (19.Balzac F. Retta S.F. Albini A. Melchiorri A. Koteliansky V.E. Geuna M. Silengo L. Tarone G. J. Cell Biol. 1994; 127: 557-565Crossref PubMed Scopus (66) Google Scholar). Cells expressing the α6A as opposed to the α6B isoform are more migratory (20.Shaw L.M. Mercurio A.M. Mol. Biol. Cell. 1994; 5: 679-690Crossref PubMed Scopus (56) Google Scholar). Thus, the distribution and expression pattern of these splice variants may enable unique functional properties to constituent cells. Whereas phenotypic differences have been ascribed to certain splice variants, the intracellular signaling pathways utilized by them are less clear. Nevertheless, limited studies with the α6 variants have identified different patterns of adhesion-stimulated protein tyrosine phosphorylation (21.Shaw L.M. Turner C.E. Mercurio A.M. J. Biol. Chem. 1995; 270: 23648-23652Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and MAP kinase activation (22.Wei J. Shaw L.M. Mercurio A.M. J. Biol. Chem. 1998; 273: 5903-5907Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In exploring the role of adaptor protein-exchange factor complexes in the regulation of the MAP kinases, we have identified a correlation between CRKII-C3G complex formation, levels of GTP-loaded RAP, and ERK activity in CHO cells. That is, in lysates of suspension cells, we observed low ERK activity but an immunoprecipitable CRKII-C3G complex and high levels of activated RAP. In contrast lysates generated from CHO cells adherent to fibronectin (fn) demonstrated increased ERK activity but little of the CRKII-C3G complex and GTP-loaded RAP. We have also observed that cells expressing α subunit cytoplasmic splice variants differentially regulated ERK1/2 in a manner that was consistent with levels of the CRKII-C3G complex. These data suggest the possibility that integrins mediate an active mechanism to suppress or maintain basal levels of MAP kinases and that this may involve the variable association of adaptor proteins with exchange factors. The characterization of the anti-αIIbβ3 antibodies D57, anti-LIBS6, and PAC1 have been described previously (23.O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (580) Google Scholar, 24.Frelinger III, A.L. Du X. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1991; 266: 17106-17111Abstract Full Text PDF PubMed Google Scholar, 25.Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar). The antibody D57 was biotinylated with biotin-N-hydrosuccinimide (Sigma) according to the manufacturer's directions. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM and FITC-conjugated goat anti-mouse IgG were from Tago (Burlingame, CA), and phycoerythrin-streptavidin was from Molecular Probes Inc. (Junction City, OR). The peptide mimetic compound Ro43-5054, which specifically inhibits binding to αIIbβ3 was a generous gift from Beat Steiner (Hoffmann-La Roche). Antibodies to ERK1, ERK2, CRKII, C3G, B-RAF, and the HA epitope were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and an anti-PTP1B antibody was from Transduction Laboratories (Lexington, KY). Phospho-ERK-specific antibodies were from Promega (Madison, WI) and Santa Cruz Biotechnology. Fibrinogen (fg) was obtained from Enzyme Research Laboratories, Inc. (South Bend, IN) and G418 (Geneticin) was obtained from Life Technologies, Inc. The construction of the chimeric integrins β3β1, αIIbα6A, and αIIbα6B has been described previously (23.O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (580) Google Scholar). pCDM8 constructs expressing the chimeric integrins αIIbα3A and αIIbα3B were created by a similar strategy. Briefly, cytoplasmic sequences of the integrin variants α3A and α3B were generated from full-length cDNA clones by polymerase chain reaction with appropriately designed oligonucleotide primers, digested with HindIII andXbaI, and subcloned into pCDM8. The resulting construct was digested with HindIII and fused to a HindIII fragment of CD2b (26.O'Toole T.E. Loftus J.C. Plow E.F. Glass A. Harper J.R. Ginsberg M.H. Blood. 1989; 74: 14-18Crossref PubMed Google Scholar) containing the extracellular and transmembrane sequences of αIIb. A cDNA construct encoding a CRKII variant with an SH3 domain mutation has been described previously (27.Matsuda M. Hashimoto Y. Muroya K. Hasegawa H. Kurata T. Tanaka S. Nakamura S. Hattori S. Mol. Cell. Biol. 1994; 14: 5495-5500Crossref PubMed Scopus (183) Google Scholar,28.Tanaka S. Hattori S. Kurata T. Nagashima K. Fukui Y. Nakamura S. Matsuda M. Mol. Cell. Biol. 1993; 13: 4409-4415Crossref PubMed Scopus (97) Google Scholar), whereas an expression construct for wild-type CRKII was obtained from Jeffrey Pessin (University of Iowa). A catalytically inactive mutant of PTP1B (C215S) was generated from the wild-type clone (gift of Nicholas Tonks; Cold Spring Harbor Laboratory) by Quick Change Mutagenesis (Stratagene) with appropriately designed oligonucleotides. A B-RAF cDNA was supplied by Phillip Stork (Vollum Institute), and expression vectors for RAS and RAP GTPases were supplied by Gary Bokoch (Scripps Research Institute). All constructs were verified before use by DNA sequencing. CHO cells were obtained from ATCC (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) containing 10% fetal bovine serum (Sigma), 1% l-glutamine (Sigma), 1% penicillin and streptomycin (Sigma), and 1% non-essential amino acids (Sigma). To establish stable lines expressing integrin chimera, plasmids encoding these sequences and a construct expressing the neomycin resistance gene were cotransfected into CHO cells. Briefly, the cells were plated at a density of 1 × 106 cells/100-mm dish, and 24 h later, 2 μg of each integrin construct and 0.1 μg of the neomycin resistance construct were mixed with 20 μl of LipofectAMINE (Life Technologies, Inc.) in a final volume of 200 μl and incubated for 10 min. At this time, the DNA-liposome complex was made up to 4 ml with unsupplemented DMEM and added to the cells. The cells were incubated for 6 h at 37 °C, washed with PBS, and put into complete media. The media were changed after 24 h, and the cells were subjected to G418 selection (700 μg/ml) after 48 h. After 2 weeks of selection, the cells were collected and single-cell sorted by flow cytometry (FACScan, Becton Dickinson) using the αIIbβ3-specific antibody, D57. Clonal lines were expanded and further analyzed. Transient transfections were also performed with the LipofectAMINE reagent. In these experiments, the cells were cotransfected with the pHOOK vector (Invitrogen) and positive cells isolated after 48 h by magnetic sorting as per manufacturer's suggestions. Integrin expression was determined by single color flow cytometry. Cells were harvested by incubation with trypsin-EDTA (Irvine Scientific, CA), washed in buffer containing soybean trypsin inhibitor (Sigma), and analyzed for D57 antibody binding. To do this, the cells were incubated in a final volume of 50 μl of DMEM, 1% BSA with 0.5 μg of D57 antibody for 30 min on ice. The cells were then diluted to 0.5 ml with DMEM-BSA, pelleted, and resuspended in 50 μl of DMEM-BSA containing 10% FITC-conjugated goat anti-mouse IgG. After 30 min on ice, the cells were diluted to 0.5 ml with PBS, centrifuged, resuspended in 0.5 ml of PBS, and analyzed on a FACScan (Becton Dickinson). The ligand binding properties of these transfectants were determined by two-color flow cytometry using the activation-specific anti-αIIbβ3 antibody (PAC1) and D57, as has been previously described (23.O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (580) Google Scholar). Adhesion studies were done by first incubating cells overnight in DMEM containing 0.5% fetal bovine serum. At this time, the cells were harvested by incubation in trypsin-EDTA and washed with serum-free DMEM containing 0.5 mg/ml soybean trypsin inhibitor and 0.2% BSA (Calbiochem, nuclease- and protease-free). The cells were then resuspended in DMEM, 0.2% BSA, and incubated for 2 h at 37 °C in suspension dishes (Corning Glass) that had been blocked overnight with 1% heat-denatured BSA. At this time, some of the cells were collected and washed in PBS (suspension cells). The remaining cells were allowed to adhere for 10 min to tissue culture dishes that had been coated with substrate proteins (overnight incubation with 15 μg/ml fg or fn) and washed once with PBS (adherent cells). Suspended and adherent cells prepared as described above were lysed on ice in M2 buffer (20 mmTris-HCl, pH 7.4, 250 mm NaCl, 0.5% Nonidet P-40, 3 mm EGTA, 5 mm EDTA, 20 mmNaPi, 3 mm β-glycerophosphate, 1 mm Na3VO4, 1 mmphenylmethylsulfonyl fluoride, 10 mm NaF, 1× complete phosphatase inhibitor (Roche Molecular Biochemicals)), the lysates clarified by centrifugation at 13,000 rpm for 10 min at 4 °C, and the protein concentration determined by the BCA assay (Pierce). For phospho-ERK immunoblotting, 60 μg of protein were separated by SDS-PAGE and transferred to nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in Tris-buffered saline (TBS: 20 mm Tris-HCl, pH 7.4, 150 mm NaCl) for 2 h at room temperature, incubated with an anti-phospho-ERK antibody (1:2000 dilution in 5% milk) for 1 h at room temperature, and then washed with TBS containing 0.5% Tween 20 (TBS-T). The membranes were then incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody (1:3000 dilution) for 1 h, washed with TBS-T, and immunoreactive bands visualized using ECL reagent (Amersham Pharmacia Biotech). To confirm equal loading of ERK1 and ERK2, the membrane was stripped in buffer containing 62.5 mmTris-HCl, pH 6.8, 2% SDS, and 100 mm β-mercaptoethanol for 30 min at 65 °C, blocked with 5% non-fat milk, and re-probed with antibodies to ERK1 and ERK2. For in vitro kinase reactions, ERK1 and ERK2 were immunoprecipitated from 150 μg of lysate by incubation with 0.4 μg each of anti-ERK1 and anti-ERK2 antibodies and 30 μl of a 50% slurry of protein-G Sepharose for 3 h at 4 °C. Immunoprecipitates were collected by centrifugation, washed with M2 buffer and with kinase buffer (KB: 20 mm HEPES, pH 7.6, 20 mmβ-glycerophosphate, 20 mm p-nitrophenyl phosphate, 10 mm DTT, 5 mmNa3VO4), and then resuspended in KB. Kinase reactions were initiated by addition of 2 μg of myelin basic protein (MBP), 20 μm cold ATP, and 5 μCi of [γ-32P]ATP and allowed to incubate for 20 min at 30 °C. Reactions were stopped by addition of SDS sample buffer, and labeled products were resolved by SDS-PAGE and visualized by autoradiography. Equal immunoprecipitation of ERK1 and ERK2 was confirmed by Western blotting with antibodies to these proteins. Suspension and adherent lysates prepared as described above were clarified by centrifugation, and the resulting supernatants were collected. For immunoprecipitations, 300 μg of cell extract was diluted to 0.4 ml in lysis buffer and incubated overnight at 4 °C with the appropriate antibodies. Proteins were recovered after a 2-h incubation with protein G-Sepharose, resolved by SDS-PAGE, and electrophoretically transferred to nitrocellulose membranes. Protein bands were identified after incubating the membranes with specific primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and finally visualized with the ECL reagent (Amersham Pharmacia Biotech). To determine the GTP-loading of RAS and RAP GTPases, we performed pull-down assays. GST fusion proteins with the minimal RAP-binding domain of RalGDS and the RAS-binding domain of RAF-1 (RBD) were generated in Escherichia colistrain BL21 following induction with 1 mmisopropyl-1-thio-β-d-galactopyranoside, and isolated by immobilizing bacterial lysates for 1 h at 4 °C with glutathione-Sepharose beads. The beads were then washed three times with RBD buffer (20 mm HEPES, pH 7.5, 120 mmNaCl, 10% glycerol, 0.5% Nonidet P-40, 2 mm EDTA, 10 μg/ml aprotinin), and then resuspended in RBD buffer to make a 50% slurry. CHO cells were transfected with expression vectors for HA-tagged wild-type RAP1 or wild-type Ha-RAS and pHOOK as described above. After 48 h, transfectants were isolated by magnetic sorting and suspended and adherent cells generated as above. These samples were then lysed in ice-cold pull-down buffer (25 mm HEPES, pH 7.5, 500 mm NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 25 mm NaF, 1 mmNa3VO4, 1 mm EDTA, 10 μg/ml aprotinin, and 1× completeTM protease inhibitors) and immediately centrifuged at 13,000 rpm for 2 min at 4 °C. The resulting supernatant was incubated with immobilized GST-RalGDS or GST-RBD beads (50 μl of 50% slurry containing about 10 μg of protein) for 1 h at 4 °C. At this time, the beads were washed three times with pull-down buffer, and isolated proteins were resolved by SDS-PAGE. Detection of pulled down RAP and RAS GTPases was accomplished by Western blotting with anti-HA and anti-RAS antibodies, respectively. As noted above, the association of SH2 and SH3-containing adaptor proteins with guanine nucleotide exchange factors can be regulated by signals arising from occupancy of growth factor receptors. To determine if integrin occupancy also regulates these associations, we have examined the properties of these molecules in suspension and adherent lysates. As expected, when CHO cells were serum-starved overnight and then kept in suspension for 2 h, we observed low levels of ERK1/2 activity that were significantly increased upon adhesion to fn (Fig.1 A). When we immunoprecipitated CRKII from CHO lysates, we consistently observed C3G in Western blots from suspension lysates but could not do so or could do so less efficiently from adherent lysates (Fig. 1 B; also Figs. 4 C and 5 C). Similar amounts of CRKII were immunoprecipitated from both lysates. In contrast we saw no change in levels of the GRB2-SOS complex from suspended or adherent lysates (data not shown). Thus low ERK1/2 activity correlated with the presence of an immunoprecipitable CRKII-C3G complex.Figure 4Disruption of CRKII-C3G complex activates ERK. CHO cells were transfected with pHOOK, and either wild-type (wt) CRKII or its W109L variant and suspended (S) and adherent (A) lysates were prepared as above.A, equal amounts of these lysates were then analyzed for levels of ERK activity by Western blotting (WB) with a phospho-ERK antibody. The same blot was stripped and reprobed with antibodies to ERK1 and ERK2 to determine levels of ERK protein.B, to quantitate ERK activity levels, we performed densitometric analysis of the blots shown in A. Relative ERK2 activity was expressed as the amount of phosphorylated ERK2 normalized to the amount of ERK2 protein. C, equal amounts of protein from the suspended and adherent lysates were immunoprecipitated (IP) with an anti-CRKII antibody and recovered proteins analyzed by Western blotting with anti-CRKII and anti-C3G antibodies as indicated. D, to determine the expression of the transfected CRKII constructs, equal amounts of lysate were resolved by SDS-PAGE and analyzed by Western blotting with an anti-CRKII antibody. The results illustrated are from a representative experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5The protein tyrosine phosphatase PTP1B regulates CRKII-C3G complex formation and ERK activity. CHO cells were transfected with pHOOK and either pCDNA3.1, wild-type (wt) PTP1B, or the PTP1B C215S variant, and suspended (S) and adherent (A) lysates were prepared as above. A, equal amounts of these lysates were then analyzed for levels of ERK activity by Western blotting (WB) with a phospho-ERK antibody. The same blot was stripped and reprobed with antibodies to ERK1 and ERK2 to determine levels of ERK protein.B, to quantitate ERK activity levels, we performed densitometric analysis of the blots shown in A. Relative ERK2 activity was expressed as the amount of phosphorylated ERK2 normalized to the amount of ERK2 protein. C, equal amounts of protein from the suspended and adherent lysates of the PTP1B-transfected cells were immunoprecipitated (IP) with an anti-CRKII antibody, and recovered proteins were analyzed by Western blotting with anti-CRKII and anti-C3G antibodies as indicated.D, to determine the expression of the transfected PTP1B constructs, equal amounts of the lysates were resolved by SDS-PAGE and analyzed by Western blotting with an anti-PTP1B antibody. The results illustrated are from a representative experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As C3G functions as an exchange factor for the RAS family member RAP1, we were interested if the state of integrin occupancy, and variable levels of the CRKII-C3G complex, affected the activation or GTP loading of this G protein. To determine this, we used a pull-down assay with a GST-RalGDS fusion protein that selectively binds the GTP as opposed to the GDP-bound form of RAP. When analyzed in this way, we found that levels of activated RAP were approximately 10-fold greater in suspension lysates than in adherent lysates (Fig.2). In analogous experiments using a GST-RBD fusion protein to isolate GTP-bound RAS, we found activated RAS was mostly present in adherent but not suspended cell lysates (Fig. 2). In both experiments we used an activated variant (G12V) of these GTPases as a positive control (data not shown). Thus, the state of integrin occupancy determines the nucleotide loading of the RAS and RAP GTPases, and GTP-loaded RAP is also found in lysates with low ERK activity. The downstream signaling consequences of activated RAP are dependent upon the predominant isoform of RAF present. As noted above, in certain cell types, GTP-loaded RAP is thought to be antagonistic to RAS-mediated ERK activation by competing for common effectors such as RAF1 (5.Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (334) Google Scholar). However, in many neuronal cell types that express B-RAF, activated RAP stimulates rather than suppresses ERK activity (29.York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J.S. Nature. 1998; 392: 622-626Crossref PubMed Scopus (757) Google Scholar, 30.Dugan L.L. Kim J.S. Zhang Y. Bart R.D. Sun Y. Holtzman D.M. Gutman D.H. J. Biol. Chem. 1999; 274: 25842-25848Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). As RAP is GTP-loaded in suspended CHO cells, we would predict that overexpression of B-RAF would activate ERK in this condition. To examine this possibility we transiently transfected CHO cells with B-RAF cDNA or a vector control and determined ERK activity levels upon suspension or adhesion to fn. While CHOs transfected with the empty vector construct demonstrated background levels of ERK activity, those transfected with B-RAF had significantly greater levels of active ERK in suspension lysates (Fig.3 A). As expected, ERK activity increased upon adhesion of both cell types. Although the absolute level of ERK activity was greater in the B-RAF transfectants, when analyzed quantitatively, adhesion stimulated a 50% increase in the vector transfectants but only a 5% increase in the B-RAF transfectants. Thus ERK phosphorylation, and presumably activity, was nearly maximal in suspension lysates of the B-RAF transfectants. These results are consistent with the idea that RAP is GTP-loaded when CHOs are in suspension and that the state of RAP loading is an important determinant of ERK activity. The above data suggested that activation of RAP and inhibition of ERK activity in CHO cells was dependent upon the regulated formation of the CRKII-C3G complex. Blocking this association would therefore be predicted to activate ERK1/2 in suspension cells. To look at this possibility we have made use of a CRKII variant (W109L) with an SH3 domain mutation that abolishes interaction with C3G. When we expressed this construct in CHO cells, we observed that levels of ERK activity in suspension lysates were increased relative to cells transfected with wild-type CRKII (Fig. 4, A andB), despite comparable expression of both CRKII constructs (Fig. 4 D). Consistent with this observation and the described functional properties of this amino acid substitution, we detected little of the CRKII-C3G complex in suspension lysates of the W109L transfectants (Fig. 4 C). In contrast, cells transfected with wild-type CRKII, like untransfected CHOs, did demonstrate an immunoprecipitable CRKII-C3G complex in suspension lysates (Fig. 4 C). Thus blocking the interaction of CRKII with C3G activates ERK1/2. Studies from another group suggested that overexpression of the protein tyrosine phosphatase, PTP1B, impaired integrin-mediated up-regulation of ERK activity in 3Y1 fibroblasts, whereas a CAS binding-deficient variant of this phosphatase reversed these effects (31.Liu F. Sells M.A. Chernoff J. Curr. Biol. 1998; 8: 173-176Abstract Full Text Full Text PDF PubMed Google Scholar). In another study, expression of a catalytically inactive form of this phosphatase resulted in altered morphology, reduced spreading, and decreased focal contacts in mouse L fibroblasts (32.Arregui C.O. Balsamo J. Lillen J. J. Cell Biol. 1998; 143: 861-873Crossref PubMed Sc" @default.
• W2003879013 created "2016-06-24" @default.
• W2003879013 creator A5040326919 @default.
• W2003879013 creator A5076431128 @default.
• W2003879013 date "2000-04-01" @default.
• W2003879013 modified "2023-10-16" @default.
• W2003879013 title "The Association of CRKII with C3G Can be Regulated by Integrins and Defines a Novel Means to Regulate the Mitogen-activated Protein Kinases" @default.
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