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• W2142477858 abstract "Stimulation of T47D cells with epidermal growth factor (EGF) results in the activation of the intrinsic tyrosine kinases of the receptor and the phosphorylation of multiple cellular proteins including the receptor, scaffold molecules such as c-Cbl, adapter molecules such as Shc, and the serine/threonine protein kinase Akt. We demonstrate that EGF stimulation of T47D cells results in the activation of the Src protein-tyrosine kinase and that the Src kinase inhibitor PP1 blocks the EGF-induced phosphorylation of c-Cbl but not the activation/phosphorylation of the EGF receptor itself. PP1 also blocks EGF-induced ubiquitination of the EGF receptor, which is presumably mediated by phosphorylated c-Cbl. Src is associated with c-Cbl, and we have previously demonstrated that the Src-like kinase Fyn can phosphorylate c-Cbl at a preferred binding site for the p85 subunit of phosphatidylinositol 3′-kinase. PP1 treatment blocks EGF-induced activation of the anti-apoptotic protein kinase Akt suggesting that Src may regulate activation of Akt, perhaps by a Src → c-Cbl → phosphatidylinositol 3′-kinase → Akt pathway. Stimulation of T47D cells with epidermal growth factor (EGF) results in the activation of the intrinsic tyrosine kinases of the receptor and the phosphorylation of multiple cellular proteins including the receptor, scaffold molecules such as c-Cbl, adapter molecules such as Shc, and the serine/threonine protein kinase Akt. We demonstrate that EGF stimulation of T47D cells results in the activation of the Src protein-tyrosine kinase and that the Src kinase inhibitor PP1 blocks the EGF-induced phosphorylation of c-Cbl but not the activation/phosphorylation of the EGF receptor itself. PP1 also blocks EGF-induced ubiquitination of the EGF receptor, which is presumably mediated by phosphorylated c-Cbl. Src is associated with c-Cbl, and we have previously demonstrated that the Src-like kinase Fyn can phosphorylate c-Cbl at a preferred binding site for the p85 subunit of phosphatidylinositol 3′-kinase. PP1 treatment blocks EGF-induced activation of the anti-apoptotic protein kinase Akt suggesting that Src may regulate activation of Akt, perhaps by a Src → c-Cbl → phosphatidylinositol 3′-kinase → Akt pathway. epidermal growth factor EGF receptor glutathioneS-transferase phosphatidylinositol phosphatidylinositol 3′-kinase Src homology 2 Src homology 3 Signal transduction by the EGF1 (1White M.F. Kahn C.R J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar) receptor requires activation of the tyrosine kinase of the receptor to activate downstream signaling molecules. Downstream signaling pathways activated by the EGF receptor include the classic Ras/Raf/mitogen-activated protein kinase pathways, phospholipase C, and PI3-kinase. Although some signaling molecules are activated by direct binding to the activated EGF receptor, mediated by the direct binding of their SH2 domains to phosphorylated tyrosine residues, other signaling molecules do not appear to bind directly to the receptor itself, yet they are activated in a receptor-dependent manner. With regard to the EGF receptor, examples of the former include phospholipase C, whereas an example of the latter type of interaction is PI3-kinase, which does not appear to bind to the EGF receptor. In recent years it has become clear that large scaffolding molecules such as insulin-regulated substrate-1, Gab1, Gab2, and perhaps c-Cbl, may function in cooperation with growth factor receptors to regulate activation of downstream signaling molecules that do not bind directly to growth factor receptors (1White M.F. Kahn C.R J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Liu Y.-C. Altman A. Cell. Signal. 1998; 10: 377-385Crossref PubMed Scopus (84) Google Scholar, 3Lupher M.L. Andoniou C.E. Bonita D.P. Miyake S. Band H. Int. J. Biochem. Cell Biol. 1998; 30: 439-444Crossref PubMed Scopus (56) Google Scholar, 4Rudd C.E. Cell. 1999; 96: 5-8Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). These molecules are all relatively large in size (M r 95,000–130,000) and contain numerous tyrosine residues that become phosphorylated following receptor engagement. In our studies we have focused upon c-Cbl, a 120,000-dalton protein with 22 tyrosine residues. c-Cbl is the first member of the Cbl family of scaffolding molecules that include c-Cbl, Cbl-b, Cbl-3, D-Cbl from Drosophila and the Caenorhabditis elegans homologue sli-1(5Yoon C.H. Lee J. Jongeward G.D. Sternberg P.W. Science. 1995; 269: 1102-1105Crossref PubMed Scopus (282) Google Scholar, 6Thien C.B.F. Langdon W.Y. Nat. Rev. Mol. Cell Biol. 2001; 2: 294-305Crossref PubMed Scopus (522) Google Scholar). Engagement of numerous receptors results in the phosphorylation of c-Cbl; these receptors include the EGF receptor (7Levkowitz G. Klapper L.N. Tzahar E. Freywald A. Sela M. Yarden Y. Oncogene. 1996; 12: 1117-1125PubMed Google Scholar, 8Soltoff S.P. Cantley L.C. J. Biol. Chem. 1996; 271: 563-567Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 9Fukazawa T. Miyake S. Band V. Band H. J. Biol. Chem. 1996; 271: 14554-14559Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), the interleukin-3 receptor (10Barber D.L. Mason J.M. Fukazawa T. Reedquist K.A. Druker B.J. Band H. D'Andrea A.D. Blood. 1997; 89: 3166-3174Crossref PubMed Google Scholar, 11Anderson S.M. Burton E.A. Koch B.L. J. Biol. Chem. 1997; 272: 739-745Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), the erythropoietin receptor (8Soltoff S.P. Cantley L.C. J. Biol. Chem. 1996; 271: 563-567Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 10Barber D.L. Mason J.M. Fukazawa T. Reedquist K.A. Druker B.J. Band H. D'Andrea A.D. Blood. 1997; 89: 3166-3174Crossref PubMed Google Scholar), the prolactin receptor (12Hunter S. Koch B.L. Anderson S.M. Mol. Endocrinol. 1997; 11: 1213-1222Crossref PubMed Scopus (41) Google Scholar), integrins (13Ojaniemi M. Martin S.S. Dolfi F. Olefsky J.M. Vuori K. J. Biol. Chem. 1997; 272: 3780-3787Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 14Sattler M. Salgia R. Shrikhande G. Verma S. Uemura N. Law S.F. Golemis E.A. Griffin J.D. J. Biol. Chem. 1997; 272: 14320-14326Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), the T-cell receptor (15Donovan J.A. Wange R.L. Langdon W.Y. Samelson L.E. J. Biol. Chem. 1994; 269: 22921-22924Abstract Full Text PDF PubMed Google Scholar, 16Fukazawa T. Reedquist K.A. Trub T. Soltoff S. Panchamoorthy G. Druker B. Cantley L. Shoelson S.E. Band H. J. Biol. Chem. 1995; 270: 19141-19150Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), and the B-cell receptor (17Panchamoorthy G. Fukazawa T. Miyake S. Soltoff S. Reedquist K. Druker B. Shoelson S. Cantley L. Band H. J. Biol. Chem. 1996; 271: 3187-3194Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 18Smit L. van Der Horst G. Borst J. Oncogene. 1996; 13: 381-389PubMed Google Scholar). c-Cbl has been shown to be associated with numerous signaling molecules (Src, Fyn, Lyn, Syk, ZAP70, and PI3-kinase) (8Soltoff S.P. Cantley L.C. J. Biol. Chem. 1996; 271: 563-567Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 11Anderson S.M. Burton E.A. Koch B.L. J. Biol. Chem. 1997; 272: 739-745Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 16Fukazawa T. Reedquist K.A. Trub T. Soltoff S. Panchamoorthy G. Druker B. Cantley L. Shoelson S.E. Band H. J. Biol. Chem. 1995; 270: 19141-19150Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 17Panchamoorthy G. Fukazawa T. Miyake S. Soltoff S. Reedquist K. Druker B. Shoelson S. Cantley L. Band H. J. Biol. Chem. 1996; 271: 3187-3194Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 19Fournel M. Davidson D. Weil R. Veillette A. J. Exp. Med. 1997; 183: 301-306Crossref Scopus (123) Google Scholar) as well as several adapter molecules (Shc, Crk, and Grb2) (9Fukazawa T. Miyake S. Band V. Band H. J. Biol. Chem. 1996; 271: 14554-14559Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 16Fukazawa T. Reedquist K.A. Trub T. Soltoff S. Panchamoorthy G. Druker B. Cantley L. Shoelson S.E. Band H. J. Biol. Chem. 1995; 270: 19141-19150Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 17Panchamoorthy G. Fukazawa T. Miyake S. Soltoff S. Reedquist K. Druker B. Shoelson S. Cantley L. Band H. J. Biol. Chem. 1996; 271: 3187-3194Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 20Galisteo M.L. Dikic I. Batzer A.G. Langdon W.Y. Schlessinger J. J. Biol. Chem. 1995; 270: 20242-20245Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 21Sawasdikosol S. Chang J.-H. Pratt J.C. Wolf G. Shoelson S.E. Burakoff S.J. J. Immunol. 1996; 157: 110-116PubMed Google Scholar, 22Khwaja A. Hallberg B. Warne P.H. Downward J. Oncogene. 1996; 12: 2491-2498PubMed Google Scholar). The association of c-Cbl with PI3-kinase suggests that c-Cbl could function as a scaffolding molecule that regulates activation of downstream signaling molecules. c-Cbl can be phosphorylated by both Src-like kinases as well as members of the Syk/ZAP70 family of tyrosine kinases (23Feshchenko E.A. Langdon W.Y. Tsygankov A.Y. J. Biol. Chem. 1998; 273: 8323-8331Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 24Ota Y. Beitz L.O. Scharenberg A.M. Donovan J.A. Kinet J.-P. Samelson L.E. J. Exp. Med. 1996; 184: 1713-1723Crossref PubMed Scopus (86) Google Scholar, 25Deckert M. Elly C. Altman A. Liu Y.-C. J. Biol. Chem. 1998; 273: 8867-8874Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 26Tanaka S. Amling M. Neff L. Peyman A. Uhlmann E. Levy J.B. Baron R. Nature. 1996; 383: 528-531Crossref PubMed Scopus (252) Google Scholar, 27Howlett C.J. Bissone S.A. Resek M.E. Tigley A.W. Robbins S.M. Biochem. Biophys. Res. Commun. 1999; 257: 129-138Crossref PubMed Scopus (26) Google Scholar, 28Fitzer-Attas C.J. Schindler D.G. Waks T. Eshhar Z. J. Biol. Chem. 1997; 272: 8551-8557Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 29Tsygankov A.Y. Mahajan S. Fincke J.E. Bolen J.B. J. Biol. Chem. 1996; 271: 27130-27137Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar); however, it is not clear which specific tyrosine residues are phosphorylated by these kinases and whether different receptors utilize different tyrosine kinases to phosphorylate different regions of c-Cbl. Complicating the view of c-Cbl as merely a molecular scaffold is the observation that the C. elegans homologue of c-Cbl,sli-1, functions as a negative regulator of theC. elegans EGF receptor homologue let-63 (5Yoon C.H. Lee J. Jongeward G.D. Sternberg P.W. Science. 1995; 269: 1102-1105Crossref PubMed Scopus (282) Google Scholar). Consistent with this observation, overexpression of c-Cbl in fibroblasts results in the down-regulation of the EGF receptor (30Lill N.L. Douillard P. Awwad R.A. Ota S. Lupher M.L. Miyake S. Meissner-Lula N. Hsu V.W. Band H. J. Biol. Chem. 2000; 275: 367-377Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar,31Miyake S. Mullane-Robinson K.P. Lill N.L. Douillard P. Band H. J. Biol. Chem. 1999; 274: 16619-16628Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). A mechanism explaining this observation was revealed when it was realized that the RING finger motif of c-Cbl could function as part of a ubiquitin ligase complex (32Joazeiro C.A.P. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.-C. Science. 1999; 286: 309-312Crossref PubMed Scopus (912) Google Scholar, 33Yokouchi M. Kondo T. Houghton A. Bartkiewicz M. Horne W.C. Zhang H. Yoshimura A. Baron R. J. Biol. Chem. 1999; 274: 31707-31712Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 34Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (716) Google Scholar) and that overexpression of c-Cbl in fibroblasts stimulated the ubiquitination of the EGF receptor leading to its degradation (33Yokouchi M. Kondo T. Houghton A. Bartkiewicz M. Horne W.C. Zhang H. Yoshimura A. Baron R. J. Biol. Chem. 1999; 274: 31707-31712Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 34Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (716) Google Scholar, 35Waterman H. Levkowitz G. Alroy I. Yarden Y. J. Biol. Chem. 1999; 274: 22151-22154Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 36Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar). This leads to the question of whether the primary cellular function of c-Cbl is as a scaffolding molecule, a ubiquitin ligase, or both. In this paper we demonstrate that EGF-stimulated phosphorylation of c-Cbl appears to require a PP1-sensitive tyrosine kinase, presumably one of the members of the Src family of tyrosine kinases. Blocking phosphorylation of c-Cbl with PP1 also blocks ubiquitination of the EGF receptor following EGF stimulation, although the EGFR is activated and autophosphorylated. Furthermore, the activation of Src family members regulates activation of the anti-apoptotic protein kinase Akt, perhaps by a Src → c-Cbl → PI3-kinase → Akt pathway. The human breast cancer cell line T47D was obtained from Dr. Dean Edwards (University of Colorado Health Sciences Center, Denver, CO). MCF12A cells were obtained from the University of Colorado Cancer Center Tissue Culture Core Facility. T47D cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1 mm l-glutamine, 1× nonessential amino acids, 0.4 μg/ml insulin, 100 units/ml penicillin, and 100 μg/ml streptomycin. MCF12A were cultured in a 50:50 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 supplemented with 5% horse serum, 1 mm l-glutamine, 10 μg/ml insulin, 0.5 μg/ml hydrocortisone, 0.1 μg/ml cholera toxin, 20 ng/ml EGF, 100 units/ml penicillin, and 100 μg/ml streptomycin. Fetal calf serum was from Summit Biotechnology (Fort Collins, CO); horse serum was from Sigma, and charcoal-stripped fetal calf serum was obtained from HyClone (Logan, UT). Murine epidermal growth factor was obtained from Collaborative Biomedical Products (Bedford, MA). All other media components were from Invitrogen. Cells to be stimulated with EGF were cultured overnight (for 16 h) in culture media lacking fetal calf serum prior to stimulation with growth factors at the indicated concentrations and for the times indicated prior to harvest. PP1 was obtained from Calbiochem or Alexis Biochemical Co. (San Diego, CA). Cells to be immunoprecipitated were lysed in EB (50 mm NaCl, 10 mm Tris, pH 7.4, 5 mm EDTA, 50 mmNaF, 1% Triton X-100, 1 mm sodium orthovanadate with 100 units/ml aprotinin), and the lysates were clarified by spinning at 10,000 rpm in a Savant RCF13K refrigerated microcentrifuge for 30 min. For the experiments involving ubiquitin, 10 mm N-ethylmaleimide and 50 μm N-acetyl-leucyl-leucyl-norleucinal were added to the extraction buffer. 1–2 μg of the indicated antibodies were added to 1 ml of cell lysate corresponding to 1 mg of total cellular protein, as determined by the Pierce BCA protein assay (Pierce), and placed on a rocking platform for 1 h at 4 °C. The immune complexes were collected by adding 30 μl of Pansorbin (Calbiochem) to each immunoprecipitate for 1 h. The bound proteins were washed three times with lysis buffer, and the immunoprecipitated proteins were resolved by SDS-polyacrylamide gel electrophoresis. The resolved proteins were electrotransfered to Immobilon membranes (Millipore, Bedford, MA). Detection of proteins by immunoblotting was conducted using the enhanced chemiluminescence lighting (ECL) system according to the manufacturer's recommendations (AmershamBiosciences). In some experiments whole cell lysates were directly analyzed by immunoblotting without immunoprecipitation. Agarose-conjugated anti-phosphotyrosine monoclonal antibody 4G10 (catalogue number 16–101) and a rabbit polyclonal antibody to the p85 subunit of PI3-kinase (catalogue number 06–195) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-Shc (catalogue number S14630), anti-EGF receptor antibody (catalogue number E12020), and a second anti-p85 antibody (catalogue number 65721A) were obtained from Transduction Laboratories (Lexington, KY). A polyclonal antibody directed against c-Cbl was obtained from Santa Cruz Biotechnology (catalogue number sc-170, Santa Cruz, CA). EGFR antibodies used in the experiments in Fig. 8 were also from Santa Cruz Biotechnology (catalogue number sc-120 for immune precipitations and sc-03 for Western blotting). Monoclonal antibody 4G10 directed against phosphotyrosine was kindly provided by Dr. Brian Druker (University of Oregon Health Sciences Center, Portland, OR). Anti-Src antibody 2-17 was produced in this laboratory as were two monoclonal antibodies directed against c-Cbl (802H6 for immunoprecipitation and A672E4 for immunoblotting). The anti-ubiquitin monoclonal antibody Ubi-1 was obtained from Zymed Laboratories Inc. (catalogue number 13–1600, South San Francisco, CA). PI kinase assay was conducted as described previously (11Anderson S.M. Burton E.A. Koch B.L. J. Biol. Chem. 1997; 272: 739-745Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The immune complex protein kinase assay for Src was conducted as described previously (37Anderson S.M. Jorgensen B. J. Immunol. 1995; 155: 1660-1670PubMed Google Scholar). A series of GST fusion proteins that include different regions of c-Cbl have been described previously (38Hunter S. Burton E.A., Wu, S.C. Anderson S.M. J. Biol. Chem. 1999; 274: 2097-2106Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The names for the fusion proteins used in this study and the amino acids included in the proteins are as follows: GST-PRO (proline-rich region, amino acids 475–694); GST-LZIP (the C-terminal end of c-Cbl which includes the leucine zipper region, amino acids 426–906, GST-LZIP-Y731F (GST-LZIP with tyrosine 731 mutated to a phenylalanine). A cDNA clone of human c-CBL was provided by W. Langdon (University of Western Australia, Perth, Australia) and was used as the template to make all of these GST fusion proteins. All primers were taken directly from the published DNA sequence of this cDNA. Mutagenesis of individual codons was performed according to the manufacturer's recommendations using the Ex-Site mutagenesis kit from Stratagene (La Jolla, CA). GST fusion proteins encoding the N- and C-terminal SH2 domains and the SH3 domain of the p85 subunit of PI3-kinase were kindly provided by Dr. Lewis Cantley (Harvard Medical School, Boston, MA). The purification of the GST fusion proteins was conducted as described previously (11Anderson S.M. Burton E.A. Koch B.L. J. Biol. Chem. 1997; 272: 739-745Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Binding assays were conducted by adding 2 nmol of the desired GST fusion protein to a cell lysate prepared in RIPA buffer as described above, in a final volume of 1 ml. Following a 1-h incubation at 4 °C on a rocking platform, 40 μl of glutathione-agarose (Amersham Biosciences) was added and incubated for 1 h. The bound proteins were washed three times with RIPA, resolved on SDS-polyacrylamide gels, and subjected to immunoblotting as described above. EGF receptor family members are thought to contribute to the development of cancer in numerous epithelial tissues including the breast, lung, ovaries, and uterus (39Slamon D.J. Clark G.M. Wong S.G. Levin W.J. Ullrich A. McGuire W.L. Science. 1987; 235: 177-182Crossref PubMed Scopus (9954) Google Scholar, 40O'Reilly S.M. Barnes D.M. Camplejohn R.S. Bartkova J. Gregory W.M. Richards M.A. Br. J. Cancer. 1991; 63: 444-446Crossref PubMed Scopus (132) Google Scholar, 41Slamon D.J. Godolphin W. Jones L.A. Holt J.A. Wong S.G. Keith D.E. Levin W.J. Stuart S.G. Udove J. Ullrich A. Press M.F. Science. 1989; 244: 707-712Crossref PubMed Scopus (6262) Google Scholar). Stimulation of cells with EGF has been reported to activate numerous signaling molecules, including the large adapter molecule c-Cbl (8Soltoff S.P. Cantley L.C. J. Biol. Chem. 1996; 271: 563-567Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 20Galisteo M.L. Dikic I. Batzer A.G. Langdon W.Y. Schlessinger J. J. Biol. Chem. 1995; 270: 20242-20245Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 42Kretzner L. Blackwood E.M. Eisenman R.N. Nature. 1992; 359: 426-429Crossref PubMed Scopus (380) Google Scholar). To examine the role of c-Cbl in the proliferation of human breast cancer cells, we first examined the effect of EGF stimulation upon c-Cbl phosphorylation in T47D cells. EGF stimulation resulted in the rapid tyrosine phosphorylation of c-Cbl and its association with multiple tyrosine-phosphorylated proteins (Fig.1). A major tyrosine-phosphorylated protein of approximately 185 kDa was observed to co-precipitate with c-Cbl, along with minor bands of 62 and 54 kDa (Fig. 1). Based upon the work of other investigators (7Levkowitz G. Klapper L.N. Tzahar E. Freywald A. Sela M. Yarden Y. Oncogene. 1996; 12: 1117-1125PubMed Google Scholar, 8Soltoff S.P. Cantley L.C. J. Biol. Chem. 1996; 271: 563-567Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 20Galisteo M.L. Dikic I. Batzer A.G. Langdon W.Y. Schlessinger J. J. Biol. Chem. 1995; 270: 20242-20245Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 42Kretzner L. Blackwood E.M. Eisenman R.N. Nature. 1992; 359: 426-429Crossref PubMed Scopus (380) Google Scholar), the major band of 185 kDa appears to be the EGF receptor. The minor bands of 62 and 54 kDa appeared to correspond to tyrosine-phosphorylated Shc as indicated by reprobing the immunoblot with anti-Shc antibody (Fig. 1 B). The position and amount of c-Cbl in the anti-phosphotyrosine immunoblot were confirmed by reprobing the blot with anti-c-Cbl antibody. Although the amount of c-Cbl protein appeared to decrease following EGF stimulation (Fig. 1 C), this appeared to be an artifact of the way this experiment was done. When a similar immunoblot was first probed with anti-Cbl antibody, there was no decrease in the amount of c-Cbl protein (data not shown), although when the blot was probed with anti-phosphotyrosine antibody first followed by the anti-Cbl antibody, it appeared that there was a transient decrease in the amount of c-Cbl protein (Fig. 1 C). This suggested to us that the extensive signal detected with anti-phosphotyrosine antibody may prevent the binding of a portion of the anti-Cbl antibody. Our previous work (11Anderson S.M. Burton E.A. Koch B.L. J. Biol. Chem. 1997; 272: 739-745Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) with the interleukin-3 and prolactin receptors indicated that there was an increase in c-Cbl-associated PI3-kinase activity following cytokine stimulation, even though we could not detect the phosphorylation of the p85 subunit of PI3-kinase. No readily apparent tyrosine-phosphorylated protein with a molecular weight corresponding to that of the p85 subunit of PI3-kinase was detected in EGF-stimulated T47D cells (Fig. 1 A); however, we did detect the rapid and transient association of the p85 subunit of PI3-kinase with c-Cbl by immunoblotting with anti-p85 antibody (Fig.1 D, lanes 2 and 3). In the study shown in Fig. 1, the amount of p85 associated with c-Cbl peaked quickly (2 min after stimulation with EGF in the study shown) and declined after that time. These data suggest that there is a rapid and transient association of c-Cbl with the EGF receptor, Shc, and the p85 subunit of PI3-kinase. Of these interactions, the complex of c-Cbl with Shc appears to be more stable. A dose-response study was conducted to determine what concentration of EGF was required to induce phosphorylation of c-Cbl. T47D cells were stimulated with 0–200 ng/ml EGF for 10 min, and the cells were lysed and immunoprecipitated with anti-Cbl antibody. The resolved proteins were immunoblotted with anti-phosphotyrosine antibody 4G10 (Fig.2). Treatment of T47D cells with EGF concentrations as low as 5 ng/ml was able to induce tyrosine phosphorylation of both c-Cbl and the EGF receptor (Fig. 2). Stimulation with concentrations greater than 100 ng/ml caused no further increase in the phosphorylation of either c-Cbl or the EGF receptor. Stimulation of T47D cells also caused a dose-dependent increase in the phosphorylation of a protein with a molecular weight corresponding to two different isoforms of Shc (Fig. 2). There was no effect of EGF stimulation upon the amount of c-Cbl protein present in the immunoprecipitates (data not shown). Studies by other investigators (43Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3721) Google Scholar, 44Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2218) Google Scholar, 45Yao R. Cooper G.M. Science. 1995; 267: 2003-2006Crossref PubMed Scopus (1293) Google Scholar, 46Kennedy S.G. Wagner A.J. Conzen S.D. Jordan J. Bellacosa A. Tsichlis P.N. Hay N. Genes Dev. 1997; 11: 701-713Crossref PubMed Scopus (980) Google Scholar, 47Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (833) Google Scholar) have suggested that the activation of PI3-kinase by growth factors is critical in the generation of either a proliferative signal by growth factor receptors and/or in the suppression of apoptosis. The transformation of mammary epithelial cells into tumor cells may involve the induction of proliferative signals, anti-apoptotic signals, or perhaps both. For this reason we wished to investigate further the data shown in Fig. 1 regarding the association of PI3-kinase with c-Cbl following stimulation of T47D cells with EGF. We first asked whether PI kinase activity was also associated with c-Cbl following EGF stimulation of T47D cells. T47D cells were stimulated with EGF for 0–20 min, and the cells were lysed and immunoprecipitated with anti-Cbl antibody. One-tenth of the immunoprecipitated protein was used in a PI kinase assay, and the remainder of the protein was subjected to immunoblotting with an anti-p85 monoclonal antibody (Fig.3 A). Consistent with the results shown in Fig. 1, the rapid phosphorylation of c-Cbl was noted, as was the co-immunoprecipitation of a 185-kDa protein corresponding to the EGF receptor (data not shown). The rapid association of the p85 subunit of PI3-kinase with c-Cbl following EGF stimulation was also detected (Fig. 3 A). The maximal amount of c-Cbl-associated p85 was detectable at 1–2 min and declined after that time (Fig.3 A). It did appear that there were two bands in the anti-p85 immunoblot, and we do not know whether this represents modification of p85 or the presence of different p85 isoforms. c-Cbl-associated PI kinase activity was also examined using a PI kinase assay. A maximal amount of PI kinase activity was also detected after 1 min of stimulation with EGF (Fig. 3 B, lane 9). Although c-Cbl-associated PI kinase activity was still detectable 2 min post-stimulation, it declined dramatically after that time (Fig.3 B). Other studies in our laboratory have shown that c-Cbl-associated PI kinase activity can be detected after 5 min of stimulation with EGF; however, kinase activity was not detectable at later time points (10–30 min) (data not shown). PhosphorImager quantitation indicated that there was 2–5-fold more c-Cbl-associated PI kinase activity at 1-min post-EGF stimulation than present 2 min after EGF stimulation. There did not appear to be a dramatic difference in the amount of c-Cbl-associated p85 protein at 1 and 2 min post-EGF stimulation when compared with the difference in PI kinase activity observed at these two time points (compare Fig. 3, A andB); however, the kinase assay should reflect the amount of activated PI3-kinase and not the total amount of p85. These data indicate that both p85 and PI kinase activity are rapidly associated with c-Cbl following stimulation of T47D cells with EGF, and the kinetics of PI kinase activation and p85 binding to c-Cbl suggests that this event may represent a very early signaling event. The binding of c-Cbl to p85 was examined in a series of binding assays utilizing GST fusion proteins that contained different regions of either p85 or c-Cbl. The p85 subunit of PI3-kinase contained two SH2 domains (referred to as the N-SH2 and C-SH2 for the N- and C-terminal SH2 domains, respectively) and a single SH3 domain (48Kapeller R. Prasad K.V.S. Janssen O. Hou W. Schaffhausen B.S. Rudd C.E. Cantley L.C. J. Biol. Chem. 1993; 269: 1927-1933Abstract Full Text PDF Google Scholar). The ability of GST fusion proteins encoding these three domains to bind to c-Cbl present in lysates of T47D cells was investigated by adding 2 nmol of the indicated GST fusion proteins to lysates of unstimulated and EGF-stimulated T47D cells (Fig. 4). The GST-p85-SH3 domain was observed to bind to c-Cbl in lysates of both unstimulated and EGF-stimulated T47D cells (Fig. 4, lanes 7and 8). When lysates of EGF-stimulated T47D cells were used in the binding assay, there was a decrease in the amount of c-Cbl that bound to the GST-p85 SH3 domain fusion protein. We do not believe that this reflects a decrease in the amount of c-Cbl present in these cells at this time point because immunoblotting of" @default.
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• W2142477858 title "Inhibition of Src Family Kinases Blocks Epidermal Growth Factor (EGF)-induced Activation of Akt, Phosphorylation of c-Cbl, and Ubiquitination of the EGF Receptor" @default.
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