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• W2004202136 abstract "The functional role of Cbl in regulating T cell receptor (TCR)-mediated signal transduction pathways is unknown. This study uses Cbl overexpression in conjunction with a Ras-sensitive AP1 reporter construct to examine its role in regulating TCR-mediated activation of the Ras pathway. Cbl overexpression in Jurkat T cells inhibited AP1 activity after TCR ligation. However, AP1 induction by 4β-phorbol 12-myristate 13-acetate, which up-regulates Ras activity in a protein kinase C-dependent, TCR/tyrosine kinase-independent manner, was not affected by Cbl overexpression. Cbl overexpression also did not affect AP1 induction by an activated Ras protein or a membrane-bound form of the guanine nucleotide exchange factor Sos. In addition, activation of the mitogen-activated protein kinase Erk2 was decreased by Cbl overexpression. Therefore, Cbl regulates events that are required for full TCR-mediated Ras activation, and data are presented to support a model whereby Cbl regulates events required for Ras activation via its association with Grb2. The functional role of Cbl in regulating T cell receptor (TCR)-mediated signal transduction pathways is unknown. This study uses Cbl overexpression in conjunction with a Ras-sensitive AP1 reporter construct to examine its role in regulating TCR-mediated activation of the Ras pathway. Cbl overexpression in Jurkat T cells inhibited AP1 activity after TCR ligation. However, AP1 induction by 4β-phorbol 12-myristate 13-acetate, which up-regulates Ras activity in a protein kinase C-dependent, TCR/tyrosine kinase-independent manner, was not affected by Cbl overexpression. Cbl overexpression also did not affect AP1 induction by an activated Ras protein or a membrane-bound form of the guanine nucleotide exchange factor Sos. In addition, activation of the mitogen-activated protein kinase Erk2 was decreased by Cbl overexpression. Therefore, Cbl regulates events that are required for full TCR-mediated Ras activation, and data are presented to support a model whereby Cbl regulates events required for Ras activation via its association with Grb2. Perturbation of the T cell receptor (TCR) 1The abbreviations used are: TCR, T cell receptor; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; IL-2, interleukin-2; PMA, 4β-phorbol 12-myristate 13-acetate; HA, hemagglutinin; GST, glutathioneS-transferase; SEAP, secreted alkaline phosphatase. 1The abbreviations used are: TCR, T cell receptor; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; IL-2, interleukin-2; PMA, 4β-phorbol 12-myristate 13-acetate; HA, hemagglutinin; GST, glutathioneS-transferase; SEAP, secreted alkaline phosphatase. by antigen in the context of the appropriate major histocompatibility complex molecule initiates a cascade of events that involves the phosphorylation of proteins by tyrosine kinases (1Perlmutter R.M. Levin S.D. Appleby M.W. Anderson S.J. Alberola J. Annu. Rev. Immunol. 1993; 11: 451-499Crossref PubMed Scopus (190) Google Scholar). Two members of the Src family of tyrosine kinases, Fyn and Lck, are constitutively associated with the TCR and/or the CD4/CD8 coreceptors. Fyn and Lck appear to be responsible for the phosphorylation of conserved immune receptor tyrosine-based activation motifs on the cytosolic tails of the TCR-associated ζ and CD3 γ, δ, and ε chains (2Burkhardt A.L. Stealey B. Rowley R.B. Mahajan S. Prendergast M. Fargnoli J. Bolen J.B. J. Biol. Chem. 1994; 269: 23642-23647Abstract Full Text PDF PubMed Google Scholar, 3Gauen L.K.T. Zhu Y. Leturneur F. Hu Q. Bolen J.B. Matis L.A. Klausner R.D. Shaw A.S. Mol. Cell. Biol. 1994; 14: 3729-3741Crossref PubMed Scopus (125) Google Scholar, 4Samelson L.E. Phillips A.F. Luong E.T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4358-4362Crossref PubMed Scopus (532) Google Scholar). Phosphorylation of immune receptor tyrosine-based activation motifs leads to the recruitment of the Syk family tyrosine kinase ZAP-70. Full activation of ZAP-70 appears to require its phosphorylation by Fyn or Lck (5Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar), and because there are multiple immune receptor tyrosine-based activation motifs on the TCR·CD3·ζ complex, signal amplification may occur at this step (6Koyasu S. Tse A.G.D. Moingeon P. Hussey R.E. Mildonian A. Hannisian J. Clayton L.K. Reinherz E.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6693-6697Crossref PubMed Scopus (67) Google Scholar, 7Irving B.A. Chan A.C. Weiss A. J. Exp. Med. 1993; 177: 1093-1103Crossref PubMed Scopus (255) Google Scholar). The targets of ZAP-70 kinase remain largely unknown, but two downstream events that are critical for T cell activation are the activation of phospholipase Cγ1 and p21 ras (Ras) (8June C.H. Fletcher M.C. Ledbetter J.A. Schieven G.L. Siegel J.N. Phillips A.F. Samelson L.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7722-7726Crossref PubMed Scopus (432) Google Scholar, 9Mustelin T. Coggeshall K.M. Isakov N. Altman A. Science. 1990; 247: 1584-1587Crossref PubMed Scopus (369) Google Scholar, 10Pastor M.I. Reif K. Cantrell D. Immunol. Today. 1995; 16: 159-164Abstract Full Text PDF PubMed Scopus (153) Google Scholar, 11Crabtree G.R. Clipstone N.A. Annu. Rev. Biochem. 1994; 63: 1045-1083Crossref PubMed Scopus (626) Google Scholar). Activation of phospholipase Cγ1 results in inositol phospholipid hydrolysis and the generation of second messengers that control protein kinase C activation and Ca2+ mobilization (12Rhee S.G. Suh P.G. Ryu S.H. Lee S.Y. Science. 1989; 244: 546-550Crossref PubMed Scopus (697) Google Scholar). Ras regulates the activity of multiple downstream effector pathways, including the mitogen-activated protein kinases Erk1 and Erk2 (10Pastor M.I. Reif K. Cantrell D. Immunol. Today. 1995; 16: 159-164Abstract Full Text PDF PubMed Scopus (153) Google Scholar, 13Deng T. Karin M. Nature. 1994; 371: 171-175Crossref PubMed Scopus (318) Google Scholar, 14Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1659) Google Scholar). Activation of the phospholipase Cγ1 and Ras signaling pathways culminates in the induction of transcription factors that regulate lymphokine production, receptor expression, and cell proliferation. Although the events that link ZAP-70 activation with these downstream events are unclear, there are several early prominently tyrosine-phosphorylated proteins observed in response to TCR/CD3 engagement that may be important for signal transduction. One of these proteins is the product of the proto-oncogene c-cbl (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,16Reedquist K.A. Fukazawa T. Druker B. Panchamoorthy G. Shoelson S.E. Band H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4135-4139Crossref PubMed Scopus (74) Google Scholar). Cbl is a cytosolic protein found in all hematopoietic cell lineages and some non-hematopoietic tissues such as testis, lung, and brain. It is a major target of tyrosine phosphorylation after the engagement of multiple receptor types, including the antigen receptors for T and B cells; Fc receptors; receptors for EGF, colony-stimulating factor-1, and granulocyte-macrophage colony-stimulating factor; and the thrombopoietin and erythropoietin receptors, suggesting that Cbl is involved in signal transduction pathways triggered by these distinct receptors (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, 16Reedquist K.A. Fukazawa T. Druker B. Panchamoorthy G. Shoelson S.E. Band H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4135-4139Crossref PubMed Scopus (74) Google Scholar, 17Tanaka S. Neff L. Baron R. Levy J.B. J. Biol. Chem. 1995; 270: 14347-14351Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 18Cory G.O.C. Lovering R.C. Hinshelwood S. MacCarthy-Morrogh L. Levinsky R.J. Kinnon C. J. Exp. Med. 1995; 182: 611-615Crossref PubMed Scopus (126) Google Scholar, 19Panchamoorthy 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, 20Odai H. Sasaki K. Iwamatsu A. Hanazono Y. Tanaka T. Mitani K. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 10800-10805Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 21Sasaki K. Odai H. Hanazono Y. Ueno H. Ogawa S. Langdon W.Y. Tanaka T. Miyagawa K. Mitani K. Yazaki Y. Biochem. Biophys. Res. Commun. 1995; 216: 338-347Crossref PubMed Scopus (58) Google Scholar, 22Wang Y. Yeung Y.-G. Langdon W.Y. Stanley E.R. J. Biol. Chem. 1996; 271: 17-20Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 23Meisner H. Czech M.P. J. Biol. Chem. 1995; 270: 25332-25335Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Cbl has no known enzymatic activity, and its function is unknown. However, the presence of multiple sites within Cbl with the potential for binding to SH2 and SH3 domains and the finding that Cbl interacts with a wide variety of proteins involved in signal transduction suggest that it may function as a complex adapter molecule (24Tsygankov 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, 25Buday L. Khwaja A. Sipeki S. Farago A. Downward J. J. Biol. Chem. 1996; 271: 6159-6163Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 26Sawasdikosol S. Chang J.-H. Pratt J.C. Wolf G. Shoelson S.E. Burakoff S.J. J. Immunol. 1996; 157: 110-116PubMed Google Scholar, 27Hartley D. Corvera S. J. Biol. Chem. 1996; 271: 21939-21943Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 28Fukazawa 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, 29Meisner H. Conway B.R. Hartley D. Czech M.P. Mol. Cell. Biol. 1995; 15: 3571-3578Crossref PubMed Scopus (212) Google Scholar, 30Ribon V. Hubbell S. Herrera R. Saltiel A.R. Mol. Cell. Biol. 1995; 16: 45-52Crossref Scopus (102) Google Scholar, 31Fournel M. Davidson D. Weil R. Veillette A. J. Exp. Med. 1996; 183: 301-306Crossref PubMed Scopus (123) Google Scholar, 32Lupher Jr., M.L. Reedquist K.A. Miyake S. Langdon W.Y. Band H. J. Biol. Chem. 1996; 271: 24063-24068Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). One protein with which Cbl interacts is the adapter Grb2, a protein consisting of two SH3 domains and one SH2 domain (25Buday L. Khwaja A. Sipeki S. Farago A. Downward J. J. Biol. Chem. 1996; 271: 6159-6163Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 28Fukazawa 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, 29Meisner H. Conway B.R. Hartley D. Czech M.P. Mol. Cell. Biol. 1995; 15: 3571-3578Crossref PubMed Scopus (212) Google Scholar, 33Donovan J.A. Ota Y. Langdon W.Y. Samelson L.E. J. Biol. Chem. 1996; 271: 26369-26374Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Grb2 has recently been implicated as a regulator of the Ras activation pathway after TCR engagement by virtue of its ability to mediate the translocation of the guanine nucleotide exchange factor Sos from the cytosol to the membrane, where it can interact with and activate Ras (34McCormick F. Nature. 1993; 363: 15-16Crossref PubMed Scopus (440) Google Scholar, 35Holsinger L.J. Spencer D.M. Austin D.J. Schreiber S.L. Crabtree G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9810-9814Crossref PubMed Scopus (125) Google Scholar, 36Egan S.E. Giddings B.W. Brooks M.W. Buday L. Sizeland A.M. Weinberg R.A. Nature. 1993; 363: 45-51Crossref PubMed Scopus (1008) Google Scholar). In Caenorhabditis elegans, a Cbl homolog, Sli-1, acts as a negative regulator of the Ras homolog, Let60, possibly by regulating the activity of Sem5, a Grb2 homolog (37Yoon C.H. Lee J. Jongeward G.D. Sternberg P.W. Science. 1995; 269: 1102-1105Crossref PubMed Scopus (281) Google Scholar). In addition, a transforming mutant Cbl protein, 70Z/3, has been implicated in the activation of Ras-dependent signaling pathways that are involved in the induction of NF-AT, a transcriptional regulator involved in interleukin-2 (IL-2) gene expression (38Liu Y.-C. Elly C. Langdon W.Y. Altman A. J. Biol. Chem. 1997; 272: 168-173Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Ras activation in T lymphocytes controls the assembly of the AP1 complex, a transcription factor complex that binds to site(s) within the IL-2 gene enhancer (11Crabtree G.R. Clipstone N.A. Annu. Rev. Biochem. 1994; 63: 1045-1083Crossref PubMed Scopus (626) Google Scholar). We have therefore evaluated whether Cbl can regulate Ras-dependent signaling in T lymphocytes by using Cbl overexpression in conjunction with an AP1 reporter gene construct. We have found that Cbl overexpression inhibited TCR-induced AP1 and Erk2 activation. We further observed a T cell activation-induced exchange of Cbl for Sos on Grb2, suggesting one potential mechanism by which Cbl could regulate Ras activation in T lymphocytes. 4β-Phorbol 12-myristate 13-acetate (PMA) was from Calbiochem Corp. (Nottingham, United Kingdom). The anti-CD3 monoclonal antibody (OKT3) was a generous gift from Ortho Biotech, Inc. (Raritan, NJ). The rabbit anti-Cbl antibody (C-15) that recognizes amino acids 892–906, the rabbit anti-Grb2 antibody (C-23), and the anti-Erk2 antibody (C-14) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The anti-active mitogen-activated protein kinase antibody was from Promega (Madison, WI). The mouse anti-Sos1 antibody was from Upstate Biotechnology, Inc. (Lake Placid, NY). The R5 anti-Cbl antibody, which is directed against the carboxyl-terminal region of the Cbl protein (amino acids 143–450), was provided by Dr. W. Langdon (University of Western Australia) (39Blake T.J. Heath K.G. Langdon W.Y. EMBO J. 1993; 12: 2017-2026Crossref PubMed Scopus (110) Google Scholar). The anti-hemagglutinin (HA) monoclonal antibody (12CA5) was used as ascites from the corresponding hyridoma. All restriction enzymes were from New England Biolabs Inc. (Beverly, MA) or Life Technologies, Inc. The AP1 reporter vector (AP1-SEAP) and the myristoylated Sos expression plasmid (MSosE) were provided by Dr. G. Crabtree (Stanford University Medical School) (35Holsinger L.J. Spencer D.M. Austin D.J. Schreiber S.L. Crabtree G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9810-9814Crossref PubMed Scopus (125) Google Scholar). The Ras expression vector pMEXneo-H-RasLys12 was provided by Dr. J. Laborda (Center for Biologics Evaluation and Research, Food and Drug Administration) (40Benito M. Porras A. Nebreda A.R. Santos E. Science. 1991; 253: 565-568Crossref PubMed Scopus (141) Google Scholar). The tagged Erk2 expression plasmid pcDNAIII HA-erk2 (Erk2-HA) was provided by Dr. S. Gutkind (National Institutes of Health). The human Cbl expression plasmid pCI-Cbl was generated by excising the Cbl cDNA from pJZen-Cbl (39Blake T.J. Heath K.G. Langdon W.Y. EMBO J. 1993; 12: 2017-2026Crossref PubMed Scopus (110) Google Scholar) with BamHI and cloning it into the BamHI site of pGEM-11Zf+(Promega). Orientation was confirmed by restriction digestion. Cbl cDNA was then excised from a pGEM-11 clone with the correct orientation using XhoI and NotI and cloned into the expression vector pCI-neo (Promega) through the XhoI andNotI sites. The pCI-c/v-Cbl expression construct was made in an identical manner except that the cDNA was excised from pJZen-c/v-Cbl (39Blake T.J. Heath K.G. Langdon W.Y. EMBO J. 1993; 12: 2017-2026Crossref PubMed Scopus (110) Google Scholar). The Jurkat cell line used was a high CD4-expressing line generated by Drs. T. Oravecz and M. Norcross (Center for Biologics Evaluation and Research, Food and Drug Administration). Jurkat cells were maintained and transfected by electroporation as described (41Rellahan B.L. Jensen J.P. Weissman A.M. J. Exp. Med. 1994; 180: 1529-1534Crossref PubMed Scopus (30) Google Scholar). Jurkat cells (107) were transfected with 4 μg of AP1-SEAP and the indicated expression vectors. Multiple transfections were pooled and allowed to rest for 24 h. The cells were then resuspended in fresh medium, divided into wells at ∼3.3 × 106 cells/well, and either left unstimulated or activated with immobilized OKT3 (20 μg/well) or 10 nm PMA for 12 h. All stimulations were done in duplicate. The supernatant was collected, heated at 65 °C for 30 min, and assayed for alkaline phosphatase activity as follows. 100 μl of supernatant was incubated with 100 μl of 2 × assay buffer (0.2 m EDTA, 2.0 m diethanolamine, 1 mm MgCl2, and 20 mml-homoarginine), pH 9.8, for 5 min, at which time 100 μl of CSPD substrate (Tropix Inc., Bedford, MA) diluted 1:80 in a solution of one-third Emerald Enhance (Tropix Inc.) and two-thirds reaction buffer (0.2 m EDTA, 100 mmdiethanolamine, and 1.0 mm MgCl2), pH 10, was added, and samples were read on an ML3000 microplate luminometer (Dynatech Laboratories Inc., Chantilly, VA). Cells were stimulated with 10 μg of soluble OKT3/107 cells in 0.5 ml of phosphate-buffered saline with 1% bovine serum albumin and lysed by adding an equal volume of 2 × lysis buffer (100 mm Tris-HCl, 4 mmvanadate, 10 mm EDTA, 10% glycerol, and 2% Triton X-100 with protease and phosphatase inhibitors), pH 7.8. Cleared lysates were precipitated with anti-Grb2 bound to protein A-Trisacryl beads (Pierce) or with the indicated GST fusion protein (100 pmol) bound to glutathione-Sepharose 4B beads (Pharmacia Biotech, Uppsala, Sweden). Washed precipitates were eluted in sample buffer, resolved by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Hybond-C Super, Amersham Corp.) in 23 mm Tris-HCl, 192 mm glycine, and 20% (v/v) methanol. Membranes were blocked and probed with primary antibodies followed by a rabbit anti-mouse antibody (Cappel, West Chester, PA), if required, and 125I-protein A (ICN). To analyze Erk activation, Jurkat cells were transfected with 5 μg of either pCI-c/v-Cbl or pCI-Cbl and 2.5 μg of Erk2-HA plasmid and allowed to rest for 24 h. Cells were then left unstimulated or stimulated for 20 min with pervanadate (0.1 mm vanadate and 0.3 mm H2O2) or PMA, lysed, and precipitated with an anti-HA antibody. Equal volumes of the precipitated proteins were then loaded onto gels and analyzed by Western blotting with both an anti-active mitogen-activated protein kinase antibody and an anti-Erk2 antibody. The degree of Erk2 activation was calculated by determining the amount of active Erk2 compared with the amount of total Erk2 in a given sample. Western blots were quantitated by a Molecular Dynamics PhosphorImager using ImageQuant™ software (Version 3.3). The plasmid pGEX-KG-Grb2-N-SH3, which encodes a GST fusion protein of the amino-terminal SH3 domain of Grb2, was provided by Dr. W. Wong (NIDR, National Institutes of Health) (42Sastri L. Lin W. Wong W.T. DiFiore P.P. Scoppa C.A. King C.R. Oncogene. 1995; 11: 1107-1112PubMed Google Scholar). The GST fusion protein of the proline-rich region of Cbl (pGEX-Cbl-polyP) corresponds to amino acids 458–669 of Cbl and was provided by Dr. M. Shapiro (Center for Biologics Evaluation and Research, Food and Drug Administration). All other GST-Grb2 fusion proteins were purchased from Santa Cruz Biotechnology Inc. The IL-2 enhancer is composed of numerous regulatory elements that work in concert to regulate IL-2 production after T cell activation (11Crabtree G.R. Clipstone N.A. Annu. Rev. Biochem. 1994; 63: 1045-1083Crossref PubMed Scopus (626) Google Scholar). One of these elements consists of a conventional AP1 site. After TCR engagement, the induction of AP1 activity is dependent on the activation of multiple effector pathways by Ras (13Deng T. Karin M. Nature. 1994; 371: 171-175Crossref PubMed Scopus (318) Google Scholar, 43Buday L. Egan S.E. Rodriguez Viciana P. Cantrell D.A. Downward J. J. Biol. Chem. 1994; 269: 9019-9023Abstract Full Text PDF PubMed Google Scholar, 44Rayter S.I. Woodrow M. Lucas S.C. Cantrell D.A. Downward J. EMBO J. 1992; 11: 4549-4556Crossref PubMed Scopus (149) Google Scholar, 45Genot E. Cleverley S. Henning S. Cantrell D. EMBO J. 1996; 15: 3923-3933Crossref PubMed Scopus (151) Google Scholar, 46Izquierdo M. Leevers S.J. Marshall C.J. Cantrell D. J. Exp. Med. 1993; 178: 1199-1208Crossref PubMed Scopus (152) Google Scholar, 47Whitehurst C.E. Boulton R.G. Cobb M.H. Geppert T.D. J. Immunol. 1992; 148: 3230-3237PubMed Google Scholar). We therefore used the reporter construct AP1-SEAP, which has the secreted alkaline phosphatase gene under the control of an IL-2 minimal promoter with upstream AP1-binding sites, to assess the effects of Cbl overexpression on TCR-induced AP1 activation. Jurkat T cells were transfected with AP1-SEAP and increasing amounts of a Cbl expression plasmid (pCI-Cbl) or a control vector (pCI-neo) and allowed to rest for 24 h. Cbl overexpression at this point in time was verified by Western blot analysis (Fig.1 A). The cells were then activated with OKT3 or the phorbol ester PMA, which stimulates the Ras/mitogen-activated protein kinase pathway in a TCR/tyrosine kinase-independent manner (48Williams D.H. Woodrow M. Cantrell D.A. Murray E.J. Eur. J. Immunol. 1995; 25: 42-47Crossref PubMed Scopus (26) Google Scholar, 49Gulbins E. Coggeshall K.M. Baier G. Telford D. Langlet C. Baier-Bitterlich G. Bonnefoy-Berard N. Burn P. Wittinghofer A. Altman A. Mol. Cell. Biol. 1994; 14: 4749-4758Crossref PubMed Scopus (124) Google Scholar). Overexpression of Cbl led to a dose-dependent decrease in AP1 activity after stimulation with OKT3 that reached a maximum at ∼30–40% of the control response (Fig. 1 B). Cbl overexpression led to a similar decrease in AP1 activity in cells stimulated with the pharmacologic activator pervanadate (Fig. 1 C), which mimics the activation events generated by TCR ligation (50Lee J.M. Fournel M. Veillette A. Branton P.E. Oncogene. 1996; 12: 253-263PubMed Google Scholar, 51O'Shea J.J. McVicar D.W. Bailey T.L. Burns C. Smyth M.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10306-10310Crossref PubMed Scopus (144) Google Scholar). However, responses to PMA were unaffected by Cbl overexpression (Fig. 1 B). Cbl overexpression also had no effect on the ability of an activated form of Ras (H-RasLys12) or an activating form of Sos (MSosE) to stimulate AP1 activity in Jurkat cells (Fig. 1 D). These data suggest that Cbl overexpression affects AP1 activation prior to or independent of the activation of Ras itself and without directly affecting the ability of Sos to mediate Ras activation. The viral homolog of Cbl, v-Cbl, is a fusion between the group-specific antigen protein of the CAS NS-1 retrovirus and the first 360 amino acids of the Cbl protein (39Blake T.J. Heath K.G. Langdon W.Y. EMBO J. 1993; 12: 2017-2026Crossref PubMed Scopus (110) Google Scholar). This fusion protein localizes predominantly to the nucleus and is capable of inducing the transformation of NIH 3T3 cells. Removal of the viral group-specific antigen protein resulted in a protein (designated c/v-Cbl) (Fig.2 A) that could be found in both the cytoplasm and nucleus, but retained the ability to transform cells (39Blake T.J. Heath K.G. Langdon W.Y. EMBO J. 1993; 12: 2017-2026Crossref PubMed Scopus (110) Google Scholar). To assess the effect that c/v-Cbl had on AP1 activation in T cells, an expression plasmid encoding the truncated protein (pCI-c/v-Cbl) was cotransfected with AP1-SEAP into Jurkat cells. Transfection of pCI-c/v-Cbl resulted in expression of the truncated protein (Fig. 2 B), but had no effect on AP1 activation after stimulation with OKT3 (Fig. 2 C) or pervanadate (data not shown). These data indicate that c/v-Cbl does not affect TCR-induced AP1 activation. In addition, these data suggest that the inhibitory effect of Cbl overexpression on AP1 activation requires the proline-rich carboxyl-terminal region of Cbl. This region contains multiple potential SH3-binding sites, including a binding site for Grb2, as demonstrated by the ability of a GST fusion protein encompassing a portion of this region to interact with and precipitate Grb2 (Fig. 3 A, lower panel) (33Donovan J.A. Ota Y. Langdon W.Y. Samelson L.E. J. Biol. Chem. 1996; 271: 26369-26374Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). During T cell activation, Grb2 is thought to mediate the translocation of Sos from the cytosol to the membrane, thus allowing Sos to interact with and activate Ras. Cbl and Sos bind preferentially and in a mutually exclusive manner to the amino-terminal SH3 domain of Grb2 (data not shown and Refs. 25Buday L. Khwaja A. Sipeki S. Farago A. Downward J. J. Biol. Chem. 1996; 271: 6159-6163Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 28Fukazawa 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, 29Meisner H. Conway B.R. Hartley D. Czech M.P. Mol. Cell. Biol. 1995; 15: 3571-3578Crossref PubMed Scopus (212) Google Scholar, 52Rozakis-Adcock M. Fernley R. Wade J. Pawson T. Bowtell D. Nature. 1993; 363: 83-85Crossref PubMed Scopus (837) Google Scholar). We therefore reasoned that Cbl could regulate AP1 complex assembly by controlling Ras activation via the Sos/Grb2 interaction.Figure 3Cbl and Sos bind to Grb2 in a reciprocal manner. A, Jurkat cells (5 × 106) were stimulated with pervanadate (Perv.; 0.1 mmvanadate and 0.3 mm H2O2) for 2 min, lysed, and precipitated with 100 pmol of the indicated GST-Grb2 fusion protein or a GST fusion protein of the proline-rich region of Cbl (pGEX-Cbl-polyP, amino acids 458–669). Eluted samples were analyzed by Western blotting with an anti-Cbl or anti-Grb2 antibody.B, Jurkat cells (5 × 106) were stimulated with OKT3, and samples were analyzed as described for A.C, Jurkat cells (3 × 107) were stimulated with pervanadate (1 mm vanadate and 3.0 mmH2O2) for 2 min, precipitated with an anti-Grb2 antibody, and analyzed by Western blotting with an anti-Sos antibody and subsequently with the C-15 anti-Cbl antibody. PFPindicates the fusion protein used for precipitation, WBindicates the antibody used for Western blotting, and IPindicates the antibody used for immunoprecipitation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For Cbl to regulate the association of Sos with Grb2, it would need to dissociate from Grb2 after activation, freeing a pool of Grb2 that could then interact with Sos. We and others (25Buday L. Khwaja A. Sipeki S. Farago A. Downward J. J. Biol. Chem. 1996; 271: 6159-6163Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 28Fukazawa 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, 33Donovan J.A. Ota Y. Langdon W.Y. Samelson L.E. J. Biol. Chem. 1996; 271: 26369-26374Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) have analyzed the ability of Cbl to associate with Grb2 under different activation conditions. In unstimulated T cells, Cbl readily associates with the amino-terminal SH3 domain of Grb2, as can be shown by the ability of Cbl to react with a GST fusion protein consisting of this Grb2 domain (Fig. 3 A, upper panel). Cbl associates very weakly with the carboxyl-terminal SH3 domain of Grb2 and not at all with the SH2 domain, even after activation through the TCR (Fig.3 A and data not shown). Activation of Jurkat cells with OKT3 or pervanadate for 2 min leads to a substantial decrease in the amount of Cbl recovered with the full-length Grb2 fusion protein and the GST-Grb2 N-terminal SH3 domain fusion protein (Fig. 3, A andB). The dissociation of Cbl from Grb2 is due to modifications of Cbl itself (either a direct post-translational modification or an induced structural change), rather than to Grb2, as demonstrated by the ability of a GST fusion protein of a fragment of the proline-rich region of Cbl to precipitate equal amounts of Grb2 from unactivated and activated Jurkat cells (Fig. 3 A,lower panel). An activation-dependent dissociation of Cbl from Grb2 was also observed when Cbl was coprecipitated with Grb2 by using a Grb2 antiserum, confirming that it is a physiologic consequence of T cell activation (Fig. 3 C). Interestingly, in unstimulated Jurkat cells, little Sos could be coprecipitated with Grb2, although the amount of Sos that associated with Grb2 increased considerably after activation (Fig. 3 C). The inducible association of Sos with Grb2 after TCR engagement is consistent with the data of Nel et al. (53Nel A.E. Gupta S. Lee L. Ledbetter J.A. Kanner S.B. J. Biol. Chem. 1995; 270: 18428-18436Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and Ravichandranet al. (54Ravichandran K.S. Lorenz U. Shoelson S.E. Burakoff S.J. Mol. Cell. Biol. 1995; 15: 593-600Crossref PubMed Google Scholar), who found it to persist for up to 30 min. Our data indicate that Cbl and Sos interact with an interchangeable pool of Grb2 and suggest that Cbl can regulate TCR-mediated, Sos-dependent activation of the Ras pathway in T lymphocytes by competing with Sos for binding to the amino-terminal SH3 domain of G" @default.
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• W2004202136 title "Cbl-mediated Regulation of T Cell Receptor-induced AP1 Activation" @default.
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