title: Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides.
abstract: We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.
authors.affiliation: Division of Cell Biology, La Jolla Institute for Allergy and Immunology, California 92037.
authors.forename: E
authors.initials: E
authors.lastname: Gulbins
authors.forename: K M
authors.initials: KM
authors.lastname: Coggeshall
authors.forename: G
authors.initials: G
authors.lastname: Baier
authors.forename: D
authors.initials: D
authors.lastname: Telford
authors.forename: C
authors.initials: C
authors.lastname: Langlet
authors.forename: G
authors.initials: G
authors.lastname: Baier-Bitterlich
authors.forename: N
authors.initials: N
authors.lastname: Bonnefoy-Berard
authors.forename: P
authors.initials: P
authors.lastname: Burn
authors.forename: A
authors.initials: A
authors.lastname: Wittinghofer
authors.forename: A
authors.initials: A
authors.lastname: Altman
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016227:Benzoquinones; D018797:Cell Cycle Proteins; D002460:Cell Line; D002518:Ceramides / Q000494:pharmacology; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004075:Diglycerides / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D047029:Lactams, Macrocyclic; D008969:Molecular Sequence Data; D016853:Muromonab-CD3 / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009281:Naphthalenes; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D017354:Point Mutation; D011083:Polycyclic Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000378:metabolism*; D051567:Proto-Oncogene Proteins c-vav; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D017828:Rifabutin / Q000031:analogs & derivatives; D019311:Staurosporine; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000470:Alkaloids; D016227:Benzoquinones; D018797:Cell Cycle Proteins; D002518:Ceramides; D017931:DNA Primers; D004075:Diglycerides; D047029:Lactams, Macrocyclic; D016853:Muromonab-CD3; D009281:Naphthalenes; D011083:Polycyclic Compounds; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; D011809:Quinones; C496618:VAV1 protein, human; C061465:calphostin complex; D006153:Guanosine Diphosphate; D017828:Rifabutin; D015240:Phorbol 12,13-Dibutyrate; C020754:herbimycin; D020794:Receptor Protein-Tyrosine Kinases; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate