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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Alkaloids / pharmacology
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Amino Acid Sequence
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Animals
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Base Sequence
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Benzoquinones
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Cell Cycle Proteins*
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Cell Line
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Ceramides / pharmacology
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Chlorocebus aethiops
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DNA Primers
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Diglycerides / pharmacology*
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GTP-Binding Proteins / metabolism*
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Guanosine Diphosphate / metabolism
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Humans
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Kinetics
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Lactams, Macrocyclic
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Molecular Sequence Data
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Muromonab-CD3 / pharmacology
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Mutagenesis, Site-Directed
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Naphthalenes*
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Phorbol 12,13-Dibutyrate / metabolism
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Point Mutation
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Polycyclic Compounds / pharmacology
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Protein Kinase C / antagonists & inhibitors
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Protein Kinase C / metabolism
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Proto-Oncogene Proteins / biosynthesis
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Proto-Oncogene Proteins / metabolism*
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Proto-Oncogene Proteins c-vav
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Proto-Oncogene Proteins p21(ras) / metabolism*
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Quinones / pharmacology
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Receptor Protein-Tyrosine Kinases / metabolism
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Rifabutin / analogs & derivatives
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Staurosporine
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T-Lymphocytes / drug effects
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T-Lymphocytes / metabolism*
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Tetradecanoylphorbol Acetate / pharmacology*
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Transfection
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Tumor Cells, Cultured
Substances
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Alkaloids
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Benzoquinones
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Cell Cycle Proteins
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Ceramides
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DNA Primers
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Diglycerides
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Lactams, Macrocyclic
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Muromonab-CD3
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Naphthalenes
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Polycyclic Compounds
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-vav
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Quinones
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VAV1 protein, human
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calphostin complex
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Guanosine Diphosphate
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Rifabutin
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Phorbol 12,13-Dibutyrate
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herbimycin
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Receptor Protein-Tyrosine Kinases
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Protein Kinase C
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GTP-Binding Proteins
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HRAS protein, human
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Proto-Oncogene Proteins p21(ras)
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Staurosporine
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Tetradecanoylphorbol Acetate