Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides

Mol Cell Biol. 1994 Jul;14(7):4749-58. doi: 10.1128/mcb.14.7.4749.

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

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alkaloids / pharmacology
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Benzoquinones
  • Cell Cycle Proteins*
  • Cell Line
  • Ceramides / pharmacology
  • Chlorocebus aethiops
  • DNA Primers
  • Diglycerides / pharmacology*
  • GTP-Binding Proteins / metabolism*
  • Guanosine Diphosphate / metabolism
  • Humans
  • Kinetics
  • Lactams, Macrocyclic
  • Molecular Sequence Data
  • Muromonab-CD3 / pharmacology
  • Mutagenesis, Site-Directed
  • Naphthalenes*
  • Phorbol 12,13-Dibutyrate / metabolism
  • Point Mutation
  • Polycyclic Compounds / pharmacology
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism
  • Proto-Oncogene Proteins / biosynthesis
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-vav
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • Quinones / pharmacology
  • Receptor Protein-Tyrosine Kinases / metabolism
  • Rifabutin / analogs & derivatives
  • Staurosporine
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / metabolism*
  • Tetradecanoylphorbol Acetate / pharmacology*
  • Transfection
  • Tumor Cells, Cultured

Substances

  • Alkaloids
  • Benzoquinones
  • Cell Cycle Proteins
  • Ceramides
  • DNA Primers
  • Diglycerides
  • Lactams, Macrocyclic
  • Muromonab-CD3
  • Naphthalenes
  • Polycyclic Compounds
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-vav
  • Quinones
  • VAV1 protein, human
  • calphostin complex
  • Guanosine Diphosphate
  • Rifabutin
  • Phorbol 12,13-Dibutyrate
  • herbimycin
  • Receptor Protein-Tyrosine Kinases
  • Protein Kinase C
  • GTP-Binding Proteins
  • HRAS protein, human
  • Proto-Oncogene Proteins p21(ras)
  • Staurosporine
  • Tetradecanoylphorbol Acetate