Transformation by pp60src or stimulation of cells with epidermal growth factor induces the stable association of tyrosine-phosphorylated cellular proteins with GTPase-activating protein.
GTPase-activating protein (GAP) is a cytosolic protein that stimulates the rate of hydrolysis of GTP (GTP to GDP) bound to normal p21ras, but does not catalyze the hydrolysis of GTP bound to oncogenic, activated forms of the ras protein. Transformation of cells with v-src or activated transforming variants of c-src or stimulation of cells with epidermal growth factor resulted in the stable association of GAP with two tyrosine-phosphorylated cellular proteins of 64 kDa (p64) and 190 kDa (p190). Analysis of GAP immune complexes isolated from extracts of metabolically labeled src-transformed cells and epidermal growth factor-stimulated cells indicated that tyrosine phosphorylation of p64 and p190 appeared to be coincident with the stable association of these proteins with GAP. Quantitation of the amount of p64 associated with GAP in v-src-transformed cells, however, indicated that only 15 to 25% of tyrosine-phosphorylated p64 was found in complex with GAP. Mutations within the SH2 region of pp60src that render activated pp60src defective for transformation inhibited the efficient formation of complexes between GAP and the tyrosine-phosphorylated forms of p64 and p190. From these data, we suggest that tyrosine phosphorylation and stable association of p64 with GAP is an important step in mediating cellular signaling through the p21ras-GAP pathway.
Bouton
A H
AH
Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, Charlottesville 22908.
Kanner
S B
SB
Vines
R R
RR
Wang
H C
HC
Gibbs
J B
JB
Parsons
J T
JT
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics*; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002642:Chick Embryo; D015180:Electrophoresis, Gel, Two-Dimensional; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D020690:GTPase-Activating Proteins; D016391:Genes, src; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics*; D051381:Rats; D014162:Transfection; D014443:Tyrosine; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D010748:Phosphopeptides; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D014443:Tyrosine; D004815:Epidermal Growth Factor; D016392:Proto-Oncogene Proteins pp60(c-src)