Phospholipase C-gamma 1 associates with viral and cellular src kinases.
A tyrosine kinase inhibitor, genistein, caused the subcellular translocation of phosphoinositide-specific phospholipase C (PLC) activity from membrane fractions to cytosolic fractions in rat 3Y1 fibroblasts and their transformants by Rous sarcoma virus, SR-3Y1. The ratio of PLC activities associated with the membrane fractions to those of the homogenate fractions was greater in SR-3Y1 (32.6%) than in 3Y1 (20.8%) whereas membrane-associated PLC activities were strikingly reduced to the same levels in both cells by treatment with genistein. Moreover, it was found by immunoblotting analyses of membrane fractions that the amounts of PLC-gamma 1 isozyme were reduced to 20.4% of initial level in SR-3Y1 and to 30.2% of that in 3Y1 cells. While the levels of PLC-delta, another detectable PLC isozyme, were not altered by genistein suggesting that tyrosine kinase plays an important role in the association of PLC-gamma 1 with membranes. PLC-gamma 1 molecules were detected in anti-p60arc antibody immunoprecipitates of both 3Y1 and SR-3Y1 cells. The amounts of PLC-gamma 1 co-immunoprecipitating with src kinases were higher in SR-3Y1 than 3Y1 cells and were reduced in both cell types by treatment with genistein. In addition, it was confirmed that PLC-gamma 1 purified from rat liver was phosphorylated at a tyrosine residue and associated with viral src kinase and that src kinases associated with the recombinant SH2 region of PLC-gamma 1, expressed in Escherichia coli, depending upon phosphorylation of tyrosine residues. These findings suggest that both viral and cellular src kinases associate with PLC-gamma 1 and may mediate cellular signaling in normal and transformant cell growth.
Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Japan.
O
O
Nakanishi
F
F
Shibasaki
M
M
Hidaka
Y
Y
Homma
T
T
Takenawa
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000201:enzymology / Q000235:genetics*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D016391:Genes, src; D019833:Genistein; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007529:Isoflavones / Q000494:pharmacology*; D007700:Kinetics; D015688:Oncogene Protein pp60(v-src) / Q000302:isolation & purification / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D007529:Isoflavones; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); D010738:Type C Phospholipases