Growth factor stimulation of phospholipase C-gamma 1 activity. Comparative properties of control and activated enzymes.
We demonstrated previously tyrosine phosphorylation-dependent modulation of phospholipase C-gamma 1 (PLC-gamma 1) catalytic activity (Nishibe, S., Wahl, M. I., Hernandez-Sotomayor, S. M. T., Tonks, N. K., Rhee, S. G., and Carpenter, G. (1990) Science 250, 1253-1256). The increase in PLC-gamma 1 catalytic activity in A-431 cells occurs rapidly, with maximal activation 5 min after epidermal growth factor (EGF) stimulation. Certain other growth factors (fibroblast growth factor, platelet-derived growth factor) also stimulate PLC-gamma 1 catalytic activity, whereas insulin does not. A similar increase in PLC-gamma 1 specific activity (2-3-fold) was observed in both soluble (cytosol) and particulate (membrane) preparations from EGF-treated cells. Tyrosine-phosphorylated PLC-gamma 1 was detected in both cytosol and membrane fractions in lysates from EGF-treated A-431 cells, but the proportion of tyrosine-phosphorylated PLC-gamma 1 was higher in the cytosol (approximately 50%) than in the membrane (approximately 20%). Because a micellar concentration of the non-ionic detergent Triton X-100 allows detection of the tyrosine phosphorylation-dependent increase in PLC-gamma 1 catalytic activity in this assay, we evaluated the kinetic properties of PLC-gamma 1, immunoprecipitated from cytosol of control or EGF-treated cells, using substrate, phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2), solubilized in Triton X-100 at various molar ratios. The behavior of the control enzyme differed from the EGF-activated enzyme with respect to both Ks and Km. The control enzyme has a 7.5-fold higher Ks value than the activated enzyme (1.5 mM as compared with 0.22 mM). Activation by EGF is also a positive allosteric modifier of PLC-gamma 1-catalyzed PtdIns 4,5-P2 hydrolysis, i.e. the activated enzyme displayed apparent Michalis-Menton kinetics, with a Km of 0.6 mol fraction PtdIns 4,5-P2, whereas the control enzyme displayed sigmoidal kinetics with respect to PtdIns 4,5-P2 hydrolysis. At low substrate mol fractions (e.g. 0.07), the reaction velocity of the control enzyme was 4-fold lower than the activated enzyme. However, at a high substrate mol fraction (e.g. 0.33), the estimated maximal reaction velocities (Vmax) for both forms of PLC-gamma 1 were equivalent. PLC-gamma 1 activity from both control and EGF-treated cells was stimulated by increasing nanomolar Ca2+ concentrations. Although the catalytic activity of PLC-gamma 1 from EGF-treated cells was greater than control PLC-gamma 1 at every Ca2+ concentration tested, the relative stimulation of activity was markedly greater at Ca2+ concentrations above approximately 300 nM.
Wahl
M I
MI
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Jones
G A
GA
Nishibe
S
S
Rhee
S G
SG
Carpenter
G
G
D016475:3T3 Cells; D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D003902:Detergents; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005346:Fibroblast Growth Factors / Q000494:pharmacology*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D017830:Octoxynol; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011092:Polyethylene Glycols; D011233:Precipitin Tests; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D003902:Detergents; D007328:Insulin; D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010982:Platelet-Derived Growth Factor; D011092:Polyethylene Glycols; D014443:Tyrosine; D005346:Fibroblast Growth Factors; D004815:Epidermal Growth Factor; D015544:Inositol 1,4,5-Trisphosphate; D017830:Octoxynol; D010738:Type C Phospholipases