Phosphatidylinositol kinase is activated in membranes derived from cells treated with epidermal growth factor.
The ability of epidermal growth factor (EGF) to stimulate phosphatidylinositol (PtdIns) kinase activity in A431 cells was examined. The incorporation of 32P from [gamma-32P]ATP into PtdIns by A431 membranes was increased in membranes prepared from cells that had been pretreated with EGF. Demonstration of a stimulation of the PtdIns kinase activity by EGF required the use of subconfluent cultures and was dependent on the inclusion of protease inhibitors in the buffers used to prepare the membranes. Stimulation of the PtdIns kinase activity was rapid. The activation peaked 2 min after the addition of EGF and declined slowly thereafter. Half-maximal stimulation of the PtdIns kinase occurred at 7 nM EGF. Kinetic analyses of the reaction indicated that treatment of the cells with EGF resulted in a decrease in the Km for PtdIns with no change in the Vmax. The kinetic parameters for the utilization of ATP were unchanged in the EGF-treated membranes compared to the control membranes. Pretreatment of the cells with the phorbol ester phorbol 12-myristate 13-acetate blocked the ability of EGF to stimulate PtdIns kinase activity. These findings demonstrate that a PtdIns kinase activity in A431 cells is regulated by EGF and provide a good system for examining the mechanism by which EGF stimulates the activity of this intracellular enzyme.
Walker
D H
DH
Howard Hughes Medical Institute, Washington University School of Medicine, Department of Biological Chemistry, St. Louis, MO 63110.
Pike
L J
LJ
D019870:1-Phosphatidylinositol 4-Kinase; D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D010770:Phosphotransferases / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase; D013755:Tetradecanoylphorbol Acetate