Platelet-derived growth factor activates membrane-associated phosphatidylinositol 3-kinase and mediates its translocation from the cytosol. Detection of enzyme activity in detergent-solubilized cell extracts.
Phosphatidylinositol 3-kinase (PI 3-kinase) activity has been detected in immune complexes with active protein tyrosine kinases, and its products have been measured in intact cells in response to growth stimuli. Both methods do not directly evaluate whole cell PI 3-kinase enzymatic activity. We have developed a sensitive method to measure PI 3-kinase activity in diluted, detergent-containing whole cell extracts and used this method to determine total, soluble, and membrane-associated PI 3-kinase activity in PDGF-stimulated NIH 3T3 fibroblasts. PDGF stimulation induced a 1.4-fold increase in total Nonidet P-40-extractable PI 3-kinase activity, which occurred within 1 min and was maintained above basal levels at 10 min. At the same time, PI 3-kinase activity in the soluble fraction decreased 30-50%. However, membrane-bound PI 3-kinase activity increased 2.4-fold at 1 min and 3.1-fold at 5 min. Translocation of the p85 PI 3-kinase subunit to the membrane was maximal at 10 min. These results suggest that PDGF-mediated activation of PI 3-kinase in membrane fraction results from initial intrinsic enzymatic activation followed by translocation from the cytosol.
Susa
M
M
St. Elizabeth's Hospital, Department of Biomedical Research, Tufts University School of Medicine, Boston, Massachusetts 02135.
Keeler
M
M
Varticovski
L
L
D016475:3T3 Cells; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D003902:Detergents; D004789:Enzyme Activation; D007700:Kinetics; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols / Q000302:isolation & purification / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*
D003902:Detergents; D010716:Phosphatidylinositols; D010982:Platelet-Derived Growth Factor; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases