Assay and stimulation of the Rab5 GTPase by the p85 alpha subunit of phosphatidylinositol 3-kinase.
Rab5 is a small monomeric GTPase involved in regulating vesicle fusion events during receptor-mediated endocytosis. During endocytosis of the activated platelet-derived growth factor receptor, phosphatidylinositol 3-kinase (PI3K) remains associated with the receptor. We have found that the p85 alpha subunit of PI3K binds directly to Rab5 and possesses GTPase-activating protein (GAP) activity toward Rab5. We describe two methods used to characterize the GAP activity of p85 toward the Rab5 protein. The first method is a steady-state GAP assay, used to show that the p85 alpha protein has GAP activity toward Rab5. The second method is a single turnover GAP assay and measures changes in the catalytic rate of Rab5 GTP hydrolysis with or without the p85 alpha protein.
Deborah H
DH
Anderson
M Dean
MD
Chamberlain
D020134:Catalytic Domain; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D019869:Phosphatidylinositol 3-Kinases; D020696:rab5 GTP-Binding Proteins