Membrane-associated GAIP is a phosphoprotein and can be phosphorylated by clathrin-coated vesicles

GAIP (G alpha interacting protein) is a member of the RCS (regulators of G protein signaling) family and accelerates the turnover of GTP bound to G al pha i, G alpha q, and G alpha 13, There are two pools of GAIP-a soluble and a membrane-anchored pool. The membrane-anchored pool is found on clathrin- coated vesicles (CCVs) and pits in rat liver and AtT-20 pituitary cells. By treatment of a GAIP-enriched rat liver fraction with alkaline phosphatase, we found that membrane-bound GAIP is phosphorylated. By immunoprecipitatio n carried out on [P-32]orthophosphate-labeled AtT-20 pituitary cells stably expressing GAIP, P-32-labeling was associated exclusively with the membran e pool of GAIP, Phosphoamino acid analysis revealed that phosphorylation of GAIP occurred largely on serine residues. Recombinant GAIP could be phosph orylated at its N terminus with purified casein kinase 2 (CK2). It could al so be phosphorylated by isolated CCVs in vitro. Phosphorylation was Mn2+-de pendent, using both purified CK2 and CCVs. Ser-24 was identified as one of the phosphorylation sites. Our results establish that GAIP is phosphorylate d and that only the membrane pool is phosphorylated, suggesting that GAIP c an he regulated by phosphorylation events taking place at the level of clat hrin-coated pits and vesicles.