Human phosphoinositide 3-kinase C2 beta, the role of calcium and the C2 domain in enzyme activity

The cDNA for a human Class II phosphoinositide 3-kinase (PI 3-kinase C2 bet a) with a C2 domain was cloned from a U937 monocyte cDNA library and the en zyme expressed in mammalian and insect cells. Like other Class II PI 3-kina ses in vitro, PI 3-kinase C2 beta utilizes phosphatidylinositol (PI) and PI 4-monophosphate but not PI 4,5-biphosphate as substrates in the presence o f Mg2+. Remarkably, and unlike other PI 3-kinases, the enzyme can use eithe r Mg-ATP or Ca-ATP to generate PI 3-monophosphate. PI 3-kinase C2 beta, lik e the Class I PI 3-kinases, but unlike PI 3-kinase C2 alpha, is sensitive t o low nanomolar levels of the inhibitor wortmannin. The enzyme is not regul ated by the small GTP-binding protein Pas. The C2 domain of the enzyme boun d anionic phospholipids such as PI and phosphatidylserine in vitro, but did not co-operatively bind Ca2+ and phospholipids. Deletion of the C2 domain increased the lipid kinase activity suggesting that it functions as a negat ive regulator of the catalytic domain. Although presently it is not known w hether PI 3-kinase C2 beta is regulated by Ca2+ in vivo, our results sugges t a novel role for Ca2+ ions in phosphate transfer reactions.