Activation of phosphoinositide 3-kinase γ by Ras

Background: Type I phosphoinositide 3-kinases are responsible for the hormone-sensitive synthesis of the lipid messenger phosphatidylinositol(3,4,5)-trisphosphate. Type IA and IB subfamily members contain a Ras binding domain and are stimulated by activated Ras proteins both in vivo and in vitro. The mechanism of Ras activation of type I PI3Ks is unknown, in part because no robust in vitro assay of this event has been established and characterized. Other Ras effectors, such as Raf and phosphoinositide-phospholipase Cepsilon, have been shown to be translocated into the plasma membrane, leading to their activation. Results: We show that posttranslationally lipid-modified, activated N-, H-, K-, and R-Ras proteins can potently and substantially activate PI3K-gamma when using a stripped neutrophil membrane fraction as a source of phospholipid substrate. We have found GTP-gammaS-loaded Ras can significantly (6- to 8-fold) activate PI3K-gamma when using artificial phospholipid vesicles containing their substrate, and this effect is a result of both a decrease in apparent Km for phosphatidylinositol(4,5)-bisphosphate and an increase in the apparent Vmax. However, neither in vivo nor in the two in vitro assays of Ras activation of PI3K-gamma could we detect any evidence of a Ras-dependent translocation of PI3Kgamma to its source of phospholipid substrate. Conclusions: Our data suggest that Ras activate PI3K-gamma at the level of the membrane, by allosteric modulation and/or reorientation of the PI3K-gamma, implying that Ras can activate PI3K-gamma without its membrane translocation. This view is supported by structural work that has suggested binding of Ras to PI3K-gamma results in a change in the structure of the catalytic pocket.