Activation of phosphoinositide 3-kinase gamma 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 PI3Kgamma when using a stripped neutrophil membrane fraction as a source of phospholipid substrate. We have found GTPgammaS-loaded Ras can significantly (6- to 8-fold) activate PI3Kgamma 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 PI3Kgamma 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 PI3Kgamma at the level of the membrane, by allosteric modulation and/or reorientation of the PI3Kgamma, implying that Ras can activate PI3Kgamma without its membrane translocation. This view is supported by structural work that has suggested binding of Ras to PI3Kgamma results in a change in the structure of the catalytic pocket.
Inositide Laboratory, The Babraham Institute, CB2 4AT Cambridge, United Kingdom.
Sabine
S
Suire
Phillip
P
Hawkins
Len
L
Stephens
D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D050356:Lipid Metabolism; D008081:Liposomes / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D013552:Swine; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D008081:Liposomes; D019869:Phosphatidylinositol 3-Kinases; D010743:Phospholipids; D020033:Protein Isoforms; D018631:ras Proteins