The ability of G protein ␣ and ␥ subunits to activate the p110␥ isoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) was examined using pure, recombinant G proteins and the p101/p110␥ form of PtdIns 3-kinase reconstituted into synthetic lipid vesicles. GTPactivated G s , G i , G q , or G o ␣ subunits were unable to activate PtdIns 3-kinase. Dimers containing G 1-4 complexed with ␥ 2 -stimulated PtdIns 3-kinase activity about 26-fold with EC 50 values ranging from 4 to 7 nM. G 5 ␥ 2 was not able to stimulate PtdIns 3-kinase despite producing a 10-fold activation of avian phospholipase C. A series of dimers with  subunits containing point mutations in the amino acids that undergo a conformational change upon interaction of ␥ with phosducin (1H311A␥2, 1R314A␥2, and 1W332A␥2) was tested, and only 1W332A␥2 inhibited the ability of the dimer to stimulate PtdIns 3-kinase. Dimers containing the  1 subunit complexed with a panel of different G␥ subunits displayed variation in their ability to stimulate PtdIns 3-kinase. The  1 ␥ 2 ,  1 ␥ 10 ,  1 ␥ 12 , and  1 ␥ 13 dimers all activated PtdIns 3-kinase about 26-fold with 4 -25 nM EC 50 values. The  1 ␥ 11 dimer, which contains the farnesyl isoprenoid group and is highly expressed in tissues containing the p101/p110␥ form of PtdIns 3-kinase, was ineffective. The role of the prenyl group on the ␥ subunit in determining the activation of PtdIns 3-kinase was examined using ␥ subunits with altered CAAX boxes directing the addition of farnesyl to the ␥ 2 subunit and geranylgeranyl to the ␥ 1 and ␥ 11 subunits. Replacement of the geranylgeranyl group of the ␥ 2 subunit with farnesyl inhibited the activity of  1 ␥ 2 on PtdIns 3-kinase. Conversely, replacement of the farnesyl group on the ␥ 1 and ␥ 11 subunit with geranylgeranyl restored almost full activity. These findings suggest that all  subunits, with the exception of  5 , interact equally well with PtdIns 3-kinase. In contrast, the composition of the ␥ subunit and its prenyl group markedly affects the ability of the ␥ dimer to stimulate PtdIns 3-kinase.The generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) 1 in the inner leaflet of the plasma membrane is critical to the regulation of cell function (1-3). The phosphorylated inositol head group provides a docking site for proteins containing pleckstrin homology domains (PH domains) and leads to activation of many enzymes including the phosphoinositol-dependent protein kinase and protein kinase B (Akt). Activation of protein kinase B regulates multiple cellular functions including differentiation, regulation of metabolic events, cell survival, and motility (1-3). In keeping with this central role, the level of PIP3 is tightly regulated, it can be elevated by multiple classes of receptors (1, 2), and there are specific phosphatidylinositol 5-phosphatases (SHIP and PTEN) that degrade the signal (4 -8).A large family of phosphatidylinositol 4,5-bisphosphate 3-kinases (PtdIns 3-kinases) is responsible for generating PIP3 by phosphorylating the D3 ...
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