Differential Sensitivity of Phosphatidylinositol 3-Kinase p110γ to Isoforms of G Protein βγ Dimers

Abstract

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 ...

RACK1 Regulates Directional Cell Migration by Acting on Gβγ at the Interface with Its Effectors PLCβ and PI3Kγ

“…For example, by substituting the charged amino acid within residues 44 -54 of G␤ with alanines, we confirmed the results from the peptide-based studies that these residues play a critical role in PLC␤2 activation (Chen et al, 2005). Although we have not yet evaluated the contribution of different amino acids of G␤ to PI3K activation, previous studies from Garrison's group demonstrated that mutation of residues H311, R314, and V315 impaired the ability of G␤␥ to activate PI3K␥ (Kerchner et al, 2004). These residues are contained in the peptide 309 -316, which inhibited ϳ20% of G␤␥-dependent PI3K activity in our studies.…”

“…However, an almost perfect fit between the simulations and empirical data for both GRK2-ct and RACK1 can be achieved when either the affinities of G␤1␥2 for RACK1 and GRK2-ct are set to be twofold lower (200 and 1040 nM, respectively), or the affinity of G␤1␥2 for PI3K␥ is set to be twofold higher (2.8 nM; Supplementary Figure S2, B and C). Because these values are well within the range of variations reported for each parameter in the literature (Pumiglia et al, 1995;Maier et al, 1999;Maier et al, 2000;Kerchner et al, 2004;Chen et al, 2005), this indicates that the experimental data we obtained for both GRK2-ct and RACK1 are comparable to the theoretical predictions. Taken together, these results indicate that like GRK2-ct, RACK1 regulates PI3K␥ activity by competitive binding to G␤␥.…”

“…G␤␥-mediated PI3K␥ activation was determined essentially as described before (Kerchner et al, 2004). To determine the effect of RACK1 and its mutants, they were preincubated with G␤1␥2 (100 nM) for 30 min before the addition of PI3K␥ (50 ng) and lipid vesicles.…”

“…To determine if the lower potency of RACK1 in inhibiting PI3K␥ reflects the relatively lower binding affinity of G␤␥ to RACK1 (EC 50 ϳ 520 nM) than PI3K␥ (EC 50 ϳ 5.6 nM) and GRK2-ct (EC 50 ϳ 100 nM; Pumiglia et al, 1995;Kerchner et al, 2004;Chen et al, 2005), we have generated a mathematical model to describe the effect of RACK1 and GRK2-ct on PI3K␥ activation (Supplementary Figure S1, A-C). We assumed that both RACK1 and GRK2-ct competitively inhibit G␤␥-dependent PI3K␥ activity.…”

Insulin Contributes to Fine-Tuning of the Pancreatic Beta-Cell Response to Glucagon-Like Peptide-1

“…Activation of Erk also affects insulin secretion and proliferation of β-cells (Sonoda et al, 2008). Activation of the phosphatidylinositol 3 kinase (PI3K) pathway by GLP-1 has also been reported either through direct activation by the β/γ-subunits of Gs (Kerchner et al, 2004) or through an indirect pathway involving c-src-mediated transactivation of the epidermal growth factor receptor (EGFR) (Buteau et al, 2003).…”