By using purified preparations we show that nanomolar concentrations of G␤␥ significantly stimulated lipid kinase activity of phosphatidylinositol 3-kinase (PI3K) ␤ and PI3K␥ in the presence as well as in the absence of non-catalytic subunits such as p85␣ or p101. Concomitantly, G␤␥ stimulated autophosphorylation of the catalytic subunit of PI3K␥ (EC 50 , 30 nM; stoichiometry >0.6 mol of P i /mol of p110␥), which also occurred in the absence of p101. Surprisingly, we found that p101 affected the lipid substrate preference of PI3K␥ in its G␤␥-stimulated state. With phosphatidylinositol as substrate, p110␥ but not p101/p110␥ was significantly stimulated by G␤␥ to form PI-3-phosphate (EC 50 , 20 nM). The opposite situation was found when PI-4,5-bisphosphate served as substrate. G␤␥ efficiently and potently (EC 50 , 5 nM) activated the p101/p110␥ heterodimer but negligibly stimulated the p110␥ monomer to form PI-3,4,5-trisphosphate. However, this weak stimulatory effect on p110␥ was overcome by excess concentrations of G␤␥ (EC 50 , 100 nM). This finding is in accordance with the in vivo situation, where activated PI3K catalyzes the formation of PI-3,4,5-trisphosphate but not PI-3-phosphate. We conclude that p101 is responsible for PI-4,5-bisphosphate substrate selectivity of PI3K␥ by sensitizing p110␥ toward G␤␥ in the presence of PI-4,5-P 2 .Phosphoinositides are integral constituents of eukaryotic lipid bilayers but also play a crucial role in transmembrane signaling (1, 2). An exponent that sets up one-third of all phosphoinositides in mammalian cells is phosphatidylinositol-4,5-bisphosphate (PI-4,5-P 2 ) 1 (3), which serves as a precursor for intracellular second messengers. On the one hand it is cleaved into inositol-1,4,5-P 3 and diacylglycerol by members of the phospholipase C family (4), which respond to receptor tyrosine kinases and G-protein-coupled receptors (GPCRs) (5, 6).On the other hand, the D-3 position of the inositol ring of PI-4,5-P 2 is sensitive to phosphorylation leading to phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P 3 ) (7). PI-3,4,5-P 3 is considered to act as a second messenger, since it is absent in quiescent cells but spikes instantly upon exposure to various stimuli (8, 9). Putative PI-3,4,5-P 3 -dependent functions include regulation of cell proliferation, survival, cytoskeletal rearrangements, and vesicle trafficking (10 -12). Hence, it is not surprising that PI-3,4,5-P 3 has also been implicated in pathophysiological processes leading to tumor growth and malignancy (13-15).PI-3,4,5-P 3 is generated from PI-4,5-P 2 by members of a considerably large family of enzymes called class I phosphoinositide 3-kinases (PI3K) (16 -20). They are heterodimers consisting of 110 -120-kDa catalytic (p110␣, -␤, -␥, and -␦) and 50 -100-kDa non-catalytic subunits (p85␣, -␤, p55␥, and p101), which are also capable of phosphorylating PI and PI-4-P in vitro, although they are assumed to exhibit a preference for PI-4,5-P 2 within the cell (21, 22). In contrast, class II and class III PI3Ks show a mor...

Class I phosphoinositide 3-kinases (PI3Ks) regulate important cellular processes such as mitogenesis, apoptosis, and cytoskeletal functions. They include PI3K␣, -␤, and -␦ isoforms coupled to receptor tyrosine kinases and a PI3K␥ isoform activated by receptor-stimulated G proteins. This study examines the direct interaction of purified recombinant PI3K␥ catalytic subunit (p110␥) and G␤␥ complexes. When phosphatidylinositol was used as a substrate, G␤␥ stimulated p110␥ lipid kinase activity more than 60-fold (EC 50 , ϳ20 nM). Stimulation was inhibited by G␣ o -GDP or wortmannin in a concentration-dependent fashion. Stoichiometric binding of a monoclonal antibody to the putative pleckstrin homology domain of p110␥ did not affect G␤␥-mediated enzymatic stimulation, whereas incubation of G␤␥ with a synthetic peptide resembling a predicted G␤␥ effector domain of type 2 adenylyl cyclase selectively inhibited activation of p110␥. G␤␥ complexes bound to N-as well as C-terminal deletion mutants of p110␥. Correspondingly, these enzymatically inactive N-and C-terminal mutants inhibited G␤␥-mediated activation of wild type p110␥. Our data suggest that (i) p110␥ directly interacts with G␤␥, (ii) the pleckstrin homology domain is not the only region important for G␤␥-mediated activation of the lipid kinase, and (iii) G␤␥ binds to at least two contact sites of p110␥, one of which is close to or within the catalytic core of the enzyme.

We have previously reported that, in venous myocytes, Gbetagamma scavengers inhibit angiotensin AT1A receptor-induced stimulation of L-type Ca2+ channels (1). Here, we demonstrate that intracellular infusion of purified Gbetagamma complexes stimulates the L-type Ca2+ channel current in a concentration-dependent manner. Additional intracellular dialysis of GDP-bound inactive Galphao or of a peptide corresponding to the Gbetagamma binding region of the beta-adrenergic receptor kinase completely inhibited the Gbetagamma-induced stimulation of Ca2+ channel currents. The gating properties of the channel were not affected by intracellular application of Gbetagamma, suggesting that Gbetagamma increased the whole-cell calcium conductance. In addition, both the angiotensin AT1A receptor- and the Gbetagamma-induced stimulation of L-type Ca2+ channels were blocked by pretreatment of the cells with wortmannin, at nanomolar concentrations. Correspondingly, intracellular infusion of an enzymatically active purified recombinant Gbetagamma-sensitive phosphoinositide 3-kinase, PI3Kgamma, mimicked Gbetagamma-induced stimulation of Ca2+ channels. Both Gbetagamma- and PI3Kgamma-induced stimulations of Ca2+ channel currents were reduced by protein kinase C inhibitors suggesting that the Gbetagamma/PI3Kgamma-activated transduction pathway involves a protein kinase C. These results indicate for the first time that Gbetagamma dimers stimulate the vascular L-type Ca2+ channels through a Gbetagamma-sensitive PI3K.

In this study, G␤ specificity in the regulation of G␤␥-sensitive phosphoinositide 3-kinases (PI3Ks) and phospholipase C␤ (PLC␤) isozymes was examined. Recombinant mammalian G␤ 1-3 ␥ 2 complexes purified from Sf9 membranes stimulated PI3K␥ lipid kinase activity with similar potency (10 -30 nM) and efficacy, whereas transducin G␤␥ was less potent. Functionally active G␤ 5 ␥ 2 dimers were purified from Sf9 cell membranes following coexpression of G␤ 5 and G␥ 2-His . This preparation as well as G␤ 1 ␥ 2-His supported pertussis toxin-mediated ADP-ribosylation of G␣ i1 . G␤ 1 ␥ 2-His stimulated PI3K␥ lipid and protein kinase activities at nanomolar concentrations, whereas G␤ 5 ␥ 2-His had no effect. Accordingly, G␤ 1 ␥ 2-His , but not G␤ 5 ␥ 2-His , significantly stimulated the lipid kinase activity of PI3K␤ in the presence or absence of tyrosine-phosphorylated peptides derived from the p85-binding domain of the platelet derived-growth factor receptor. Conversely, both preparations were able to stimulate PLC␤ 2 and PLC␤ 1 . However, G␤ 1 ␥ 2-His , but not G␤ 5 ␥ 2-His , activated PLC␤ 3 . Experimental evidence suggests that the mechanism of G␤ 5 -dependent effector selectivity may differ between PI3K and PLC␤. In conclusion, these data indicate that G␤ subunits are able to discriminate among effectors independently of G␣ due to selective protein-protein interaction.

High Content Screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modelling systems. In this work, we established a medium to high throughput HCS assay in the 384-well format to measure cellular type I phosphoinositide 3 kinase (PI3K) activity. Type I PI3K is involved in several intracellular pathways such as cell survival, growth and differentiation as well as immunological responses. As a cellular model system we used Chinese Hamster Ovary (CHO) cells that had been stably transfected with human insulin receptor (hIR) and an AKT1-enhanced green fluorescent protein (EGFP) fusion construct. Upon stimulation of the hIR with insulin-like growth factor-1 (IGF-1), PI3K was activated to phosphorylate phosphatidylinositol (PtdIns)-4,5-bisphosphate at the 3-position, resulting in the recruitment of AKT1-EGFP to the plasma membrane. The AKT1-EGFP redistribution assay was robust and displayed little day-to-day variability, the quantification of the fluorescence intensity associated with plasma membrane spots delivered good Z' statistics. A novel format of compound dose-response testing was employed using serial dilutions of test compounds across consecutive microtiter plates (MTPs). The dose response testing of a PI3K inhibitor series provided reproducible IC50 values. The profiling of the redistribution assay with isoform-selective inhibitors indicates that PI3Kalpha is the main isoform activated in the CHO host cells after IGF-1 stimulation. Toxic compound side effects could be determined using automated image analysis. We conclude that the AKT1-EGFP redistribution assay represents a solid medium/high throughput screening (MTS/HTS) format to determine the cellular activity of PI3K inhibitors under conditions of growth factor stimulation.