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...

The proposed International Linear Collider (ILC) is well-suited for discovering physics beyond the Standard Model and for precisely unraveling the structure of the underlying physics. The physics return can be maximized by the use of polarized beams. This report shows the paramount role of polarized beams and summarizes the benefits obtained from polarizing the positron beam, as well as the electron beam. The physics case for this option is illustrated explicitly by analyzing reference reactions in different physics scenarios. The results show that positron polarization, combined with the clean experimental environment provided by the linear collider, allows to improve strongly the potential of searches for new particles and the identification of their dynamics, which opens the road to resolve shortcomings of the Standard Model. The report also presents an overview of possible designs for polarizing both beams at the ILC, as well as for measuring their polarization.2

Previous results have shown that in rat portal vein myocytes the ␤␥ dimer of the G 13 protein transduces the angiotensin II-induced stimulation of calcium channels and increase in intracellular Ca 2؉ concentration through activation of phosphoinositide 3-kinase (PI3K). In the present work we determined which class I PI3K isoforms were involved in this regulation. Western blot analysis indicated that rat portal vein myocytes expressed only PI3K␣ and PI3K␥ and no other class I PI3K isoforms. In the intracellular presence of an anti-p110␥ antibody infused by the patch clamp pipette, both angiotensin II-and G␤␥-mediated stimulation of Ca 2؉ channel current were inhibited, whereas intracellular application of an anti-p110␣ antibody had no effect. The anti-PI3K␥ antibody also inhibited the angiotensin IIand G␤␥-induced production of phosphatidylinositol 3,4,5-trisphosphate. In Indo-1 loaded cells, the angiotensin II-induced increase in [Ca 2؉ ] i was inhibited by intracellular application of the anti-PI3K␥ antibody, whereas the anti-PI3K␣ antibody had no effect. The specificity of the anti-PI3K␥ antibody used in functional experiments was ascertained by showing that this antibody did not recognize recombinant PI3K␣ in Western blot experiments. Moreover, anti-PI3K␥ antibody inhibited the stimulatory effect of intracellularly infused recombinant PI3K␥ on Ca 2؉ channel current without altering the effect of recombinant PI3K␣. Our results show that, although both PI3K␥ and PI3K␣ are expressed in vascular myocytes, the angiotensin II-induced stimulation of vascular L-type calcium channel and increase of [Ca 2؉ ] i involves only the PI3K␥ isoform.

Abstract-Heterodimeric class I phosphoinositide 3-kinase (PI3K) has been shown to be involved in the stimulation of voltage-gated Ca 2ϩ channels by various mediators. In this study, we bring evidences that vascular L-type Ca 2ϩ channels can be modulated by both tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks. Purified recombinant PI3Ks increased the peak Ca 2ϩ channel current density when applied intracellularly. Furthermore, PI3K␣-, ␤-, and ␦-mediated stimulations of Ca 2ϩ channel currents were increased by preactivation by a phosphotyrosyl peptide, whereas PI3K␥-and ␤-mediated effects were increased by G␤␥. In freshly isolated and cultured vascular myocytes, angiotensin II and G␤␥ stimulated L-type Ca 2ϩ channel current. In contrast, platelet-derived growth factor (PDGF)-BB and the phosphotyrosyl peptide did not stimulate Ca 2ϩ channel current in freshly isolated cells despite the presence of endogenous PDGF receptors and PI3K␣ and PI3K␥. Interestingly, when endogenous PI3K␤ expression arose in cultured myocytes, both PDGF and phosphotyrosyl peptide stimulated Ca 2ϩ channels through PI3K␤, as revealed by the inhibitory effect of an anti-PI3K␤ antibody. These results suggest that endogenous PI3K␤ but not PI3K␣ is specifically involved in PDGF receptor-