The mammalian Ptdlns 3-kinase is shown to be inhibited by low nanomolar concentrations of demethoxyviridin, an antifungal agent structurally related to wortmannin. The inhibitory potency of both compounds could be observed in purified Ptdlns 3-kinase whether or not the regulatory subunit @85x) was present, suggesting that the inhibitors bind to the catalytic subunit fpl 10) of the Ptdlns 3kinase. These ~hibitors also show similar potency against the intrinsic p85-phosphorylating activity of the pl IO-kinase. However, the structurally related Ptdlns 3-kinase from Succharomyces cerevisae (Vps34p) is not inhibited by either compound. Both inhibitors target the mammalian Ptdlns 3-kinase in vitro and in vivo, implying that these compounds should be useful in suppressing Ptdlns 3-kinase in rna~ali~ systems. The ~~bitors did not affect the m~alian Ptdlns 4-kinase, but they are able to inhibit a membrane-associated Ptdlns 4-kinase from Schizosacchromyces pombe.
Key words: Demethoxyviridin;Wortmannin; Lipid kinase; Phosphatidylinositol 3-kinase
Introductii.ulThe phosphatidylinositol 3kinase (Ptdlns 3-kinase) seems to play an important role in growth factor mediated cell transformation and mitogenesis (reviewed in [l-6]. The enzyme exists as a heterodimer, which contains a regulatory subunit (p8.5) and a catalytic subunit (pll0). The regulatory subunit (p85) is responsible for the association of the complex with other signal transduction elements a process mediated by either the intrinsic SH2 or SH3 domains [7-lo]. The catalytic subunit (pll0) shows homology to the VPS34 gene product (Vps34p) from Saccharomyces cerevisae, which is involved in protein sorting [11,12]. The Vps34p has been shown recently to be a Ptdlns 3-kinase [13].Recent evidence has shown that wortmannin can inhibit the mammalian Ptdlns 3-kinase [14,15] [17,18] and has been employed as a potent inhibitor of neutrophil activation 1191, where it blocks phospholi~ ase C and phospholipase D activation. Studies from this laboratory have shown that wortmannin does not significantly affect partially purified mammalian phospholipase D [20] consistent with the notion that wortmannin targets some upstream signalling component [ 191. In line with the effect of wortmannin on the Ptdlns 3-kinase, it has been reported to affect Ptdlns metabolism but not at the level of the Ptdlns 4-kinase, Ptdlns 4-phosphate 5-kinase nor diacylgly~rol kinase [Zl]. Like wor~a~in the structurally related but distinct compound demethoxyviridin (DMV) has potent effects on neutrophi1 activation that resemble those induced by wortmannin [19]. While DMV has some inhibitory effects upon partially purified phospholipase D, these are seen in the mM range and are not consistent with the efficacy of the compound in vivo [ZO].If Ptdlns 3-kinase is indeed the physiological target of wortmannin, DMV may well act at the same target enzyme. In this paper we compare the ability of wortmannin and DMV to inhibit complexed and free catalytic forms of the Ptdlns 3kinase as well as the related Vps34p fro...
In this paper we present evidence that phosphatidylinositol 4-phosphate induces curvature in biological membranes. The phase behaviour of mixtures of distearoylphosphatidylinositol 4-phosphate (DSPIP) and dioleoylphosphatidylcholine (DOPC) as a function of pressure and temperature has been studied using small-angle X-ray scattering and in the presence of biologically relevant magnesium concentrations. Our results demonstrate that at physiologically relevant concentrations (2 mol%), DSPIP is capable of inducing the formation of the inverse hexagonal phase (H II ) over a wide range of conditions. This result has implications for the structural role of phosphatidylinositol lipids in vivo.
The substrate specificity of the purified, mammalian phosphatidylinositol 3-kinase is subject to modulation by detergents, which are able to switch substrate specificity in vitro in favor of PtdInsP2. This effect of the detergents is due to an activation of the phosphatidylinositol biphosphate 3-kinase activity, while the phosphatidylinositol 3-kinase activity is inhibited. The selective inhibition of the phosphatidylinositol 3-kinase activity (p110 alpha/p85 alpha) is shown here also to be observed by employing cholesterol sulfate or sulfatide at low micromolar concentrations, whereas cholesterol and androsterone sulfate fail to inhibit. These naturally occurring sulfated lipids have at these concentrations no effect on the phosphatidylinositol bisphosphate 3-kinase activity but inhibit the manganese-dependent intrinsic protein kinase activity, thus switching substrate specificity toward the more highly phosphorylated inositol lipids. Cholesterol sulfate and sulfatide inhibit the free catalytic subunit p110 alpha but fail to inhibit the homologous phosphatidylinositol 3-kinase from Saccharomyces cerevisiae (Vps34p), suggesting that these sulfated lipids act specifically on the mammalian phosphatidylinositol 3-kinase. Consistent with this specificity, the regulatory subunit (p85), which is not conserved in the yeast enzyme, is found to play an important role for the affinity of these inhibitors. The implications for the phosphatidylinositol 3-kinase activity in vivo are discussed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.