title: Lipid and peptide control of phosphatidylinositol 4-kinase IIalpha activity on Golgi-endosomal Rafts.
abstract: The most abundant and widely expressed mammalian phosphoinositide kinase activity is contributed by phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha). In this study we demonstrate that PI4KIIalpha is a novel GTP-independent target of the wasp venom tetradecapeptide mastoparan and that different mechanisms of activation occur in different subcellular membranes. Following cell membrane fractionation mastoparan specifically stimulated a high activity Golgi/endosomal pool of PI4KIIalpha independently of exogenous guanine nucleotides. Conversely, GTPgammaS stimulated a low activity pool of PI4KIIalpha in a separable dense membrane fraction and this response was further enhanced by mastoparan. Overexpression of PI4KIIalpha increased the basal phosphatidylinositol 4-kinase activity of each membrane pool, as well as the mastoparan-dependent activities, thereby demonstrating that mastoparan specifically activates this isozyme. Both mastoparan and M7, at concentrations known to invoke secretion, stimulated PI4KIIalpha with similar efficacies, resulting in an increase in the apparent V(max) and decrease in K(m) for exogenously added PI. Mastoparan also stimulated PI4KIIalpha immunoprecipitated from the raft fraction, indicating that PI4KIIalpha is a direct target of mastoparan. Finally we reveal a striking dependence of both basal and mastoparan-stimulated PI4KIIalpha activity on endogenous cholesterol concentration and therefore conclude that changes in membrane environment can regulate PI4KIIalpha activity.
authors.affiliation: Centre for Molecular Cell Biology, Department of Medicine, Royal Free and University College Medical School, University College London, UK.
authors.forename: Mark G
authors.initials: MG
authors.lastname: Waugh
authors.forename: Shane
authors.initials: S
authors.lastname: Minogue
authors.forename: Dipti
authors.initials: D
authors.lastname: Chotai
authors.forename: Fedor
authors.initials: F
authors.lastname: Berditchevski
authors.forename: J Justin
authors.initials: JJ
authors.lastname: Hsuan
mesh_terms: D002460:Cell Line; D002784:Cholesterol / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D011992:Endosomes / Q000201:enzymology*; D006056:Golgi Apparatus / Q000201:enzymology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008274:Magnesium / Q000494:pharmacology; D021962:Membrane Microdomains / Q000201:enzymology*; D015778:Minor Histocompatibility Antigens; D010455:Peptides / Q000494:pharmacology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D014862:Wasp Venoms / Q000494:pharmacology*
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D015778:Minor Histocompatibility Antigens; D010455:Peptides; D014862:Wasp Venoms; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C037500:mastoparan; D002784:Cholesterol; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase; D008274:Magnesium