Recombinant mouse phosphatidylinostol transfer protein β has a major and a minor site of phosphorylation

Abstract

We have tested the reliability of two models for the action of phosphatidylinositol transfer proteins (PITPs) on the synthesis of phosphatidylinositol4,5-bisphosphate by phosphatidylinositol4 phosphate 5-kinase [PI(4)P 5-kinaseI. Here we describe a cellfree, minimal reconstitution system, consisting only of phospholipids, ATP and recombinant forms of both PITPs and PI(4)P 5kinase. We find that both mammalian PITP isoforms alpha and beta can enhance the apparent activity of alpha, beta and gamma isoforms of mammalian PI(4)P 5-kinase. Enhanced kinase activities of up to seventeen times basal levels were observed. PITPs were also shown to co-activate the kinase with previously established activators of these proteins, in some cases this activation is synergistic. Further experiments in permeabilised HL60 cells indicate that these two families of proteins can collaborate in the efficient G-protein-driven synthesis of inositol phosphates.Recombinant mouse phosphatidylinostol transfer protein B has a major C,Maad&Recombinant mouse PI-TPB is a substrate for protein kinase C-dependent phosphorylation in vitro. Phosphoamino acid analysis of in vitro phosphorylated PI-TPP showed that phosphorylation of this protein is restricted to serine residues. Based on a prediction analysis for consensus PKC-dependent phosphorylation sites, these serine residues may be Ser-165 and Ser-262. Replacement of either serinc residue with alanine yielded PI-TPB mutants that were still phosphorylated by PKC. Only when both serine residues were replaced with alanine was the mutant protein no longer a substrate for PKC. The [32P]-labeled forms of wtPI-TPB and of PI-TPB(S165A) and PI-TPB(S262A) were analyzed by two-dimensional tryptic peptide mapping. The tryptic peptide maps of both wtPI-TPp and PI-TPB(S165A) showed three major spots at identical positions. The peptide map of PI-TPB(S262A) showed only one spot which was absent from the peptide map of PI-TPB(S165A) and present as a minor spot in the peptide map of wtPI-TPB. From this we conclude that the major site of phosphorylation is Ser-262. This was confirmed by the Michaelis-Menten analysis of the phosphorylation reaction of wtPI-TPP and of the mutants, yielding a KM of 0.06 PM and a V,, of approximately 23 nmol/min for wtPI-TPp and PI-TPP(S165A) as compared to a KM of 0.30 p,M and a V,, of 5.2 nmoVmin for PI-TPP(S262A). Under optimal conditions of PKC activity (1 mM ATP) wtPI-TPP was phosphorylated for 48%.The PIand PC-transfer activities of wtPI-TPB and DI-TPP(S262A) were identical, whereas PI-TPP(S165A) and PI-TPB(S165A/S262A) lacked both activities. Localization studies in NIH3T3 cells expressing enhanced yellow fluorescent protein (EYFP) PI-TPB fusion proteins indiutcd that in the absence and presence of phorbol esters PI-TPB(S262A) was associated with perinuclear Golgi StfllCNrCS, similar to wtPI-TPB.'279 and a minor site of phosphorylation 1 280 Rab geranylgeranyltransferase in wheat seedlings Rab proteins, members of small GTP-binding proteins family, are required for the targeted m...