The charge isomers of bovine brain PI-TP␣ (i.e. PI-TP␣I containing a phosphatidylinositol (PI) molecule and PI-TP␣II containing a phosphatidylcholine (PC) molecule) were phosphorylated in vitro by rat brain protein kinase C (PKC) at different rates. From the doublereciprocal plot, it was estimated that the V max values for PI-TP␣I and II were 2.0 and 6.0 nmol/min, respectively; the K m values for both charge isomers were about equal, i.e. 0.7 M. Phosphorylation of charge isomers of recombinant mouse PI-TP␣ confirmed that the PC-containing isomer was the better substrate. Phosphoamino acid analysis of in vitro and in vivo 32 P-labeled PI-TP␣s showed that serine was the major site of phosphorylation. Degradation of 32 P-labeled PI-TP␣ by cyanogen bromide followed by high pressure liquid chromatography and sequence analysis yielded one 32 P-labeled peptide (amino acids 104 -190). This peptide contained Ser-148, Ser-152, and the consensus PKC phosphorylation site Ser-166. Replacement of Ser-166 with an alanine residue confirmed that indeed this residue was the site of phosphorylation. This mutation completely abolished PI and PC transfer activity. This was also observed when Ser-166 was replaced with Asp, implying that this is a key amino acid residue in regulating the function of PI-TP␣. Stimulation of NIH3T3 fibroblasts by phorbol ester or platelet-derived growth factor induced the rapid relocalization of PI-TP␣ to perinuclear Golgi structures concomitant with a 2-3-fold increase in lysophosphatidylinositol levels. This relocalization was also observed for Myc-tagged wtPI-TP␣ expressed in NIH3T3 cells. In contrast, the distribution of Myc-tagged PI-TP␣(S166A) and Myc-tagged PI-TP␣(S166D) were not affected by phorbol ester, suggesting that phosphorylation of Ser-166 was a prerequisite for the relocalization to the Golgi. A model is proposed in which the PKC-dependent phosphorylation of PI-TP␣ is linked to the degradation of PI.

Recombinant mouse phosphatidylinositol transfer protein (PI-TP)␤ is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser 262 was identified as the major site of phosphorylation and Ser 165 as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP␤ and PI-TP␤(S262A) were identical, whereas PI-TP␤(S165A) was completely inactive. PKC-dependent phosphorylation of Ser 262 also had no effect on the transfer activity of PI-TP␤. To investigate the role of Ser 262 in the functioning of PI-TP␤, wtPI-TP␤ and PI-TP␤(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP␤ from its phosphorylated form. After Western blotting, wtPI-TP␤ was found to be 85% phosphorylated, whereas PI-TP␤(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP␤ was strongly reduced. Immunolocalization showed that wtPI-TP␤ was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP␤ was distributed throughout the cell similar to what was observed for PI-TP␤(S262A). In contrast to wtPI-TP␤ overexpressors, cells overexpressing PI-TP␤(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP␤ to express its effect on sphingomyelin metabolism.Eukaryotic phosphatidylinositol transfer proteins (PI-TPs) 1 belong to a family of highly conserved proteins that are able to transfer phospholipids between membranes or from membrane to enzyme (1). In mammalian tissues at least two isoforms, PI-TP␣ and PI-TP␤, are found. PI-TP␣ is able to transfer phosphatidylinositol (PI) and, to a lesser extent, phosphatidylcholine (PC) (2-6) and is mainly localized in the cytosol and in the nucleus (7). Similar to PI-TP␣, PI-TP␤ is able to transfer PI and PC but is also able to transfer sphingomyelin (SM) (8). PI-TP␤ is mainly associated with the Golgi apparatus (7). The primary sequences of PI-TP␣ and PI-TP␤ are very similar, with an identity of 77% and a similarity of 94% (9).To date, little is known about the exact cellular function of PI-TP␣ and PI-TP␤. In a cell-free system containing transGolgi membranes, both PI-TP␣ and PI-TP␤ stimulated the formation of constitutive secretory vesicles and immature granules (10). In permeabilized, cytosol-depleted HL60 cells, both isoforms restored GTP␥S-stimulated protein secretion and phospholipase C-mediated inositol lipid signaling (11,12). In these assays, PI-TP␣ and PI-TP␤ were found to function equally well despite their different biochemical properties and cellular localizations. On the other hand, NIH3T3 cells with increased expression of either PI-TP␣ or PI-TP␤ demonstrated remarkable differences in lipid metabolic pathways. Cells overexpressing PI-TP␣ (SPI␣ cells) showed a...

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...