A Sphingomyelin-transferring Protein from Chicken Liver. USE OF PYRENE-LABELED PHOSPHOLIPID

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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.

mentioning

confidence: 82%

“…(11,17). Measurements were performed using a fluorimeter (Photon Technology International) equipped with a thermostated cuvette holder and a stirring device.…”

Section: Methodsmentioning

confidence: 99%

The Protein Kinase C-dependent Phosphorylation of Serine 166 Is Controlled by the Phospholipid Species Bound to the Phosphatidylinositol Transfer Protein α

Tiel

Westerman

Paasman

et al. 2000

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“…Phospholipid Transfer Activity Assay-PI, PC, and SM transfer activities were determined in a continuous fluorescence assay using donor vesicles consisting of either Pyr-PI, Pyr-PC or Pyr-SM and PA, egg-PC, and TNP-PE (10:10:70:10, mol%) and acceptor vesicles consisting of PC and PA (95:5, mol%) (3,20). Measurements were performed using a fluorimeter (Photon Technology International) equipped with a thermostated cuvette holder and a stirring device.…”

Section: Methodsmentioning

confidence: 99%

The Golgi Localization of Phosphatidylinositol Transfer Protein β Requires the Protein Kinase C-dependent Phosphorylation of Serine 262 and Is Essential for Maintaining Plasma Membrane Sphingomyelin Levels

Tiel

Westerman

Paasman

et al. 2002

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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...

“…Recently, two isoforms of PI-TP have been identified (i.e. PI-TP␣ and PI-TP␤) that demonstrate differences in cellular localization and in specific lipid transfer activity (2)(3)(4)(5).…”

Section: The Addition Of Pi-tp␣ To a Total Lysate Of Myo-[mentioning

confidence: 99%

Overexpression of Phosphatidylinositol Transfer Protein α in NIH3T3 Cells Activates a Phospholipase A

Snoek¹,

Berrie²,

Geijtenbeek³

et al. 1999

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In order to investigate the cellular function of the mammalian phosphatidylinositol transfer protein ␣ (PI-TP␣), NIH3T3 fibroblast cells were transfected with the cDNA encoding mouse PI-TP␣. Two stable cell lines, i.e. SPI6 and SPI8, were isolated, which showed a 2-and 3-fold increase, respectively, in the level of PI-TP␣. Overexpression of PI-TP␣ resulted in a decrease in the duration of the cell cycle from 21 h for the wild type Upon equilibrium labeling of the cells with myo-[ 3 H] inositol, the relative incorporation of radioactivity in the total inositol phosphate fraction was 2-3-fold increased in SPI6 and SPI8 cells when compared with wtNIH3T3 cells. A detailed analysis of the inositol metabolites showed increased levels of glycerophosphoinositol, Ins(1)P, Ins(2)P, and lysophosphatidylinositol (lyso-PtdIns) in SPI8 cells, whereas the levels of phosphatidylinositol (PtdIns) and phosphatidylinositol 4,5-bisphosphate were the same as those in control cells. The addition of PI-TP␣ to a total lysate of myo-[3 H]inositol-labeled wtNIH3T3 cells stimulated the formation of lyso-PtdIns. The addition of Ca 2؉ further increased this formation. Based on these observations, we propose that PI-TP␣ is involved in the production of lyso-PtdIns by activating a phospholipase A acting on PtdIns. The increased level of lyso-PtdIns that is produced in this reaction could be responsible for the increased growth rate and the partial loss of contact inhibition in SPI8 and SPI6 cells. The addition of growth factors (plateletderived growth factor, bombesin) to these overexpressers did not activate the phospholipase C-dependent degradation of phosphatidylinositol 4,5-bisphosphate.Phospholipid transfer proteins are proteins that are able to transfer phospholipids between membranes in vitro. A major phospholipid transfer protein in mammalian tissues is the phosphatidylinositol transfer protein (PI-TP) 1 (1). Recently, two isoforms of PI-TP have been identified (i.e. PI-TP␣ and PI-TP␤) that demonstrate differences in cellular localization and in specific lipid transfer activity (2-5).PI-TP␣ has been purified from both rat and bovine brain (6, 7). Cloning of the cDNA encoding rat brain PI-TP␣ showed that the protein consists of 271 amino acid residues (8). The subsequent isolation of the cDNAs encoding mouse and human PI-TP␣ revealed a high homology between the different mammalian PI-TPs (about 99% amino acid sequence identity) (9, 10). Furthermore, the cross-reactivity of the antibodies raised against bovine PI-TP␣ with a 35-kDa protein from other animals (e.g. rat, mouse, chicken, frog, and lizard) indicates an extensive conservation of the amino acid sequence between species (11). An exception is PI-TP from yeast (i.e. SEC14p) that has the same molecular weight as mammalian PI-TP and comparable phospholipid transfer activities yet shows no homology in the amino acid sequence (12)(13)(14).So far, very little is known about the precise cellular role of mammalian PI-TP␣. Since PI-TP␣ is able to transfer in vitro PtdIns between membrane...