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

Abstract

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...

Rottlerin Inhibits Chlamydial Intracellular Growth and Blocks Chlamydial Acquisition of Sphingolipids from Host Cells

“…Although the inclusion recruitment of PKC␦ may prevent PKC␦ from exerting its proapoptotic activity in the mitochondria (20,27), the localization of PKC␦ near the inclusions may also serve as a platform for Chlamydia to utilize the PKC␦ activity to facilitate chlamydial acquisition of host lipids. This hypothesis is supported by the finding that PKC␦ plays an essential role in various exocytosis processes, including antigen receptor-induced lytic degranulation (19), insulin secretion (28), and sphingomyelin transportation from the Golgi apparatus to the plasma membrane (29). In addition, PKC␦ can also activate the Raf-MEK-ERK-cPLA2 signaling pathway (17), which is required for chlamydial acquisition of glycerophospholipids from host cells (25).…”

Phosphatidylinositol transfer proteins: From closed for transport to open for exchange

“…Since the phosphomimetic mutants (S166D, S166E) fail to express transfer activity we may assume that phosphorylated PI-TPa is inactive as well. This also holds for PI-TPb where the mutant PI-TPb(S165A) lacks transfer activity (Van Tiel et al, 2002). As can be seen from Fig.…”

“…These serine residues were positively identified as the phosphorylation sites by using mutant PI-TPa (S166A) and mutant PI-TPbs (S165A, S262A, S165A/S262A) as substrates for PKC. As for PI-TPb, Ser165 was identified as the minor and Ser262 as the major phosphorylation site (van Tiel et al, 2002). Also in PI-TPa Ser166 was phosphorylated to a very limited extent.…”

“…PI-TPa and PI-TPb are substrates for protein kinase C (van Tiel et al, 2000(van Tiel et al, , 2002. Phosphoamino acid analysis of in vitro 32 P-labeled PI-TPs demonstrated that both isoforms were exclusively phosphorylated on a serine residue.…”

“…In contrast to the wtPI-TPb overexpressors, the PI-TPb(S262A) overexpressors were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that the association with the Golgi body is a prerequisite for PI-TPb to express its effect on sphingomyelin metabolism (van Tiel et al, 2002).…”