The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels.
Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta 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 beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(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 beta to express its effect on sphingomyelin metabolism.
van Tiel
Claudia M
CM
Center for Biomembranes and Lipid Enzymology, Department of Lipid Biochemistry, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. c.vantiel@chem.uu.nl
Westerman
Jan
J
Paasman
Marten A
MA
Hoebens
Martha M
MM
Wirtz
Karel W A
KW
Snoek
Gerry T
GT
D016475:3T3 Cells; D000409:Alanine / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008565:Membrane Proteins; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D049294:Phospholipid Transfer Proteins; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D013109:Sphingomyelins / Q000096:biosynthesis / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D008565:Membrane Proteins; D010455:Peptides; D049294:Phospholipid Transfer Proteins; D013109:Sphingomyelins; D012694:Serine; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D000409:Alanine