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Papers In Press, published online ahead of print April 12, 2002
Dept. of Lipid Biochemistry, Centre for Biomembranes and Lipid Enzymology, Utrecht, Utrecht 3584 CH
Corresponding Author: c.vantiel{at}chem.uu.nl
Recombinant mouse PI-TP
J. Biol. Chem, 10.1074/jbc.M201532200
Submitted on February 14, 2002
Revised on April 8, 2002
Accepted on April 12, 2002
The Golgi localization of phosphatidylinositol transfer protein
requires the protein kinase C-dependent phosphorylation of serine-262 and is essential for maintaining plasma membrane sphingomyelin levels
is a substrate for protein kinase C-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 site. The phospholipid transfer activities of wtPI-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. 2D-PAGE analysis of cell lysates was used to separate PI-TP from its phosphorylated form. After Western blotting wtPI-TP was found to be phosphorylated for 85% 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 the PKC-dependent association with the Golgi complex is a prerequisite for PI-TP to express its effect on sphingomyelin metabolism.
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