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)β is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser262 was identified as the major site of phosphorylation and Ser165 as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TPβ and PI-TPβ(S262A) were identical, whereas PI-TPβ(S165A) was completely inactive. PKC-dependent phosphorylation of Ser262 also had no effect on the transfer activity of PI-TPβ. To investigate the role of Ser262 in the functioning of PI-TPβ, wtPI-TPβ and PI-TPβ(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TPβ from its phosphorylated form. After Western blotting, wtPI-TPβ was found to be 85% phosphorylated, whereas PI-TPβ(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TPβ was strongly reduced. Immunolocalization showed that wtPI-TPβ was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TPβ was distributed throughout the cell similar to what was observed for PI-TPβ(S262A). In contrast to wtPI-TPβ overexpressors, cells overexpressing PI-TPβ(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-TPβ to express its effect on sphingomyelin metabolism.