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J. Biol. Chem., Vol. 277, Issue 25, 22447-22452, June 21, 2002
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Requires the Protein Kinase C-dependent Phosphorylation
of Serine 262 and Is Essential for Maintaining Plasma Membrane
Sphingomyelin Levels*
,
From the Center for Biomembranes and Lipid Enzymology, Department
of Lipid Biochemistry, Institute of Biomembranes, Utrecht University,
Padualaan 8, 3584 CH Utrecht, The Netherlands
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, Ser262 was identified as the major site of
phosphorylation and Ser165 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
Ser262 also had no effect on the transfer activity of
PI-TP. To investigate the role of Ser262 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.
To whom correspondence should be addressed. Tel.: 31-30-2533952;
Fax: 31-30-2533151; E-mail: c.vantiel@chem.uu.nl.
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