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| Content Provider | Journal of Biological Chemistry (JBC) |
|---|---|
| Author | Tiel, Claudia M. Van Westerman, Jan Paasman, Marten A. Hoebens, Martha M. Wirtz, Karel W. A. Snoek, Gerry T. |
| 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. |
| Related Links | http://www.jbc.org/content/277/25/22447.abstract |
| Ending Page | 22452 |
| Starting Page | 22447 |
| Page Count | 6 |
| File Format | HTM / HTML PDF |
| ISSN | 00219258 |
| Journal | Journal of Biological Chemistry (JBC) |
| Issue Number | 25 |
| Volume Number | 277 |
| DOI | 10.1074/jbc.M201532200 |
| e-ISSN | 1083351X |
| Language | English |
| Publisher | American Society for Biochemistry and Molecular Biology |
| Publisher Date | 2002-06-21 |
| Access Restriction | Open |
| Subject Keyword | Phosphatidylinositol transfer protein (PI-TP) Wild-type PI-TP (wtPI-TP) Phosphatidylinositol (PI) Phosphatidylcholine (PC) Inositol (Ins) Sphingomyelin (SM) Protein kinase C (PKC) Phosphatidic acid (PA) Phosphatidylserine (PS) Trinitrophenyl phosphatidylethanolamine (TNP-PE) Goat anti-rabbit peroxidase (GAR-PO) Bacterial sphingomyelinase (bSMase) Phosphate-buffered saline (PBS) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) Phospholipase A2 (PLA2) Pyrene (Pyr) Guanosine 5′-O-(thiotriphosphate) (GTPγS) L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) MECHANISMS OF SIGNAL TRANSDUCTION |
| Content Type | Text |
| Resource Type | Article |
| Subject | Cell Biology Biochemistry Molecular Biology |
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