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      Regulation of Oxysterol-binding Protein Golgi Localization through Protein Kinase D–mediated Phosphorylation

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          Protein kinase D (PKD) is a critical regulator of Golgi structure and function. Biochemical evidence is presented that demonstrates the oxysterol-binding protein OSBP as a novel PKD substrate. Phosphorylation inhibits OSBP Golgi localization, impairs CERT Golgi localization, and promotes Golgi fragmentation.


          Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIβ and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.

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          Most cited references 47

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          Molecular machinery for non-vesicular trafficking of ceramide.

          Synthesis and sorting of lipids are essential for membrane biogenesis; however, the mechanisms underlying the transport of membrane lipids remain little understood. Ceramide is synthesized at the endoplasmic reticulum and translocated to the Golgi compartment for conversion to sphingomyelin. The main pathway of ceramide transport to the Golgi is genetically impaired in a mammalian mutant cell line, LY-A. Here we identify CERT as the factor defective in LY-A cells. CERT, which is identical to a splicing variant of Goodpasture antigen-binding protein, is a cytoplasmic protein with a phosphatidylinositol-4-monophosphate-binding (PtdIns4P) domain and a putative domain for catalysing lipid transfer. In vitro assays show that this lipid-transfer-catalysing domain specifically extracts ceramide from phospholipid bilayers. CERT expressed in LY-A cells has an amino acid substitution that destroys its PtdIns4P-binding activity, thereby impairing its Golgi-targeting function. We conclude that CERT mediates the intracellular trafficking of ceramide in a non-vesicular manner.
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            Large-scale phosphorylation analysis of mouse liver.

            Protein phosphorylation is a complex network of signaling and regulatory events that affects virtually every cellular process. Our understanding of the nature of this network as a whole remains limited, largely because of an array of technical challenges in the isolation and high-throughput sequencing of phosphorylated species. In the present work, we demonstrate that a combination of tandem phosphopeptide enrichment methods, high performance MS, and optimized database search/data filtering strategies is a powerful tool for surveying the phosphoproteome. Using our integrated analytical platform, we report the identification of 5,635 nonredundant phosphorylation sites from 2,328 proteins from mouse liver. From this list of sites, we extracted both novel and known motifs for specific Ser/Thr kinases including a "dipolar" motif. We also found that C-terminal phosphorylation was more frequent than at any other location and that the distribution of potential kinases for these sites was unique. Finally, we identified double phosphorylation motifs that may be involved in ordered phosphorylation.
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              A conserved ER targeting motif in three families of lipid binding proteins and in Opi1p binds VAP.

              Intracellular lipid traffic is mediated both by membrane vesicles and by a number of non-vesicular pathways facilitated by cytoplasmic lipid binding proteins. For these proteins to act effectively they must be targeted accurately to specific membranes. Here we identify a novel short conserved determinant called the FFAT motif that is shared by several seemingly unrelated lipid binding proteins and is also found in Opi1p, a transcriptional regulator of phospholipid synthesis in yeast. FFAT motifs act as membrane- targeting determinants by their direct interaction with homologues of VAMP-associated protein (VAP), a conserved endoplasmic reticulum (ER) protein. In budding yeast, all four proteins with FFAT motifs interact with Scs2p, a homologue of VAP, to target the ER to some extent. The precise intracellular distribution of each of these proteins depends on the integration of the FFAT-Scs2p interaction with other targeting determinants, and the interaction is functionally significant. We conclude that binding to a VAP homologue is a common mechanism by which proteins with FFAT motifs, most of which are involved in lipid metabolism, target ER membranes.

                Author and article information

                Role: Monitoring Editor
                Mol Biol Cell
                Mol. Bio. Cell
                Molecular Biology of the Cell
                The American Society for Cell Biology
                1 July 2010
                : 21
                : 13
                : 2327-2337
                *Departments of Pathology and
                Signal Transduction, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215;
                Department of Pediatrics, Atlantic Research Centre, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada,
                §Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA 92093
                Author notes
                Address correspondence to: Alex Toker ( atoker@ 123456bidmc.harvard.edu )
                © 2010 by The American Society for Cell Biology

                Molecular biology


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