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
2002; Elsevier BV; Volume: 277; Issue: 25 Linguagem: Inglês
10.1074/jbc.m201532200
ISSN1083-351X
AutoresClaudia M. van Tiel, Jan Westerman, Marten Paasman, Martha M. Hoebens, K.W.A. Wirtz, Gerry T. Snoek,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoRecombinant 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. 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. phosphatidylinositol transfer protein wild-type PI-TP phosphatidylinositol phosphatidylcholine inositol sphingomyelin protein kinase C phosphatidic acid phosphatidylserine trinitrophenyl phosphatidylethanolamine goat anti-rabbit peroxidase bacterial sphingomyelinase phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid phospholipase A2 pyrene guanosine 5′-O-(thiotriphosphate) L-1-tosylamido-2-phenylethyl chloromethyl ketone Eukaryotic phosphatidylinositol transfer proteins (PI-TPs)1 belong to a family of highly conserved proteins that are able to transfer phospholipids between membranes or from membrane to enzyme (1Wirtz K.W.A. Biochem. J. 1997; 324: 353-360Crossref PubMed Scopus (174) Google Scholar). In mammalian tissues at least two isoforms, PI-TPα and PI-TPβ, are found. PI-TPα is able to transfer phosphatidylinositol (PI) and, to a lesser extent, phosphatidylcholine (PC) (2Wirtz K.W.A. Annu. Rev. Biochem. 1991; 60: 73-99Crossref PubMed Scopus (377) Google Scholar, 3van Paridon P.A. Visser A.J. Wirtz K.W.A. Biochim. Biophys. Acta. 1987; 898: 172-180Crossref PubMed Scopus (64) Google Scholar, 4Helmkamp Jr G.M. Harvey M.S. Wirtz K.W.A. Van Deenen L.L.M. J. Biol. Chem. 1974; 249: 6382-6389Abstract Full Text PDF PubMed Google Scholar, 5Helmkamp Jr G.M. Chem. Phys. Lipids. 1985; 38: 3-16Crossref PubMed Scopus (34) Google Scholar, 6Helmkamp Jr G.M. J. Bioenerg. Biomembr. 1986; 18: 71-91Crossref PubMed Scopus (56) Google Scholar) and is mainly localized in the cytosol and in the nucleus (7de Vries K.J. Westerman J. Bastiaens P.I. Jovin T.M. Wirtz K.W.A. Snoek G.T. Exp. Cell Res. 1996; 227: 33-39Crossref PubMed Scopus (81) Google Scholar). Similar to PI-TPα, PI-TPβ is able to transfer PI and PC but is also able to transfer sphingomyelin (SM) (8de Vries K.J. Heinrichs A.A. Cunningham E. Brunink F. Westerman J. Somerharju P.J. Cockcroft S. Wirtz K.W.A. Snoek G.T. Biochem. J. 1995; 310: 643-649Crossref PubMed Scopus (89) Google Scholar). PI-TPβ is mainly associated with the Golgi apparatus (7de Vries K.J. Westerman J. Bastiaens P.I. Jovin T.M. Wirtz K.W.A. Snoek G.T. Exp. Cell Res. 1996; 227: 33-39Crossref PubMed Scopus (81) Google Scholar). The primary sequences of PI-TPα and PI-TPβ are very similar, with an identity of 77% and a similarity of 94% (9Tanaka S. Hosaka K. J. Biochem. (Tokyo). 1994; 115: 981-984Crossref PubMed Scopus (84) Google Scholar).To date, little is known about the exact cellular function of PI-TPα and PI-TPβ. In a cell-free system containing trans-Golgi membranes, both PI-TPα and PI-TPβ stimulated the formation of constitutive secretory vesicles and immature granules (10Ohashi M. de Vries K.J. Frank R. Snoek G.T. Bankaitis V. Wirtz K.W.A. Huttner W.B. Nature. 1995; 377: 544-547Crossref PubMed Scopus (168) Google Scholar). In permeabilized, cytosol-depleted HL60 cells, both isoforms restored GTPγS-stimulated protein secretion and phospholipase C-mediated inositol lipid signaling (11Fensome A. Cunningham E. Prosser S. Tan S.K. Swigart P. Thomas G. Hsuan J. Cockcroft S. Curr. Biol. 1996; 6: 730-738Abstract Full Text Full Text PDF PubMed Google Scholar, 12Cunningham E. Tan S.K. Swigart P. Hsuan J. Bankaitis V. Cockcroft S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6589-6593Crossref PubMed Scopus (99) Google Scholar). In these assays, PI-TPα and PI-TPβ were found to function equally well despite their different biochemical properties and cellular localizations. On the other hand, NIH3T3 cells with increased expression of either PI-TPα or PI-TPβ demonstrated remarkable differences in lipid metabolic pathways. Cells overexpressing PI-TPα (SPIα cells) showed an enhanced PLA2-mediated PI degradation resulting in increased levels of lyso-PI, glycerophosphoinositol, Ins(1)P, and Ins(2)P (13Snoek G.T. Berrie C.P. Geijtenbeek T.B.H. van der Helm H.A. Cadee J.A. Iurisci C. Corda D. Wirtz K.W.A. J. Biol. Chem. 1999; 274: 35393-35399Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). This was not observed in cells overexpressing PI-TPβ (SPIβ cells). However, in SPIβ cells (but not in SPIα cells) it was shown that under conditions in which plasma membrane SM was hydrolyzed to ceramide by exogenous sphingomyelinase, PI-TPβ was involved in maintaining the steady-state levels of SM (14van Tiel C.M. Luberto C. Snoek G.T. Hannun Y.A. Wirtz K.W.A. Biochem. J. 2000; 346: 537-543Crossref PubMed Scopus (31) Google Scholar). It was recently postulated that PI-TPβ plays a key role in SM metabolism, making it a potential regulator of pathways for diacylglycerol production and consumption in the mammalian trans-Golgi network (15Bankaitis V.A. Science. 2002; 295: 290-291Crossref PubMed Scopus (46) Google Scholar). Disruption of the PI-TPβ gene in mice leads to early failure in embryonic development (16Alb J.G. Phillips S.E. Rostand K. Cui X. Pinxteren J. Cotlin L. Manning T. Guo S. York J.D. Sontheimer H. Collawn J.F. Bankaitis V.A. Mol. Biol. Cell. 2002; 13: 739-754Crossref PubMed Scopus (60) Google Scholar).In search of mechanisms by which PI-TP activity is regulated, PI-TPα was shown to be phosphorylated by protein kinase C in vitroas well as in vivo (17Snoek G.T. Westerman J. Wouters F.S. Wirtz K.W.A. Biochem. J. 1993; 291: 649-656Crossref PubMed Scopus (20) Google Scholar, 18van Tiel C.M. Westerman J. Paasman M. Wirtz K.W.A. Snoek G.T. J. Biol. Chem. 2000; 275: 21532-21538Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). PI-TPα was exclusively phosphorylated on Ser166, with the PC-carrying form of PI-TPα more readily phosphorylated than the PI-carrying form (18van Tiel C.M. Westerman J. Paasman M. Wirtz K.W.A. Snoek G.T. J. Biol. Chem. 2000; 275: 21532-21538Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Furthermore, in NIH3T3 cells, PI-TPα was translocated from the cytosol to the Golgi membranes upon phosphorylation after stimulation with phorbol ester. This relocalization of PI-TPα coincided with an increased level of intracellular lyso-PI, indicating the activation of a PI-specific PLA2 (17Snoek G.T. Westerman J. Wouters F.S. Wirtz K.W.A. Biochem. J. 1993; 291: 649-656Crossref PubMed Scopus (20) Google Scholar, 18van Tiel C.M. Westerman J. Paasman M. Wirtz K.W.A. Snoek G.T. J. Biol. Chem. 2000; 275: 21532-21538Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Based on these findings, a model was proposed for the regulation of PI-TPα by PKC-dependent phosphorylation. In contrast to PI-TPα, PI-TPβ purified from bovine or rat brain could not be phosphorylated despite the fact that it contains the same serine residue (Ser165) and an additional putative PKC phosphorylation site (Ser262) not present in PI-TPα (8de Vries K.J. Heinrichs A.A. Cunningham E. Brunink F. Westerman J. Somerharju P.J. Cockcroft S. Wirtz K.W.A. Snoek G.T. Biochem. J. 1995; 310: 643-649Crossref PubMed Scopus (89) Google Scholar, 17Snoek G.T. Westerman J. Wouters F.S. Wirtz K.W.A. Biochem. J. 1993; 291: 649-656Crossref PubMed Scopus (20) Google Scholar, 19Geijtenbeek T.B. de Groot E. van Baal J. Brunink F. Westerman J. Snoek G.T. Wirtz K.W.A. Biochim. Biophys. Acta. 1994; 1213: 309-318Crossref PubMed Scopus (27) Google Scholar).In this study, we report that murine PI-TPβ can be phosphorylated by PKC in vitro as well as in situ. The major site of phosphorylation was Ser262, whereas Ser165was a minor site. By site-directed mutagenesis we have shown that Ser165 is essential for the lipid transfer activity of the protein, whereas phosphorylation of Ser262 is required for the association of PI-TPβ with the Golgi membranes. This latter residue is also essential for the ability of PI-TPβ to maintain steady-state levels of SM in the plasma membrane.DISCUSSIONIn this study we have shown that PI-TPβ is a substrate for PKC. By mutation analysis we could establish that Ser262 was the main phosphorylation site. However, Ser165, which is analogous to Ser166 in PI-TPα, was also phosphorylated although to a very limited extent. In a previous study we had shown that the PKC-dependent phosphorylation of PI-TPα was restricted to Ser166 (18van Tiel C.M. Westerman J. Paasman M. Wirtz K.W.A. Snoek G.T. J. Biol. Chem. 2000; 275: 21532-21538Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The PI-TPα isoform, which is highly homologous to PI-TPβ (similarity of 94%), lacks Ser262 (9Tanaka S. Hosaka K. J. Biochem. (Tokyo). 1994; 115: 981-984Crossref PubMed Scopus (84) Google Scholar). Assuming that phosphorylation of the PI-TP isoforms is important for the regulation of their function, we were interested to establish the role of Ser262 in the function of PI-TPβ.The peptide maps of wtPI-TPβ and PI-TPβ(S165A) show three major spots, which are absent from the map of PI-TPβ(S262A) (Fig. 3). This indicates that these three spots represent peptides containing phosphorylated Ser262. The formation of these peptides is probably due to the presence of multiple tryptic cleavage sites in the amino acid sequence of the peptide Met-Arg-Lys-Lys-Gly-Ser262-Val-Arg. Partial digestion would yield the peptides Lys-Lys-Gly-Ser-Val-Arg, Lys-Gly-Ser-Val-Arg, and Gly-Ser-Val-Arg. According to the method described in Ref. 22Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1273) Google Scholar, we could assign Lys-Lys-Gly-Ser-Val-Arg to spot 1, Lys-Gly-Ser-Val-Arg to spot 2, and Gly-Ser-Val-Arg to spot 3. The tryptic map of PI-TPβ(S262A) showed one spot representing a peptide containing phosphorylated Ser165. This spot was barely visible in the peptide map of wtPI-TPβ, indicating that phosphorylation was almost exclusively restricted to Ser262.Replacement of Ser165 with Ala yielded PI-TPβ(S165A) and the double mutated PI-TPβ(S165A/S262A), both of which in vitro completely lacked PI, PC, and SM transfer activity. On the other hand, PI-TPβ(S262A) was fully active. Mutation of the corresponding serine (Ser166) in PI-TPα also yielded an inactive protein (18van Tiel C.M. Westerman J. Paasman M. Wirtz K.W.A. Snoek G.T. J. Biol. Chem. 2000; 275: 21532-21538Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). From the three-dimensional structure it can be inferred that Ser166 is exposed at the surface as part of the regulatory loop of PI-TPα (26Yoder M.D., T.L.M. Tremblay J.M. Oliver R.L. Yarbrough L.R. Helmkamp Jr G.M. J. Biol. Chem. 2001; 276: 9246-9252Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Hence it is possible that the loss of transfer activity is due to the inability of PI-TPα(S166A) to properly interact with the membrane interface. However, at this stage we cannot exclude the possibility that replacement of Ser166 with Ala affects the proper folding of the protein during synthesis in E. coli. The same explanations may hold for the lack of transfer activity observed in PI-TPβ(S165A) and PI-TPβ(S165A/S262A). After PKC-dependent phosphorylation, the phospholipid transfer activity of PI-TPβ was unchanged, indicating that phosphorylation of Ser262 had no effect (Fig. 4).The lysates from SPIβ cells contained two forms of PI-TPβ that could be separated by isoelectric focusing. From densitometric analysis it was estimated that 85% of the PI-TPβ collected at pH 6.2 and 15% at pH 6.5. Treatment of the cells with the PKC inhibitor GF 109203X shifted PI-TPβ to pH 6.5, strongly suggesting that the spot at pH 6.2 represented the phosphorylated form of PI-TPβ. Because the lysate from the SPIβ(S262A) cells contained predominantly PI-TPβ at pH 6.5, we conclude that in situ PI-TPβ is constitutively phosphorylated at Ser262. Given that GF 109203X inhibits conventional and novel type PKCs (27Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charons D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 28Mwanjewe J. Spitaler M. Ebner M. Windegger M. Geiger M. Kampfer S. Hofmann J. Uberall F. Grunicke H.H. Biochem. J. 2001; 359: 211-217Crossref PubMed Scopus (21) Google Scholar), we do not know which PKC isoform is involved in the phosphorylation of PI-TPβ. Because it is unlikely that PKC is constitutively active in these cells, it appears that phosphorylated PI-TPβ in association with the Golgi is a poor substrate for protein phosphatase. In a previous study PI-TPβ isolated from bovine brain could not be phosphorylated by PKC (8de Vries K.J. Heinrichs A.A. Cunningham E. Brunink F. Westerman J. Somerharju P.J. Cockcroft S. Wirtz K.W.A. Snoek G.T. Biochem. J. 1995; 310: 643-649Crossref PubMed Scopus (89) Google Scholar). Because bovine brain protein is 99% identical to murine PI-TPβ, we consider it likely that in this case also PI-TPβ is mainly present in its phosphorylated form.It has previously been reported that in Swiss mouse 3T3 fibroblasts, PI-TPβ was predominantly associated with the Golgi (8de Vries K.J. Heinrichs A.A. Cunningham E. Brunink F. Westerman J. Somerharju P.J. Cockcroft S. Wirtz K.W.A. Snoek G.T. Biochem. J. 1995; 310: 643-649Crossref PubMed Scopus (89) Google Scholar). In the present study we have confirmed that PI-TPβ was associated with the Golgi complex in the SPIβ cells (Fig. 6, panel A). By incubating SPIβ cells with GF 109203X, a relocation of PI-TPβ from the Golgi to the cytoplasm was observed (panel B). A similar distribution throughout the cytoplasm was observed for PI-TPβ(S262A) expressed in NIH3T3 cells (panel C). These observations indicate that Ser262 has to be phosphorylated for PI-TPβ to be associated with the Golgi system. It is to be noted that the phosphorylation site Ser262 is only present in PI-TPβ, whereas the phosphorylation site Ser165/166 is conserved in all PI-TPs identified so far, with the exception of PI-TP fromCaenorhabditis elegans (26Yoder M.D., T.L.M. Tremblay J.M. Oliver R.L. Yarbrough L.R. Helmkamp Jr G.M. J. Biol. Chem. 2001; 276: 9246-9252Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar).In contrast to wtPI-TPβ, PI-TPβ(S262A) that is overexpressed in mouse fibroblasts is not able to stimulate the resynthesis of SM after the breakdown of this lipid by sphingomyelinase. Because the mutant protein expresses full lipid transfer activity in vitro, we infer that the association of PI-TPβ with the Golgi is a prerequisite for PI-TPβ to stimulate rapid SM replenishment. SM and cholesterol regulation in the Golgi has also been linked to the Golgi localization and phosphorylation of the oxysterol-binding protein (29Ridgway N.D. Lagace T.A. Cook H.W. Byers D.M. J. Biol. Chem. 1998; 273: 31621-31628Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Similar to its yeast analog Sec14p, PI-TPβ may play a role in the budding of SM-containing vesicles from the Golgi (10Ohashi M. de Vries K.J. Frank R. Snoek G.T. Bankaitis V. Wirtz K.W.A. Huttner W.B. Nature. 1995; 377: 544-547Crossref PubMed Scopus (168) Google Scholar, 30Bankaitis V.A. Malehorn D.E. Emr S.D. Greene R. J. Cell Biol. 1989; 108: 1271-1281Crossref PubMed Scopus (311) Google Scholar). It has been well established that the intracellular transport of SM is linked to the assembly and dynamics of lipid rafts (31Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5091) Google Scholar). We are currently investigating whether PI-TPβ is involved in this process. Eukaryotic phosphatidylinositol transfer proteins (PI-TPs)1 belong to a family of highly conserved proteins that are able to transfer phospholipids between membranes or from membrane to enzyme (1Wirtz K.W.A. Biochem. J. 1997; 324: 353-360Crossref PubMed Scopus (174) Google Scholar). In mammalian tissues at least two isoforms, PI-TPα and PI-TPβ, are found. PI-TPα is able to transfer phosphatidylinositol (PI) and, to a lesser extent, phosphatidylcholine (PC) (2Wirtz K.W.A. Annu. Rev. Biochem. 1991; 60: 73-99Crossref PubMed Scopus (377) Google Scholar, 3van Paridon P.A. Visser A.J. Wirtz K.W.A. Biochim. Biophys. Acta. 1987; 898: 172-180Crossref PubMed Scopus (64) Google Scholar, 4Helmkamp Jr G.M. Harvey M.S. Wirtz K.W.A. Van Deenen L.L.M. J. Biol. Chem. 1974; 249: 6382-6389Abstract Full Text PDF PubMed Google Scholar, 5Helmkamp Jr G.M. Chem. Phys. Lipids. 1985; 38: 3-16Crossref PubMed Scopus (34) Google Scholar, 6Helmkamp Jr G.M. J. Bioenerg. Biomembr. 1986; 18: 71-91Crossref PubMed Scopus (56) Google Scholar) and is mainly localized in the cytosol and in the nucleus (7de Vries K.J. Westerman J. Bastiaens P.I. Jovin T.M. Wirtz K.W.A. Snoek G.T. Exp. Cell Res. 1996; 227: 33-39Crossref PubMed Scopus (81) Google Scholar). Similar to PI-TPα, PI-TPβ is able to transfer PI and PC but is also able to transfer sphingomyelin (SM) (8de Vries K.J. Heinrichs A.A. Cunningham E. Brunink F. Westerman J. Somerharju P.J. Cockcroft S. Wirtz K.W.A. Snoek G.T. Biochem. J. 1995; 310: 643-649Crossref PubMed Scopus (89) Google Scholar). PI-TPβ is mainly associated with the Golgi apparatus (7de Vries K.J. Westerman J. Bastiaens P.I. Jovin T.M. Wirtz K.W.A. Snoek G.T. Exp. Cell Res. 1996; 227: 33-39Crossref PubMed Scopus (81) Google Scholar). The primary sequences of PI-TPα and PI-TPβ are very similar, with an identity of 77% and a similarity of 94% (9Tanaka S. Hosaka K. J. Biochem. (Tokyo). 1994; 115: 981-984Crossref PubMed Scopus (84) Google Scholar). To date, little is known about the exact cellular function of PI-TPα and PI-TPβ. In a cell-free system containing trans-Golgi membranes, both PI-TPα and PI-TPβ stimulated the formation of constitutive secretory vesicles and immature granules (10Ohashi M. de Vries K.J. Frank R. Snoek G.T. Bankaitis V. Wirtz K.W.A. Huttner W.B. Nature. 1995; 377: 544-547Crossref PubMed Scopus (168) Google Scholar). In permeabilized, cytosol-depleted HL60 cells, both isoforms restored GTPγS-stimulated protein secretion and phospholipase C-mediated inositol lipid signaling (11Fensome A. Cunningham E. Prosser S. Tan S.K. Swigart P. Thomas G. Hsuan J. Cockcroft S. Curr. Biol. 1996; 6: 730-738Abstract Full Text Full Text PDF PubMed Google Scholar, 12Cunningham E. Tan S.K. Swigart P. Hsuan J. Bankaitis V. Cockcroft S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6589-6593Crossref PubMed Scopus (99) Google Scholar). In these assays, PI-TPα and PI-TPβ were found to function equally well despite their different biochemical properties and cellular localizations. On the other hand, NIH3T3 cells with increased expression of either PI-TPα or PI-TPβ demonstrated remarkable differences in lipid metabolic pathways. Cells overexpressing PI-TPα (SPIα cells) showed an enhanced PLA2-mediated PI degradation resulting in increased levels of lyso-PI, glycerophosphoinositol, Ins(1)P, and Ins(2)P (13Snoek G.T. Berrie C.P. Geijtenbeek T.B.H. van der Helm H.A. Cadee J.A. Iurisci C. Corda D. Wirtz K.W.A. J. Biol. Chem. 1999; 274: 35393-35399Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). This was not observed in cells overexpressing PI-TPβ (SPIβ cells). However, in SPIβ cells (but not in SPIα cells) it was shown that under conditions in which plasma membrane SM was hydrolyzed to ceramide by exogenous sphingomyelinase, PI-TPβ was involved in maintaining the steady-state levels of SM (14van Tiel C.M. Luberto C. Snoek G.T. Hannun Y.A. Wirtz K.W.A. Biochem. J. 2000; 346: 537-543Crossref PubMed Scopus (31) Google Scholar). It was recently postulated that PI-TPβ plays a key role in SM metabolism, making it a potential regulator of pathways for diacylglycerol production and consumption in the mammalian trans-Golgi network (15Bankaitis V.A. Science. 2002; 295: 290-291Crossref PubMed Scopus (46) Google Scholar). Disruption of the PI-TPβ gene in mice leads to early failure in embryonic development (16Alb J.G. Phillips S.E. Rostand K. Cui X. Pinxteren J. Cotlin L. Manning T. Guo S. York J.D. Sontheimer H. Collawn J.F. Bankaitis V.A. Mol. Biol. Cell. 2002; 13: 739-754Crossref PubMed Scopus (60) Google Scholar). In search of mechanisms by which PI-TP activity is regulated, PI-TPα was shown to be phosphorylated by protein kinase C in vitroas well as in vivo (17Snoek G.T. Westerman J. Wouters F.S. Wirtz K.W.A. Biochem. J. 1993; 291: 649-656Crossref PubMed Scopus (20) Google Scholar, 18van Tiel C.M. Westerman J. Paasman M. Wirtz K.W.A. Snoek G.T. J. Biol. Chem. 2000; 275: 21532-21538Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). PI-TPα was exclusively phosphorylated on Ser166, with the PC-carrying form of PI-TPα more readily phosphorylated than the PI-carrying form (18van Tiel C.M. Westerman J. Paasman M. Wirtz K.W.A. Snoek G.T. J. Biol. Chem. 2000; 275: 21532-21538Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Furthermore, in NIH3T3 cells, PI-TPα was translocated from the cytosol to the Golgi membranes upon phosphorylation after stimulation with phorbol ester. This relocalization of PI-TPα coincided with an increased level of intracellular lyso-PI, indicating the activation of a PI-specific PLA2 (17Snoek G.T. Westerman J. Wouters F.S. Wirtz K.W.A. Biochem. J. 1993; 291: 649-656Crossref PubMed Scopus (20) Google Scholar, 18van Tiel C.M. Westerman J. Paasman M. Wirtz K.W.A. Snoek G.T. J. Biol. Chem. 2000; 275: 21532-21538Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Based on these findings, a model was proposed for the regulation of PI-TPα by PKC-dependent phosphorylation. In contrast to PI-TPα, PI-TPβ purified from bovine or rat brain could not be phosphorylated despite the fact that it contains the same serine residue (Ser165) and an additional putative PKC phosphorylation site (Ser262) not present in PI-TPα (8de Vries K.J. Heinrichs A.A. Cunningham E. Brunink F. Westerman J. Somerharju P.J. Cockcroft S. Wirtz K.W.A. Snoek G.T. Biochem. J. 1995; 310: 643-649Crossref PubMed Scopus (89) Google Scholar, 17Snoek G.T. Westerman J. Wouters F.S. Wirtz K.W.A. Biochem. J. 1993; 291: 649-656Crossref PubMed Scopus (20) Google Scholar, 19Geijtenbeek T.B. de Groot E. van Baal J. Brunink F. Westerman J. Snoek G.T. Wirtz K.W.A. Biochim. Biophys. Acta. 1994; 1213: 309-318Crossref PubMed Scopus (27) Google Scholar). In this study, we report that murine PI-TPβ can be phosphorylated by PKC in vitro as well as in situ. The major site of phosphorylation was Ser262, whereas Ser165was a minor site. By site-directed mutagenesis we have shown that Ser165 is essential for the lipid transfer activity of the protein, whereas phosphorylation of Ser262 is required for the association of PI-TPβ with the Golgi membranes. This latter residue is also essential for the ability of PI-TPβ to maintain steady-state levels of SM in the plasma membrane. DISCUSSIONIn this study we have shown that PI-TPβ is a substrate for PKC. By mutation analysis we could establish that Ser262 was the main phosphorylation site. However, Ser165, which is analogous to Ser166 in PI-TPα, was also phosphorylated although to a very limited extent. In a previous study we had shown that the PKC-dependent phosphorylation of PI-TPα was restricted to Ser166 (18van Tiel C.M. Westerman J. Paasman M. Wirtz K.W.A. Snoek G.T. J. Biol. Chem. 2000; 275: 21532-21538Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The PI-TPα isoform, which is highly homologous to PI-TPβ (similarity of 94%), lacks Ser262 (9Tanaka S. Hosaka K. J. Biochem. (Tokyo). 1994; 115: 981-984Crossref PubMed Scopus (84) Google Scholar). Assuming that phosphorylation of the PI-TP isoforms is important for the regulation of their function, we were interested to establish the role of Ser262 in the function of PI-TPβ.The peptide maps of wtPI-TPβ and PI-TPβ(S165A) show three major spots, which are absent from the map of PI-TPβ(S262A) (Fig. 3). This indicates that these three spots represent peptides containing phosphorylated Ser262. The formation of these peptides is probably due to the presence of multiple tryptic cleavage sites in the amino acid sequence of the peptide Met-Arg-Lys-Lys-Gly-Ser262-Val-Arg. Partial digestion would yield the peptides Lys-Lys-Gly-Ser-Val-Arg, Lys-Gly-Ser-Val-Arg, and Gly-Ser-Val-Arg. According to the method described in Ref. 22Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1273) Google Scholar, we could assign Lys-Lys-Gly-Ser-Val-Arg to spot 1, Lys-Gly-Ser-Val-Arg to spot 2, and Gly-Ser-Val-Arg to spot 3. The tryptic map of PI-TPβ(S262A) showed one spot representing a peptide containing phosphorylated Ser165. This spot was barely visible in the peptide map of wtPI-TPβ, indicating that phosphorylation was almost exclusively restricted to Ser262.Replacement of Ser165 with Ala yielded PI-TPβ(S165A) and the double mutated PI-TPβ(S165A/S262A), both of which in vitro completely lacked PI, PC, and SM transfer activity. On the other hand, PI-TPβ(S262A) was fully active. Mutation of the corresponding serine (Ser166) in PI-TPα also yielded an inactive protein (18van Tiel C.M. Westerman J. Paasman M. Wirtz K.W.A. Snoek G.T. J. Biol. Chem. 2000; 275: 21532-21538Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). From the three-dimensional structure it can be inferred that Ser166 is exposed at the surface as part of the regulatory loop of PI-TPα (26Yoder M.D., T.L.M. Tremblay J.M. Oliver R.L. Yarbrough L.R. Helmkamp Jr G.M. J. Biol. Chem. 2001; 276: 9246-9252Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Hence it is possible that the loss of transfer activity is due to the inability of PI-TPα(S166A) to properly interact with the membrane interface. However, at this stage we cannot exclude the possibility that replacement of Ser166 with Ala affects the proper folding of the protein during synthesis in E. coli. The same explanations may hold for the lack of transfer activity observed in PI-TPβ(S165A) and PI-TPβ(S165A/S262A). After PKC-dependent phosphorylation, the phospholipid transfer activity of PI-TPβ was unchanged, indicating that phosphorylation of Ser262 had no effect (Fig. 4).The lysates from SPIβ cells contained two forms of PI-TPβ that could be separated by isoelectric focusing. From densitometric analysis it was estimated that 85% of the PI-TPβ collected at pH 6.2 and 15% at pH 6.5. Treatment of the cells with the PKC inhibitor GF 109203X shifted PI-TPβ to pH 6.5, strongly suggesting that the spot at pH 6.2 represented the phosphorylated form of PI-TPβ. Because the lysate from the SPIβ(S262A) cells contained predominantly PI-TPβ at pH 6.5, we conclude that in situ PI-TPβ is constitutively phosphorylated at Ser262. Given that GF 109203X inhibits conventional and novel type PKCs (27Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charons D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 28Mwanjewe J. Spitaler M. Ebner M. Windegger M. Geiger M. Kampfer S. Hofmann J. Uberall F. Grunicke H.H. Biochem. J. 2001; 359: 211-217Crossref PubMed Scopus (21) Google Scholar), we do not know which PKC isoform is involved in the phosphorylation of PI-TPβ. Because it is unlikely that PKC is constitutively active in these cells, it appears that phosphorylated PI-TPβ in association with the Golgi is a poor substrate for protein phosphatase. In a previous study PI-TPβ isolated from bovine brain could not be phosphorylated by PKC (8de Vries K.J. Heinrichs A.A. Cunningham E. Brunink F. Westerman J. Somerharju P.J. Cockcroft S. Wirtz K.W.A. Snoek G.T. Biochem. J. 1995; 310: 643-649Crossref PubMed Scopus (89) Google Scholar). Because bovine brain protein is 99% identical to murine PI-TPβ, we consider it likely that in this case also PI-TPβ is mainly present in its phosphorylated form.It has previously been reported that in Swiss mouse 3T3 fibroblasts, PI-TPβ was predominantly associated with the Golgi (8de Vries K.J. Heinrichs A.A. Cunningham E. Brunink F. Westerman J. Somerharju P.J. Cockcroft S. Wirtz K.W.A. Snoek G.T. Biochem. J. 1995; 310: 643-649Crossref PubMed Scopus (89) Google Scholar). In the present study we have confirmed that PI-TPβ was associated with the Golgi complex in the SPIβ cells (Fig. 6, panel A). By incubating SPIβ cells with GF 109203X, a relocation of PI-TPβ from the Golgi to the cytoplasm was observed (panel B). A similar distribution throughout the cytoplasm was observed for PI-TPβ(S262A) expressed in NIH3T3 cells (panel C). These observations indicate that Ser262 has to be phosphorylated for PI-TPβ to be associated with the Golgi system. It is to be noted that the phosphorylation site Ser262 is only present in PI-TPβ, whereas the phosphorylation site Ser165/166 is conserved in all PI-TPs identified so far, with the exception of PI-TP fromCaenorhabditis elegans (26Yoder M.D., T.L.M. Tremblay J.M. Oliver R.L. Yarbrough L.R. Helmkamp Jr G.M. J. Biol. Chem. 2001; 276: 9246-9252Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar).In contrast to wtPI-TPβ, PI-TPβ(S262A) that is overexpressed in mouse fibroblasts is not able to stimulate the resynthesis of SM after the breakdown of this lipid by sphingomyelinase. Because the mutant protein expresses full lipid transfer activity in vitro, we infer that the association of PI-TPβ with the Golgi is a prerequisite for PI-TPβ to stimulate rapid SM replenishment. SM and cholesterol regulation in the Golgi has also been linked to the Golgi localization and phosphorylation of the oxysterol-binding protein (29Ridgway N.D. Lagace T.A. Cook H.W. Byers D.M. J. Biol. Chem. 1998; 273: 31621-31628Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Similar to its yeast analog Sec14p, PI-TPβ may play a role in the budding of SM-containing vesicles from the Golgi (10Ohashi M. de Vries K.J. Frank R. Snoek G.T. Bankaitis V. Wirtz K.W.A. Huttner W.B. Nature. 1995; 377: 544-547Crossref PubMed Scopus (168) Google Scholar, 30Bankaitis V.A. Malehorn D.E. Emr S.D. Greene R. J. Cell Biol. 1989; 108: 1271-1281Crossref PubMed Scopus (311) Google Scholar). It has been well established that the intracellular transport of SM is linked to the assembly and dynamics of lipid rafts (31Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5091) Google Scholar). We are currently investigating whether PI-TPβ is involved in this process. In this study we have shown that PI-TPβ is a substrate for PKC. By mutation analysis we could establish that Ser262 was the main phosphorylation site. However, Ser165, which is analogous to Ser166 in PI-TPα, was also phosphorylated although to a very limited extent. In a previous study we had shown that the PKC-dependent phosphorylation of PI-TPα was restricted to Ser166 (18van Tiel C.M. Westerman J. Paasman M. Wirtz K.W.A. Snoek G.T. J. Biol. Chem. 2000; 275: 21532-21538Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The PI-TPα isoform, which is highly homologous to PI-TPβ (similarity of 94%), lacks Ser262 (9Tanaka S. Hosaka K. J. Biochem. (Tokyo). 1994; 115: 981-984Crossref PubMed Scopus (84) Google Scholar). Assuming that phosphorylation of the PI-TP isoforms is important for the regulation of their function, we were interested to establish the role of Ser262 in the function of PI-TPβ. The peptide maps of wtPI-TPβ and PI-TPβ(S165A) show three major spots, which are absent from the map of PI-TPβ(S262A) (Fig. 3). This indicates that these three spots represent peptides containing phosphorylated Ser262. The formation of these peptides is probably due to the presence of multiple tryptic cleavage sites in the amino acid sequence of the peptide Met-Arg-Lys-Lys-Gly-Ser262-Val-Arg. Partial digestion would yield the peptides Lys-Lys-Gly-Ser-Val-Arg, Lys-Gly-Ser-Val-Arg, and Gly-Ser-Val-Arg. According to the method described in Ref. 22Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1273) Google Scholar, we could assign Lys-Lys-Gly-Ser-Val-Arg to spot 1, Lys-Gly-Ser-Val-Arg to spot 2, and Gly-Ser-Val-Arg to spot 3. The tryptic map of PI-TPβ(S262A) showed one spot representing a peptide containing phosphorylated Ser165. This spot was barely visible in the peptide map of wtPI-TPβ, indicating that phosphorylation was almost exclusively restricted to Ser262. Replacement of Ser165 with Ala yielded PI-TPβ(S165A) and the double mutated PI-TPβ(S165A/S262A), both of which in vitro completely lacked PI, PC, and SM transfer activity. On the other hand, PI-TPβ(S262A) was fully active. Mutation of the corresponding serine (Ser166) in PI-TPα also yielded an inactive protein (18van Tiel C.M. Westerman J. Paasman M. Wirtz K.W.A. Snoek G.T. J. Biol. Chem. 2000; 275: 21532-21538Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). From the three-dimensional structure it can be inferred that Ser166 is exposed at the surface as part of the regulatory loop of PI-TPα (26Yoder M.D., T.L.M. Tremblay J.M. Oliver R.L. Yarbrough L.R. Helmkamp Jr G.M. J. Biol. Chem. 2001; 276: 9246-9252Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Hence it is possible that the loss of transfer activity is due to the inability of PI-TPα(S166A) to properly interact with the membrane interface. However, at this stage we cannot exclude the possibility that replacement of Ser166 with Ala affects the proper folding of the protein during synthesis in E. coli. The same explanations may hold for the lack of transfer activity observed in PI-TPβ(S165A) and PI-TPβ(S165A/S262A). After PKC-dependent phosphorylation, the phospholipid transfer activity of PI-TPβ was unchanged, indicating that phosphorylation of Ser262 had no effect (Fig. 4). The lysates from SPIβ cells contained two forms of PI-TPβ that could be separated by isoelectric focusing. From densitometric analysis it was estimated that 85% of the PI-TPβ collected at pH 6.2 and 15% at pH 6.5. Treatment of the cells with the PKC inhibitor GF 109203X shifted PI-TPβ to pH 6.5, strongly suggesting that the spot at pH 6.2 represented the phosphorylated form of PI-TPβ. Because the lysate from the SPIβ(S262A) cells contained predominantly PI-TPβ at pH 6.5, we conclude that in situ PI-TPβ is constitutively phosphorylated at Ser262. Given that GF 109203X inhibits conventional and novel type PKCs (27Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charons D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 28Mwanjewe J. Spitaler M. Ebner M. Windegger M. Geiger M. Kampfer S. Hofmann J. Uberall F. Grunicke H.H. Biochem. J. 2001; 359: 211-217Crossref PubMed Scopus (21) Google Scholar), we do not know which PKC isoform is involved in the phosphorylation of PI-TPβ. Because it is unlikely that PKC is constitutively active in these cells, it appears that phosphorylated PI-TPβ in association with the Golgi is a poor substrate for protein phosphatase. In a previous study PI-TPβ isolated from bovine brain could not be phosphorylated by PKC (8de Vries K.J. Heinrichs A.A. Cunningham E. Brunink F. Westerman J. Somerharju P.J. Cockcroft S. Wirtz K.W.A. Snoek G.T. Biochem. J. 1995; 310: 643-649Crossref PubMed Scopus (89) Google Scholar). Because bovine brain protein is 99% identical to murine PI-TPβ, we consider it likely that in this case also PI-TPβ is mainly present in its phosphorylated form. It has previously been reported that in Swiss mouse 3T3 fibroblasts, PI-TPβ was predominantly associated with the Golgi (8de Vries K.J. Heinrichs A.A. Cunningham E. Brunink F. Westerman J. Somerharju P.J. Cockcroft S. Wirtz K.W.A. Snoek G.T. Biochem. J. 1995; 310: 643-649Crossref PubMed Scopus (89) Google Scholar). In the present study we have confirmed that PI-TPβ was associated with the Golgi complex in the SPIβ cells (Fig. 6, panel A). By incubating SPIβ cells with GF 109203X, a relocation of PI-TPβ from the Golgi to the cytoplasm was observed (panel B). A similar distribution throughout the cytoplasm was observed for PI-TPβ(S262A) expressed in NIH3T3 cells (panel C). These observations indicate that Ser262 has to be phosphorylated for PI-TPβ to be associated with the Golgi system. It is to be noted that the phosphorylation site Ser262 is only present in PI-TPβ, whereas the phosphorylation site Ser165/166 is conserved in all PI-TPs identified so far, with the exception of PI-TP fromCaenorhabditis elegans (26Yoder M.D., T.L.M. Tremblay J.M. Oliver R.L. Yarbrough L.R. Helmkamp Jr G.M. J. Biol. Chem. 2001; 276: 9246-9252Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In contrast to wtPI-TPβ, PI-TPβ(S262A) that is overexpressed in mouse fibroblasts is not able to stimulate the resynthesis of SM after the breakdown of this lipid by sphingomyelinase. Because the mutant protein expresses full lipid transfer activity in vitro, we infer that the association of PI-TPβ with the Golgi is a prerequisite for PI-TPβ to stimulate rapid SM replenishment. SM and cholesterol regulation in the Golgi has also been linked to the Golgi localization and phosphorylation of the oxysterol-binding protein (29Ridgway N.D. Lagace T.A. Cook H.W. Byers D.M. J. Biol. Chem. 1998; 273: 31621-31628Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Similar to its yeast analog Sec14p, PI-TPβ may play a role in the budding of SM-containing vesicles from the Golgi (10Ohashi M. de Vries K.J. Frank R. Snoek G.T. Bankaitis V. Wirtz K.W.A. Huttner W.B. Nature. 1995; 377: 544-547Crossref PubMed Scopus (168) Google Scholar, 30Bankaitis V.A. Malehorn D.E. Emr S.D. Greene R. J. Cell Biol. 1989; 108: 1271-1281Crossref PubMed Scopus (311) Google Scholar). It has been well established that the intracellular transport of SM is linked to the assembly and dynamics of lipid rafts (31Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5091) Google Scholar). We are currently investigating whether PI-TPβ is involved in this process.
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