title: FAS activation induces dephosphorylation of SR proteins; dependence on the de novo generation of ceramide and activation of protein phosphatase 1.
abstract: The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases (CAPP). To date, two serine/threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), have been demonstrated to function as ceramide-activated protein phosphatases. In this study, we show that treatment with either anti-FAS IgM (CH-11) (150 ng/ml) or exogenous d-(e)-C(6-)ceramide (20 microm) induces the dephosphorylation of the PP1 substrates, serine/arginine-rich (SR) proteins, in Jurkat acute leukemia T-cells. The serine/threonine protein phosphatase inhibitor, calyculin A, but not the PP2A-specific inhibitor, okadaic acid, inhibited both FAS- and ceramide-induced dephosphorylation of SR proteins. Anti-FAS IgM treatment of Jurkat cells led to a significant increase in levels of endogenous ceramide beginning at 2 h with a maximal increase of 10-fold after 7 h. A 2-h pretreatment of Jurkat cells with fumonisin B(1) (100 microm), a specific inhibitor of CoA-dependent ceramide synthase, blocked 80% of the ceramide generated and completely inhibited the dephosphorylation of SR proteins in response to anti-FAS IgM. Moreover, pretreatment of Jurkat cells with myriocin, a specific inhibitor of serine-palmitoyl transferase (the first step in de novo synthesis of ceramide), also blocked FAS-induced SR protein dephosphorylation, thus demonstrating a role for de novo ceramide. These results were further supported using A549 lung adenocarcinoma cells treated with d-(e)-C(6-)ceramide. Dephosphorylation of SR proteins was inhibited by fumonisin B(1) and by overexpression of glucosylceramide synthase; again implicating endogenous ceramide generated de novo in regulating the dephosphorylation of SR proteins in response to FAS activation. These results establish a specific intracellular pathway involving both de novo ceramide generation and activation of PP1 to mediate the effects of FAS activation on SR proteins.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
authors.forename: C E
authors.initials: CE
authors.lastname: Chalfant
authors.forename: B
authors.initials: B
authors.lastname: Ogretmen
authors.forename: S
authors.initials: S
authors.lastname: Galadari
authors.forename: B J
authors.initials: BJ
authors.lastname: Kroesen
authors.forename: B J
authors.initials: BJ
authors.lastname: Pettus
authors.forename: Y A
authors.initials: YA
authors.lastname: Hannun
mesh_terms: D015153:Blotting, Western; D002264:Carboxylic Acids / Q000494:pharmacology; D002460:Cell Line; D002518:Ceramides / Q000096:biosynthesis*; D019852:Diacylglycerol Kinase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D037341:Fumonisins; D006801:Humans; D019169:Jurkat Cells; D019308:Palmitic Acid / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013107:Sphingolipids / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D013997:Time Factors; D019014:fas Receptor / Q000378:metabolism*
chemical_list: D002264:Carboxylic Acids; D002518:Ceramides; D004791:Enzyme Inhibitors; D037341:Fumonisins; D013107:Sphingolipids; D019014:fas Receptor; D019308:Palmitic Acid; D013912:Threonine; C056933:fumonisin B1; D012694:Serine; D019852:Diacylglycerol Kinase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D010744:Phosphoric Monoester Hydrolases