FAS Activation Induces Dephosphorylation of SR Proteins: Dependence on the de novo generation of ceramide and activation of protein phosphatase 1

Charles E. Chalfant, Besim Ogretmen, Sehamuddin Galadari, Bart Jan Kroesen, Benjamin J. Pettus, Yusuf A. Hannun

Research output: Contribution to journalArticle

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)-C6- ceramide (20 μM) 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 B1 (100 μM), 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)-C6-ceramide. Dephosphorylation of SR proteins was inhibited by fumonisin B1 and by over-expression 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.

Original languageEnglish (US)
Pages (from-to)44848-44855
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number48
DOIs
StatePublished - Nov 30 2001

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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