FAS activation induces dephosphorylation of SR proteins - Dependence on the de novo generation of ceramide and activation of protein phosphatase 1

The search for potential targets for ceramide action led to the identificat ion of ceramide-activated protein phosphatases (CAPP). To date, two serine/ threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein p hosphatase 1 (PP1), have been demonstrated to function as ceramide-activate d protein phosphatases. In this study, we show that treatment with either a nti-FAS IgM (CH-11) (150 ng/ml) or exogenous D-(e)-C-6-ceramide (20 muM) in duces 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, ok adaic acid, inhibited both FAS- and ceramide-induced dephosphorylation of S R proteins. Anti-FAS IgM treatment of Jurkat cells led to a significant inc rease in levels of endogenous ceramide beginning at 2 h with a maximal incr ease of 10-fold after 7 h. A 2-h pretreatment of Jurkat cells with fumonisi n B-1 (100 muM), a specific inhibitor of CoA-dependent ceramide synthase, b locked 80% of the ceramide generated and completely inhibited the dephospho rylation of SR proteins in response to anti-FAS IgM. Moreover, pretreatment of Jurkat cells with myriocin, a specific inhibitor of serine-palmitoyl tr ansferase (the first step in de novo synthesis of ceramide), also blocked F AS-induced SR protein dephosphorylation, thus demonstrating a role for de n ovo ceramide. These results were further supported using A549 lung adenocar cinoma cells treated with D-(e)-C-6-ceramide. Dephosphorylation of SR prote ins was inhibited by fumonisin B1 and by overexpression of glucosylceramide synthase; again implicating endogenous ceramide generated de novo in regul ating the dephosphorylation of SR proteins in response to FAS activation. T hese results establish a specific intracellular pathway involving both de n ovo ceramide generation and activation of PP1 to mediate the effects of FAS activation on SR proteins.