FAS activation induces dephosphorylation of SR proteins -: Dependence on the de novo generation of ceramide and activation of protein phosphatase 1
FAS activation induces dephosphorylation of SR proteins -: Dependence on the de novo generation of ceramide and activation of protein phosphatase 1
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DOI：
10.1074/jbc.m106291200
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发表时间：
2001-11-30
影响因子：
4.8
通讯作者：
Hannun, YA
中科院分区：
文献类型：
--
作者：
Chalfant, CE;Ogretmen, B;Hannun, YA
寻找神经酰胺作用的潜在靶点导致了神经酰胺活化蛋白磷酸酶（CAPP）的鉴定。迄今为止，已经证明两种丝氨酸/苏氨酸蛋白磷酸酶，蛋白磷酸酶2A（PP 2A）和蛋白磷酸酶1（PP 1）作为神经酰胺活化的蛋白磷酸酶起作用。在这项研究中，我们表明，无论是抗FAS IgM（CH-11）（150 ng/ml）或外源性D-（e）-C-6-神经酰胺（20 μ M）的治疗诱导的PP 1底物，丝氨酸/丝氨酸丰富（SR）蛋白的去磷酸化，在Jurkat急性白血病T细胞。丝氨酸/苏氨酸蛋白磷酸酶抑制剂，calyculin A，但不是PP 2A特异性抑制剂，冈田酸，抑制FAS和神经酰胺诱导的SR蛋白的去磷酸化。Jurkat细胞的抗FAS IgM处理导致内源性神经酰胺水平从2小时开始显著增加，7小时后最大增加10倍。用伏马菌素B-1（100 μ M）预处理Jurkat细胞2小时，伏马菌素B-1是CoA依赖性神经酰胺合成酶的特异性抑制剂，阻断了80%的神经酰胺产生，并完全抑制了SR蛋白响应于抗FAS IgM的去磷酸化。此外，预处理Jurkat细胞与myriocin，丝氨酸-棕榈酰转移酶（神经酰胺从头合成的第一步）的特异性抑制剂，也阻止FAS诱导的SR蛋白去磷酸化，从而证明了从头神经酰胺的作用。使用D-（e）-C-6-神经酰胺处理的A549肺腺癌细胞进一步支持这些结果。SR蛋白的去磷酸化被伏马菌素B1和葡萄糖神经酰胺合酶的过表达抑制;再次暗示内源性神经酰胺从头产生调节响应FAS激活的SR蛋白的去磷酸化。这些结果建立了一个特定的细胞内途径，涉及从头神经酰胺的产生和激活PP 1介导的影响，FAS激活SR蛋白。
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 muM) 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 muM), 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 B1 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.