Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A - Activation is stereospecific and regulated by phosphatidic acid
Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A - Activation is stereospecific and regulated by phosphatidic acid
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DOI：
10.1074/jbc.274.29.20313
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发表时间：
1999-07-16
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;Kishikawa, K;Hannun, YA
对神经酰胺作用的潜在靶点的研究导致了神经酰胺活化的蛋白磷酸酶的鉴定，其包括蛋白磷酸酶-2A（PP 2A）和蛋白磷酸酶-1（PP 1），其在调节细胞凋亡和细胞生长中起作用。到目前为止，在体外研究神经酰胺激活的蛋白磷酸酶已被限制使用短链神经酰胺，限制了程度的机制的见解。在这项研究中，我们发现长链D-β-C-18-神经酰胺激活PP 2A，（AB 'C三聚体），PP 2Ac（PP 2A的催化亚基）和PP 1 γ c和-α c（分别为PP 1 γ和-1 α同种型的催化亚基）在十二烷（一种脂质增溶剂）存在下的2-6倍，在约10 μ M D-β-C-18-神经酰胺时达到50%的最大活化。D-邻位-C-18-神经酰胺的非对映异构体、D-苏型-和L-苏型-C-18-神经酰胺以及对映异构体L-邻位-C-18-神经酰胺不激活PP 1或PP 2A，但它们抑制PP 1和PP 2A活性。加入磷脂酸降低了PP 1c的基础活性，但也使D-赤型-C-18-神经酰胺的刺激作用从1.8倍增加到2.8倍，并使D-赤型-C-18-神经酰胺的EC 50降低到4.45 μ M。加入150 mM KCl降低了PP 1的基础活性和激活PP 1c所需的D-β-C-18-神经酰胺的剂量（EC 50 = 6.25 μ M），并使神经酰胺的反应性增加了10-17倍。这些研究公开了在体外近生理离子强度下长链天然神经酰胺对PP 1和PP 2A的立体特异性激活。这些研究的神经酰胺作用机制的影响进行了讨论。
The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases, which include protein phosphatase-2A (PP2A) and protein phosphatase-1 (PP1) with roles in regulating apoptosis and cell growth. Thus far, in vitro studies on ceramide-activated protein phosphatases have been restricted to the use of short chain ceramides, limiting the extent of mechanistic insight. In this study, we show that the long chain D-erythro-C-18-ceramide activated PP2A (AB'C trimer), PP2Ac (catalytic subunit of PP2A), and PP1 gamma c and -alpha c (catalytic subunits of PP1 gamma and -1 alpha isoforms, respectively) 2-6-fold in the presence of dodecane, a lipid-solubilizing agent, with 50% maximal activation achieved at approximately 10 mu M D-erythro-C-18-ceramide. The diastereoisomers of D-erythro-C-18-ceramide, D-threo-, and L-threo-C-18-ceramide, as well as the enantiomeric L-erythro-C-18-ceramide, did not activate PP1 or PP2A, but they inhibited PP1 and PP2A activity. The addition of phosphatidic acid decreased the basal activity of PP1c but also increased the stimulation by D-erythro-C-18-ceramide from 1.8- to 2.8 fold and decreased the EC50 of D erythro-C-18-ceramide to 4.45 mu M. The addition of 150 mM KCI decreased the basal activity of PP1 and the dose of D-erythro-C-18-ceramide necessary to activate PP1c (EC50 = 6.25 mu M) and increased the ceramide responsiveness up to 10-17-fold. These studies disclose stereospecific activation of PP1 and PP2A by long chain natural ceramides under near physiologic ionic strengths in vitro. The implications of these studies for mechanisms of ceramide action are discussed.