Ahnak protein activates protein kinase C (PKC) through dissociation of the PKC-protein phosphatase 2A complex
Ahnak protein activates protein kinase C (PKC) through dissociation of the PKC-protein phosphatase 2A complex
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DOI：
10.1074/jbc.m706878200
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发表时间：
2008-03-07
影响因子：
4.8
通讯作者：
Bae, Yun Soo
中科院分区：
文献类型：
--
作者：
Lee, In Hye;Lim, Hee Jung;Bae, Yun Soo
我们之前报道过Ahnak的中心重复单元（CRUs）作为脚手架蛋白网络磷脂酶C γ和蛋白激酶C （PKC）。在这里，我们证明了由四个中心重复单元组成的Ahnak衍生物以不依赖于磷脂酰丝氨酸/1,2-二油基-sn-甘油的方式结合并激活pkc - α。此外，与亲本细胞相比，表达Ahnak的4个cru的NIH3T3细胞在phorbol 12-肉豆蔻酸13-乙酸酯（PMA）的作用下，c-Raf、MEK和Erk的磷酸化增强。为了评估Ahnak功能缺失对细胞信号传导的影响，我们研究了Ahnak基因敲除（Ahnak(-/-)）小鼠胚胎成纤维细胞（mef）的PKC激活和Raf磷酸化。与野生型MEF相比，Ahnak阴性MEF细胞中pkc - α的膜易位和Raf的磷酸化对PMA或血小板衍生生长因子的反应减少。一些证据表明pkc - α的活性通过与蛋白磷酸酶2A （PP2A）的关联而受到调节。共免疫沉淀实验表明，在表达Ahnak蛋白4个cru的NIH3T3细胞中，pkc - α与PP2A的关联在PMA的作用下被破坏。与此一致的是，PMA刺激的Ahnak无MEF细胞显示PKC-PP2A复合物的形成增强，并且在Ahnak无MEF细胞中添加Ahnak后，PMA会抑制PKC-PP2A复合物的形成。这些数据表明，Ahnak通过抑制PKC与PP2A的相互作用来增强PKC的激活。
We have previously reported that central repeated units (CRUs) of Ahnak act as a scaffolding protein networking phospholipase C gamma and protein kinase C (PKC). Here, we demonstrate that an Ahnak derivative consisting of four central repeated units binds and activates PKC-alpha in a phosphatidylserine/1,2-dioleoyl-sn-glycerol-independent manner. Moreover, NIH3T3 cells expressing the 4 CRUs of Ahnak showed enhanced c-Raf, MEK, and Erk phosphorylation in response to phorbol 12-myristate 13-acetate (PMA) compared with parental cells. To evaluate the effect of loss-of-function of Ahnak in cell signaling, we investigated PKC activation and Raf phosphorylation in embryonic fibroblast cells (MEFs) of the Ahnak knock-out (Ahnak(-/-)) mouse. Membrane translocation of PKC-alpha and phosphorylation of Raf in response to PMA or platelet-derived growth factor were decreased in Ahnak null MEF cells compared with wild type MEFs. Several lines of evidence suggest that PKC-alpha activity is regulated through association with protein phosphatase 2A (PP2A). A co-immunoprecipitation assay indicated that the association of PKC-alpha with PP2A was disrupted in NIH3T3 cells expressing 4 CRUs of Ahnak in response to PMA. Consistently, Ahnak null MEF cells stimulated by PMA showed enhanced PKC-PP2A complex formation, and add-back expression of Ahnak into Ahnak null MEF cells abolished the PKC-PP2A complex formation in response to PMA. These data indicate that Ahnak potentiates PKC activation through inhibiting the interaction of PKC with PP2A.