Functional analysis of C-TAK1 substrate binding and identification of PKP2 as a new C-TAK1 substrate
Functional analysis of C-TAK1 substrate binding and identification of PKP2 as a new C-TAK1 substrate
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DOI：
10.1093/emboj/cdg426
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发表时间：
2003-09-01
期刊：
影响因子：
11.4
通讯作者：
Morrison, DK
中科院分区：
文献类型：
--
作者：
Müller, J;Ritt, DA;Morrison, DK
Cdc25C相关激酶1 （C-TAK1）通过与两种假定的底物Cdc25C磷酸酶和MAPK支架KSR1相互作用，参与细胞周期调节和Ras信号传导。在这里，我们确定了稳定的C-TAK1结合和底物磷酸化所需的序列基序。利用突变方法破坏C-TAK1与KSR1和Cdc25C的结合，我们证明C-TAK1通过产生14-3-3结合位点在体内对这些蛋白进行调节。在体内，C-TAK1结合缺陷的KSR1蛋白严重降低了14-3-3结合位点的磷酸化，组成性地定位于质膜上，并增加了生物活性。Cdc25C- c - tak1相互作用的破坏导致14-3-3结合位点磷酸化减少和间期细胞中Cdc25C的核积累。最后，利用获得的C-TAK1结合和底物磷酸化数据，我们确定了嗜血小板蛋白2 （PKP2）是一种新的C-TAK1底物。C-TAK1对PKP2的磷酸化也会产生14-3-3结合位点，影响PKP2的定位。这些发现强调了C-TAK1作为14-3-3结合和蛋白定位调节因子的重要性。
Cdc25C-associated kinase 1 (C-TAK1) has been implicated in cell cycle regulation and Ras signaling through its interactions with two putative substrates, the Cdc25C phosphatase and the MAPK scaffold KSR1. Here, we identify sequence motifs required for stable C-TAK1 association and substrate phosphorylation. Using a mutational approach to disrupt binding of C-TAK1 to KSR1 and Cdc25C, we demonstrate that C-TAK1 contributes to the regulation of these proteins in vivo through the generation of 14-3-3-binding sites. KSR1 proteins defective in C-TAK1 binding had severely reduced phosphorylation at the 14-3-3-binding site in vivo, were constitutively localized to the plasma membrane and had increased biological activity. Disruption of the Cdc25C-C-TAK1 interaction resulted in reduced 14-3-3-binding site phosphorylation and nuclear accumulation of Cdc25C in interphase cells. Finally, utilizing the acquired C-TAK1 binding and substrate phosphorylation data, we identify plakophilin 2 (PKP2) as a novel C-TAK1 substrate. Phosphorylation of PKP2 by C-TAK1 also generates a 14-3-3-binding site that influences PKP2 localization. These findings underscore the importance of C-TAK1 as a regulator of 14-3-3 binding and protein localization.