Regulation of the interaction of Pak2 with Cdc42 via autophosphorylation of serine 141
Regulation of the interaction of Pak2 with Cdc42 via autophosphorylation of serine 141
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DOI：
10.1074/jbc.m509075200
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发表时间：
2005-12-02
影响因子：
4.8
通讯作者：
Traugh, JA
中科院分区：
文献类型：
--
作者：
Jung, JH;Traugh, JA
Pak2是p21激活蛋白激酶(Pak)家族的一员，在各种应激反应中被激活，并直接参与诱导细胞停滞。在分子水平上，pak2结合了cdc42(GTP)，将pak2转移到内质网，在那里它被自动磷酸化并被激活。Ak2在8个位点被自动磷酸化，调节域中的Ser-141和Ser-165以及激活环中的Thr-402被确定为蛋白激酶激活的关键位点。用野生型(WT)和pak2突变体分析Ser-141和Ser-165的磷酸化在激活中的作用。与WT和S141D相比，S141A的自磷酸化水平降低到65%，底物磷酸化水平降低45%，表明Ser-141的磷酸化是最佳活性所必需的。自身磷酸化抑制WT pak2与Cdc42(GTP)之间的相互作用。在293T细胞中，WT pak2、S141A和S141D与成分活性突变体CDC42 L61形成稳定的复合体，但不与显性负性CDC42 N17形成复合体。谷胱甘肽S转移酶下拉实验表明，S141a与CdC42(GTP)的结合水平是S141D的6倍。相反，S165A和S165D突变体对自身磷酸化、与CDC42的结合或Pak2的激活没有影响。综上所述，Ser-141的自动磷酸化对于Pak2的激活是必需的，并且下调了Pak2与Cdc42的相互作用。提出了一个模型，认为CDC42的结合使pak2定位于内质网，其中自动磷酸化改变了这两种蛋白的结合。
Pak2, a member of the p21-activated protein kinase ( Pak) family, is activated in response to a variety of stresses and is directly involved in the induction of cytostasis. At the molecular level Pak2 binds Cdc42( GTP), translocating Pak2 to the endoplasmic reticulum where it is autophosphorylated and activated. Pak2 is autophosphorylated at eight sites; Ser-141 and Ser-165 in the regulatory domain and Thr-402 in the activation loop are identified as key sites in activation of the protein kinase. The function of phosphorylation of Ser-141 and Ser-165 on the activation was analyzed with wildtype ( WT) and mutants of Pak2. With S141A, the level of autophosphorylation was reduced to 65% as compared with that of WT and S141D with a concomitant 45% reduction in substrate phosphorylation, indicating that phosphorylation at Ser-141 is required for optimal activity. Autophosphorylation inhibited the interaction between WT Pak2 and Cdc42( GTP). In 293T cells, WT Pak2, S141A, and S141D formed a stable complex with the constitutively active mutant Cdc42 L61, but not with the dominant negative Cdc42 N17. As shown in glutathione S-transferase pull-down assays, S141A bound to Cdc42( GTP) at a 6-fold higher level than that of S141D. In contrast, the S165A and S165D mutants had no effect on autophosphorylation, binding to Cdc42, or activation of Pak2. In summary, autophosphorylation of Ser-141 was required for activation of Pak2 and down-regulated the interaction of Pak2 with Cdc42. A model is proposed suggesting that binding of Cdc42 localizes Pak2 to the endoplasmic reticulum, where autophosphorylation alters association of the two proteins.