Trihydrophobin 1 Interacts with PAK1 and Regulates ERK/MAPK Activation and Cell Migration
Trihydrophobin 1 Interacts with PAK1 and Regulates ERK/MAPK Activation and Cell Migration
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DOI：
10.1074/jbc.m806144200
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发表时间：
2009-03-27
影响因子：
4.8
通讯作者：
Gu, Jianxin
中科院分区：
文献类型：
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作者：
Cheng, Chunming;Kong, Xiangfei;Gu, Jianxin
Rac1/Cdc42 效应器 p21 激活激酶 (PAK) 由各种信号级联反应激活，包括受体酪氨酸激酶和整合素，并调节细胞增殖和运动等许多过程。 PAK 活性已被证明是典型 RAF-MEK-MAPK 信号级联最大激活所必需的，可能是因为 PAK 与 RAF 和 MEK 共同激活。在这里，我们发现三疏水蛋白 1 (TH1) 最初被确定为 A-RAF 激酶的负调节因子，在培养细胞中也与 PAK1 相互作用。共聚焦显微镜检测表明TH1与PAK1在细胞质和细胞核中共定位，这与我们之前的结果一致。 GST Pulldown和免疫共沉淀实验表明TH1直接与PAK1相互作用并选择性结合PAK1的羧基末端激酶结构域，并且结合能力随着PAK1的激活而增强。 PAK1 的结合模式表明这种相互作用部分是由 PAK1 激酶活性介导的。体外激酶活性测定和Western blot检测表明，TH1抑制PAK1激酶活性并负向调节MAPK信号转导。有趣的是，TH1 在细胞内和体外与 MEK1/ERK 结合，而不直接抑制其激酶活性。此外，我们观察到 TH1 定位于细胞前缘的粘着斑和丝状伪足，其中 TH1 通过影响肌动蛋白和粘附动力学来减少细胞迁移。基于这些观察，我们提出了一个模型，其中 TH1 与 PAK1 相互作用，并通过 MAPK 途径的上游区域特异性限制 MAPK 模块的激活，从而影响细胞迁移。
The Rac1/Cdc42 effector, p21-activated kinase (PAK), is activated by various signaling cascades, including receptor-tyrosine kinases and integrins, and regulates a number of processes such as cell proliferation and motility. PAK activity has been shown to be required for maximal activation of the canonical RAF-MEK-MAPK signaling cascade, possibly because of PAK co-activation of RAF and MEK. Here we have shown that trihydrophobin 1 (TH1), originally identified as a negative regulator of A-RAF kinase, also interacted with PAK1 in cultured cells. Confocal microscopy assay indicated that TH1 colocalized with PAK1 in both the cytoplasm and nucleus, which is consistent with our previous results. GST pulldown and coimmunoprecipitation experiments demonstrated that TH1 interacted directly with PAK1 and bound selectively to the carboxyl-terminal kinase domain of PAK1, and the ability of the binding was enhanced along with activation of PAK1. The binding pattern of PAK1 implies that this interaction was mediated in part by PAK1 kinase activity. As indicated by in vitro kinase activity assays and Western blot detections, TH1 inhibited PAK1 kinase activity and negatively regulated MAPK signal transduction. Interestingly, TH1 bound with MEK1/ERK in cells and in vitro without directly suppressing their kinase activity. Furthermore, we observed that TH1 localized to focal adhesions and filopodia in the leading edge of cells, where TH1 reduced cell migration through affecting actin and adhesion dynamics. Based on these observations, we propose a model in which TH1 interacts with PAK1 and specifically restricts the activation of MAPK modules through the upstream region of the MAPK pathway, thereby influencing cell migration.