PAK1 negatively regulates the activity of the Rho exchange factor NET1
PAK1 negatively regulates the activity of the Rho exchange factor NET1
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DOI：
10.1074/jbc.m405073200
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发表时间：
2005-04-01
影响因子：
4.8
通讯作者：
Frost, JA
中科院分区：
文献类型：
--
作者：
Alberts, AS;Qin, HJ;Frost, JA
Rho 家族小 G 蛋白活性由刺激 GDP 释放的鸟嘌呤核苷酸交换因子控制，从而允许 GTP 结合。一旦激活，Rho 蛋白就会通过与下游效应蛋白的相互作用来控制细胞信号传导，从而导致细胞骨架组织和基因表达的变化。 Rho 家族成员调节其他 Rho 蛋白活性的能力也是这些过程所固有的。在这项工作中，我们证明 Rac/Cdc42hs 调节的蛋白激酶 PAK1 下调 RhoA 特异性鸟嘌呤核苷酸交换因子 NET1 的活性。具体来说，PAK1 在体外磷酸化 NET1 的三个位点：丝氨酸 152、153 和 538。用谷氨酸残基替换丝氨酸 152 和 153 会下调 NET1 作为体外交换因子的活性及其刺激细胞中肌动蛋白应力纤维形成的能力。使用识别丝氨酸 152 上磷酸化的 NET1 的磷酸化特异性抗体，我们发现 PAK1 在细胞中的该位点上磷酸化 NET1，并且 Rac1 以 PAK1 依赖性方式刺激丝氨酸 152 磷酸化。此外，仅当丝氨酸 152 和 153 存在时，组成型活性 PAK1 的共表达才会抑制 NET1 刺激肌动蛋白聚合的能力。这些数据为 Rac1 通过 NET1 的 PAK 依赖性磷酸化来控制 RhoA 活性提供了一种新机制，以降低其作为鸟嘌呤核苷酸交换因子的活性。
Rho family small G-protein activity is controlled by guanine nucleotide exchange factors that stimulate the release of GDP, thus allowing GTP binding. Once activated, Rho proteins control cell signaling through interactions with downstream effector proteins, leading to changes in cytoskeletal organization and gene expression. The ability of Rho family members to modulate the activity of other Rho proteins is also intrinsic to these processes. In this work we show that the Rac/Cdc42hs-regulated protein kinase PAK1 down-regulates the activity of the RhoA-specific guanine nucleotide exchange factor NET1. Specifically, PAK1 phosphorylates NET1 on three sites in vitro: serines 152, 153, and 538. Replacement of serines 152 and 153 with glutamate residues down-regulates the activity of NET1 as an exchange factor in vitro and its ability to stimulate actin stress fiber formation in cells. Using a phospho-specific antibody that recognizes NET1 phosphorylated on serine 152, we show that PAK1 phosphorylates NET1 on this site in cells and that Rac1 stimulates serine 152 phosphorylation in a PAK1-dependent manner. Furthermore, coexpression of constitutively active PAK1 inhibits the ability of NET1 to stimulate actin polymerization only when serines 152 and 153 are present. These data provide a novel mechanism for the control of RhoA activity by Rac1 through the PAK-dependent phosphorylation of NET1 to reduce its activity as a guanine nucleotide exchange factor.