Reeder M K, Serebriiskii I G, Golemis E A, Chernoff J
Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
J Biol Chem. 2001 Nov 2;276(44):40606-13. doi: 10.1074/jbc.M103925200. Epub 2001 Aug 20.
p21-activated kinase 1 (Pak1) is an effector for the small GTPases Cdc42 and Rac. Because Pak1 binds to and is activated by both these GTPases, it has been difficult to precisely delineate the signaling pathways that link extracellular stimuli to Pak1 activation. To separate activation of Pak1 by Cdc42 versus activation by Rac, we devised a genetic screen in yeast that enabled us to create and identify Pak1 mutants that selectively couple to Cdc42 but not Rac1. We recovered several such Pak1 mutants and found that the residues most often affected lie within the p21 binding domain, a region previously known to mediate Pak1 binding to GTPases, but that several mutations also map outside the borders of the p21 binding domain. Pak1 mutants that associate with Cdc42 but not Rac1 were also activated by Cdc42 but not Rac1. In rat 3Y1 cells expressing oncogenic Ha-Ras, the Pak1 mutants defective in Rac1 binding are not activated, suggesting that Ras signals through a GTPase other than Cdc42 to activate Pakl. Similar results were obtained when epidermal growth factor was used to activate Pak1. However, Pak1 mutants that are unable to bind Rac are nonetheless well activated by calf serum, implying that this stimulus may induce Pak activation independent of Rac.
p21激活激酶1（Pak1）是小GTP酶Cdc42和Rac的效应器。由于Pak1与这两种GTP酶都结合并被其激活，因此很难精确描绘将细胞外刺激与Pak1激活联系起来的信号通路。为了区分Cdc42介导的Pak1激活与Rac介导的激活，我们在酵母中设计了一个遗传筛选方法，使我们能够创建并鉴定出选择性地与Cdc42而非Rac1偶联的Pak1突变体。我们获得了几个这样的Pak1突变体，发现最常受影响的残基位于p21结合域内，该区域以前已知介导Pak1与GTP酶的结合，但也有几个突变位于p21结合域边界之外。与Cdc42而非Rac1结合的Pak1突变体也被Cdc42而非Rac1激活。在表达致癌性Ha-Ras的大鼠3Y1细胞中，Rac1结合缺陷的Pak1突变体未被激活，这表明Ras通过Cdc42以外的GTP酶发出信号来激活Pak1。当使用表皮生长因子激活Pak1时也获得了类似的结果。然而，无法结合Rac的Pak1突变体仍然能被小牛血清很好地激活，这意味着这种刺激可能独立于Rac诱导Pak激活。
