Function of the Rho family GTPases in Ras-stimulated Raf activation
Function of the Rho family GTPases in Ras-stimulated Raf activation
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DOI：
10.1074/jbc.m103496200
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发表时间：
2001-09-14
影响因子：
4.8
通讯作者：
Guan, KL
中科院分区：
文献类型：
--
作者：
Li, WQ;Chong, HR;Guan, KL
Ras在Raf激酶的活化中起重要作用，Raf激酶直接负责MEK-ERK激酶途径的活化。Ras和Raf的N-末端调节结构域之间的直接蛋白质-蛋白质相互作用对于Raf活化是至关重要的。然而，与Ras的关联不足以在体外激活Raf，表明Ras必须激活一些其他生化事件，导致Raf的激活。我们已经观察到RasV 12 Y32 F和RasV 12 T35 S突变体不能激活Raf，但保留了与Raf相互作用的能力。在本报告中，我们发现RasV 12 Y32 F和RasV 12 T35 S可以与Rho家族GTPases的成员合作激活Raf，而单独的Rho家族GTPases在Raf激活中无效。Rac或RhoA的显性失活突变体可以阻断Ras对Raf的激活。Rac或Cdc 42的作用可以被Pak激酶取代，Pak激酶是Rac/Cdc 42的直接下游靶标。此外，表达Pak的激酶失活突变体或Pak 1的N-末端抑制结构域可以阻断Rac或Cdc 42的作用。相比之下，Pak似乎在将信号从RhoA传递到Raf中没有直接作用，表明RhoA利用了与Rac/Cdc 42不同的机制。膜相关的Raf而不是细胞质Raf可以被Rac或RhoA激活。我们的数据支持一个模型，通过该模型Rho家族小GTP酶在介导Ras激活Raf中发挥重要作用。Ras至少在Raf活化中具有两种不同的功能，通过直接结合将Raf募集到质膜，以及通过Rho家族GTP酶刺激Raf活化激酶。
Ras plays an essential role in activation of Raf kinase which is directly responsible for activation of the MEK-ERK kinase pathway. A direct protein-protein interaction between Ras and the N-terminal regulatory domain of Raf is critical for Raf activation. However, association with Ras is not sufficient to activate Raf in vitro, indicating that Ras must activate some other biochemical events leading to activation of Raf. We have observed that RasV12Y32F and RasV12T35S mutants fail to activate Raf, yet retain the ability to interact with Raf. In this report, we showed that RasV12Y32F and RasV12T35S can cooperate with members of the Rho family GTPases to activate Raf while alone the Rho family GTPase is not effective in Raf activation. A dominant negative mutant of Rac or RhoA can block Raf activation by Ras. The effect of Rac or Cdc42 can be substituted by the Pak kinase, which is a direct downstream target of Rac/Cdc42. Furthermore, expression of a kinase inactive mutant of Pak or the N-terminal inhibitory domain of Pak1 can block the effect of Rac or Cdc42. In contrast, Pak appears to play no direct role in relaying the signal from RhoA to Raf, indicating that RhoA utilizes a different mechanism than Rac/Cdc42. Membrane-associated but not cytoplasmic Raf can be activated by Rac or RhoA. Our data support a model by which the Rho family small GTPases play an important role to mediate the activation of Raf by Ras. Ras, at least, has two distinct functions in Raf activation, recruitment of Raf to the plasma membrane by direct binding and stimulation of Raf activating kinases via the Rho family GTPases.