Daniels R H, Zenke F T, Bokoch G M
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.
J Biol Chem. 1999 Mar 5;274(10):6047-50. doi: 10.1074/jbc.274.10.6047.
Activation of p21-activated kinases (Paks) is achieved through binding of the GTPases Rac or Cdc42 to a conserved domain in the N-terminal regulatory region of Pak. Additional signaling components are also likely to be important in regulating Pak activation. Recently, a family of Pak-interacting guanine nucleotide exchange factors (Pix) have been identified and which are good candidates for regulating Pak activity. Using an active, truncated form of alphaPix (amino acids 155-545), we observe stimulation of Pak1 kinase activity when alphaPix155-545 is co-expressed with Cdc42 and wild-type Pak1 in COS-1 cells. This activation does not occur when we co-express a Pak1 mutant unable to bind alphaPix. The activation of wild-type Pak1 by alphaPix155-545 also requires that alphaPix155-545 retain functional exchange factor activity. However, the Pak1(H83,86L) mutant that does not bind Rac or Cdc42 is activated in the absence of GTPase by alphaPix155-545 and by a mutant of alphaPix155-545 that no longer has exchange factor activity. Pak1 activity stimulated in vitro using GTPgammaS-loaded Cdc42 was also enhanced by recombinant alphaPix155-545 in a binding-dependent manner. These data suggest that Pak activity can be modulated by physical interaction with alphaPix and that this specific effect involves both exchange factor-dependent and -independent mechanisms.
p21激活激酶（Paks）的激活是通过GTP酶Rac或Cdc42与Pak N端调节区域的保守结构域结合来实现的。其他信号成分在调节Pak激活中可能也很重要。最近，已鉴定出一类与Pak相互作用的鸟嘌呤核苷酸交换因子（Pix），它们是调节Pak活性的良好候选者。使用活性截短形式的αPix（氨基酸155 - 545），当αPix155 - 545与Cdc42和野生型Pak1在COS - 1细胞中共表达时，我们观察到Pak1激酶活性受到刺激。当我们共表达无法结合αPix的Pak1突变体时，这种激活不会发生。αPix155 - 545对野生型Pak1的激活还要求αPix155 - 545保留功能性交换因子活性。然而，不结合Rac或Cdc42的Pak1（H83,86L）突变体在没有GTP酶的情况下被αPix155 - 545和不再具有交换因子活性的αPix155 - 545突变体激活。使用加载GTPγS的Cdc42在体外刺激的Pak1活性也以结合依赖的方式被重组αPix155 - 545增强。这些数据表明，Pak活性可以通过与αPix的物理相互作用来调节，并且这种特定效应涉及交换因子依赖性和非依赖性机制。
