Cdc42 regulation of kinase activity and signaling by the yeast p21-activated kinase Ste20
Cdc42 regulation of kinase activity and signaling by the yeast p21-activated kinase Ste20
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DOI：
10.1128/mcb.22.9.2939-2951.2002
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发表时间：
2002-05-01
影响因子：
5.3
通讯作者：
Pryciak, PM
中科院分区：
文献类型：
--
作者：
Lamson, RE;Winters, MJ;Pryciak, PM
酿酒酵母STE20是p21激活蛋白激酶(PAK)家族的一员，具有多种功能，包括信息素反应信号转导。虽然PAK通常被小G蛋白激活，而Ste20与CDC42结合，但CDC42-Ste20结合的作用一直存在争议，主要是因为Ste20缺乏完整的CDC42结合(CRIB)结构域，保持了激酶活性和信息素反应。在这里，我们表明，与婴儿床缺失不同的是，Ste20婴儿床结构域上的点突变破坏了CDC42结合，也破坏了信息素信号。我们还发现，在体内，GTP结合的CDC42可以刺激Ste20激酶的活性，并且这种作用被婴儿床基因突变所阻断。此外，Ste20的婴儿床和激动域相互结合，而破坏这种相互作用的突变会导致高活性的激酶活性，并绕过CDC42结合的要求。这些观察表明，Ste20的Krb结构域是自我抑制的，这种负面影响可以被Cdc42拮抗，以促进Ste20激酶的活性和信号转导。丝化途径的信号传递也观察到了类似的结果，表明交配和丝化途径对Cdc42-Ste20相互作用的要求没有本质上的不同。虽然对信息素信号是必要的，但CDC42-Ste20相互作用的作用不需要信息素或信息素激活的Gbetagamma复合体的调节，因为KRIB点突变也通过激活形式的激酶级联支架蛋白Ste5来扰乱信号。总之，我们的观察表明，CDC42将Ste20转化为活性形式，而途径刺激调节这种活性Ste20通过特定途径触发信号的能力。
The Saccharomyces cerevisiae kinase Ste20 is a member of the p21-activated kinase (PAK) family with several functions, including pheromone-responsive signal transduction. While PAKs are usually activated by small G proteins and Ste20 binds Cdc42, the role of Cdc42-Ste20 binding has been controversial, largely because Ste20 lacking its entire Cdc42-binding (CRIB) domain retains kinase activity and pheromone response. Here we show that, unlike CRIB deletion, point mutations in the Ste20 CRIB domain that disrupt Cdc42 binding also disrupt pheromone signaling. We also found that Ste20 kinase activity is stimulated by GTP-bound Cdc42 in vivo and this effect is blocked by the CRIB point mutations. Moreover, the Ste20 CRIB and kinase domains bind each other, and mutations that disrupt this interaction cause hyperactive kinase activity and bypass the requirement for Cdc42 binding. These observations demonstrate that the Ste20 CRIB domain is autoinhibitory and that this negative effect is antagonized by Cdc42 to promote Ste20 kinase activity and signaling. Parallel results were observed for filamentation pathway signaling, suggesting that the requirement for Cdc42-Ste20 interaction is not qualitatively different between the mating and filamentation pathways. While necessary for pheromone signaling, the role of the Cdc42-Ste20 interaction does not require regulation by pheromone or the pheromone-activated Gbetagamma complex, because the CRIB point mutations also disrupt signaling by activated forms of the kinase cascade scaffold protein Ste5. In total, our observations indicate that Cdc42 converts Ste20 to an active form, while pathway stimuli regulate the ability of this active Ste20 to trigger signaling through a particular pathway.