Direct binding and In vivo regulation of the fission yeast p21-activated kinase shk1 by the SH3 domain protein scd2.
Direct binding and In vivo regulation of the fission yeast p21-activated kinase shk1 by the SH3 domain protein scd2.
复制标题
SH3 结构域蛋白 scd2 对裂殖酵母 p21 激活激酶 shk1 的直接结合和体内调节。
DOI：
10.1128/mcb.19.12.8066
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发表时间：
1999
影响因子：
5.3
通讯作者：
Marcus,S
中科院分区：
文献类型：
--
作者：
Chang,E;Bartholomeusz,G;Pimental,R;Chen,J;Lai,H;Wang,Lh;Yang,P;Marcus,S
Ste 20/p21激活的激酶同源物Shk 1对裂殖酵母裂殖酵母的生存力和正常形态、交配和细胞周期控制是必不可少的。Shk 1受p21 G蛋白Cdc 42调节，Cdc 42已显示与SH 3结构域蛋白Scd 2（也称为Ral 3）形成复合物。在这项研究中，我们调查是否Scd 2在调节Shk 1功能中发挥作用。我们发现，重组Scd 2和Shk 1直接在体外相互作用，它们在体内相互作用，确定在裂殖酵母的双杂交试验和遗传分析。Scd 2的两个N-末端SH 3结构域中的第二个对于与Shk 1的相互作用是必要且充分的。虽然全长Scd 2相互作用，只有R1的N-末端调节亚结构域的Shk 1，C-末端缺失突变体的Scd 2相互作用的R1和R3亚结构域的Shk 1，这表明非SH 3的Scd 2的C-末端结构域可能参与定义特异性SH 3结合结构域识别。Scd 2的过表达刺激了裂殖酵母中野生型Shk 1的自磷酸化活性，但与遗传分析的结果一致，没有刺激缺乏R1亚结构域的Shk 1蛋白的活性。额外的双杂交实验的结果表明，Scd 2可以刺激Shk 1的催化功能，至少部分，通过积极调节Cdc 42和Shk 1之间的蛋白质-蛋白质相互作用。我们建议，Scd 2功能作为一个组织中心，或支架，为Cdc 42复合体在裂变酵母，它的行为与Cdc 42的音乐会积极调节Shk 1功能。
The Ste20/p21-activated kinase homolog Shk1 is essential for viability and required for normal morphology, mating, and cell cycle control in the fission yeastSchizosaccharomyces pombe. Shk1 is regulated by the p21 G protein Cdc42, which has been shown to form a complex with the SH3 domain protein Scd2 (also called Ral3). In this study, we investigated whether Scd2 plays a role in regulating Shk1 function. We found that recombinant Scd2 and Shk1 interact directly in vitro and that they interact in vivo, as determined by the two-hybrid assay and genetic analyses in fission yeast. The second of two N-terminal SH3 domains of Scd2 is both necessary and sufficient for interaction with Shk1. While full-length Scd2 interacted with only the R1 N-terminal regulatory subdomain of Shk1, a C-terminal deletion mutant of Scd2 interacted with both the R1 and R3 subdomains of Shk1, suggesting that the non-SH3 C-terminal domain of Scd2 may be involved in defining specificity in SH3 binding domain recognition. Overexpression of Scd2 stimulated the autophosphorylation activity of wild-type Shk1 in fission yeast but, consistent with results of genetic analyses, did not stimulate the activity of a Shk1 protein lacking the R1 subdomain. Results of additional two-hybrid experiments suggest that Scd2 may stimulate Shk1 catalytic function, at least in part, by positively modulating protein-protein interaction between Cdc42 and Shk1. We propose that Scd2 functions as an organizing center, or scaffold, for the Cdc42 complex in fission yeast and that it acts in concert with Cdc42 to positively regulate Shk1 function.