Cdc42-induced activation of the mixed-lineage kinase SPRK in vivo -: Requirement of the Cdc42/Rac interactive binding motif and changes in phosphorylation
Cdc42-induced activation of the mixed-lineage kinase SPRK in vivo -: Requirement of the Cdc42/Rac interactive binding motif and changes in phosphorylation
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DOI：
10.1074/jbc.275.19.14231
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发表时间：
2000-05-12
影响因子：
4.8
通讯作者：
Gallo, KA
中科院分区：
文献类型：
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作者：
Böck, BC;Vacratsis, PO;Gallo, KA
Src同源3结构域(SH3)-containing proline-rich protein kinase (SPRK)/mix -lineage kinase (MLK)-3是一种丝氨酸/苏氨酸激酶，在哺乳动物细胞中过表达可激活c-Jun nh2末端激酶途径。调控SPRK活性的机制尚不清楚。Rho家族的小gtpase， Rac和Cdc42，已被证明结合并调节信号蛋白的活性，包括SPRK，其中包含Cdc42/Rac相互作用结合基序。SPRK和活化Cdc42的共表达增加了SPRK的活性。SPRKs的Cdc42/Rac相互结合样基序包含8个共识残基中的6个。使用位点定向诱变方法，我们发现SPRK包含一个功能性Cdc42/Rac相互作用结合基序，这是SPRK与Cdc42关联和被Cdc42激活所必需的。然而，使用缺乏cooh末端拉链区/基本拉伸的SPRK变体进行的实验表明，该区域也可能有助于Cdc42的结合。与PAK家族蛋白激酶不同，我们发现Cdc42对SPRK的激活不能在体外系统中使用纯化的重组蛋白重现。比较磷酸肽图谱显示，活化的Cdc42与SPRK共表达改变了SPRK的体内丝氨酸/苏氨酸磷酸化模式，提示Cdc42增加SPRK催化活性的机制涉及到SPRK的体内磷酸化的改变。据我们所知，这是cdc42介导的蛋白激酶体内磷酸化变化的第一个例子。这些研究表明，Cdc42激活SPRK需要一个额外的成分或细胞环境。
Src homology 3 domain (SH3)-containing proline-rich protein kinase (SPRK)/mixed-lineage kinase (MLK)-3 is a serine/threonine kinase that upon overexpression in mammalian cells activates the c-Jun NH2-terminal kinase pathway. The mechanisms by which SPRK activity is regulated are not well understood. The small Rho family GTPases, Rac and Cdc42, have been shown to bind and modulate the activities of signaling proteins, including SPRK, which contain Cdc42/Rac interactive binding motifs. Coexpression of SPRK and activated Cdc42 increases SPRKs activity. SPRKs Cdc42/Rac interactive binding-like motif contains six of the eight consensus residues. Using a site-directed mutagenesis approach, we show that SPRK contains a functional Cdc42/Rac interactive binding motif that is required for SPRKs association with and activation by Cdc42. However, experiments using a SPRK variant that lacks the COOH-terminal zipper region/basic stretch suggest that this region may also contribute to Cdc42 binding. Unlike the PAK family of protein kinases, we find that the activation of SPRK by Cdc42 cannot be recapitulated in an in vitro system using purified, recombinant proteins. Comparative phosphopeptide mapping demonstrates that coexpression of activated Cdc42 with SPRK alters the in vivo serine/threonine phosphorylation pattern of SPRK suggesting that the mechanism by which Cdc42 increases SPRKs catalytic activity involves a change in the in vivo phosphorylation of SPRK. This is, to the best of our knowledge, the first demonstrated example of a Cdc42-mediated change in the in vivo phosphorylation of a protein kinase. These studies suggest an additional component or cellular environment is required for SPRK activation by Cdc42.