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Functional insights into the activation mechanism of Ste20-related kinases

机译:Ste20相关激酶激活机制的功能见解

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Mammalian Ste20-related kinases modulate salt transport and ion homeostasis through physical interaction and phosphorylation of cation-chloride cotransporters. Identification of a sea urchin (Strongylocentrotus purpuratus) ortholog of the mouse Oxidative Stress Response 1 (OSR1) kinase prompted the cloning and testing of the functional effect of a non-mammalian kinase on a mammalian cotransporter. Heterologous expression of sea urchin OSR1 (suOSR1) cRNA with mouse WNK4 cRNA and mouse NKCC1 cRNA in Xenopus laevisoocytes activated the cotransporter indicating evolutionary conservation of the WNK4-OSR1-NKCC signaling pathway. However, expression of a suOSR1 kinase mutated to confer constitutive activity did not result in stimulation of the cotransporter. Using a chimeric strategy, we determined that both the mutated catalytic and regulatory domains of the suOSR1 kinase were functional, suggesting that the tertiary structure of full-length mutated suOSR1 must somehow adopt an inactive conformation. In order to identify the regions or residues which lock the suOSR1 kinase in an inactive conformation, we created and tested several additional chimeras by replacing specific portions of the suOSR1 gene with complimentary mouse OSR1 sequences. Co-expression of these chimeras identified several regions in both the catalytic and regulatory domain of suOSR1 which possibly prevented the kinase from acquiring an active conformation. Interestingly, non-functional suOSR1 chimeras were able to activate mouse NKCC1 when a mouse scaffolding protein, Cab39, was co-expressed in frog oocytes. Sea urchin/mouse OSR1 chimeras and kinase stabilization with mouse Cab39 has provided some novel insights into the activation mechanism of the Ste20-related kinases.
机译:哺乳动物Ste20相关的激酶通过阳离子氯化物共转运蛋白的物理相互作用和磷酸化来调节盐的转运和离子稳态。小鼠氧化应激反应1(OSR1)激酶的海胆(Strongylocentrotus purpuratus)直系同源物的鉴定促使克隆和测试非哺乳动物激酶对哺乳动物协同转运蛋白的功能作用。在非洲爪蟾幼稚细胞中,海胆OSR1(suOSR1)cRNA与小鼠WNK4 cRNA和小鼠NKCC1 cRNA的异源表达激活了共转运蛋白,表明WNK4-OSR1-NKCC信号通路的进化保守性。但是,suOSR1激酶的表达发生突变以赋予其组成性活性,并未刺激该共转运蛋白。使用嵌合策略,我们确定suOSR1激酶的突变催化域和调节域均具有功能,这表明全长突变suOSR1的三级结构必须以某种方式采用非活性构象。为了鉴定将suOSR1激酶锁定在非活性构象的区域或残基,我们通过用互补的小鼠OSR1序列替换suOSR1基因的特定部分来创建和测试了几种其他的嵌合体。这些嵌合体的共表达在suOSR1的催化结构域和调节结构域中确定了几个区域,这可能阻止了该激酶获得活性构象。有趣的是,当在青蛙卵母细胞中共表达小鼠支架蛋白Cab39时,非功能性suOSR1嵌合体能够激活小鼠NKCC1。海胆/小鼠OSR1嵌合体和小鼠Cab39的激酶稳定作用为Ste20相关激酶的激活机制提供了一些新颖的见解。

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