Rck2 kinase is a substrate for the osmotic stress-activated mitogen-activated protein kinase Hog1
Rck2 kinase is a substrate for the osmotic stress-activated mitogen-activated protein kinase Hog1
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DOI：
10.1128/mcb.20.11.3887-3895.2000
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发表时间：
2000-06-01
影响因子：
5.3
通讯作者：
Posas, F
中科院分区：
文献类型：
--
作者：
Bilsland-Marchesan, E;Ariño, J;Posas, F
酵母细胞暴露于细胞外渗透压的增加激活Hog 1丝裂原活化蛋白激酶（MAPK）。Hog 1 MAPK的激活导致诱导一系列适应性反应，这使得细胞能够在高渗透压环境中存活。很少有人知道如何MAPK激活的结果在诱导这些反应，主要是因为没有直接底物Hog 1已被报道。我们进行了一个双杂交筛选使用Hog 1作为诱饵，以确定底物的MAPK，和Rck 2蛋白激酶被确定为Hog 1的相互作用。双杂交分析和共沉淀分析表明，Hog 1强烈结合的C-末端区域的Rck 2。在渗透胁迫下，Rck 2在体内以Hog 1依赖的方式磷酸化。此外，纯化的Hog 1能够在体内和体外激活时磷酸化Rck 2。Rck 2磷酸化特异性地发生在Ser 519处，Ser 519是位于C-末端推定的自身抑制结构域内的残基。有趣的是，Hog 1在Ser 519的磷酸化导致Rck 2激酶活性的增加。Rck 2的过表达部分抑制了hog 1 Delta和pbs 2 Delta细胞的细胞毒性敏感表型，表明Rck 2在Hog 1的下游起作用。一致地，由Hog 1 MAPK的过度激活引起的生长停滞通过RCK 2基因的缺失而被消除。此外，催化受损（推测显性抑制）Rck 2激酶的过度表达导致野生型细胞中的抗氧化剂耐受性降低，但在hog 1 Delta细胞中则不然。两者合计，我们的数据表明，Rck 2的行为下游的Hog 1，控制一个子集的MAPK诱导的反应后渗透压。
Exposure of yeast cells to increases in extracellular osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK). Activation of Hog1 MAPK results in induction of a set of osmoadaptive responses, which allow cells to survive in high-osmolarity environments. Little is known about how the MAPK activation results in induction of these responses, mainly because no direct substrates for Hog1 have been reported. We conducted a two-hybrid screening using Hog1 as a bait to identify substrates for the MAPK, and the Rck2 protein kinase was identified as an interactor for Hog1. Both two-hybrid analyses and coprecipitation assays demonstrated that Hog1 binds strongly to the C-terminal region of Rck2. Upon osmotic stress, Rck2 was phosphorylated in vivo in a Hog1-dependent manner. Furthermore, purified Hog1 was able to phosphorylate Rck2 when activated both in vivo and in vitro. Rck2 phosphorylation occurred specifically at Ser519, a residue located within the C-terminal putative autoinhibitory domain. Interestingly, phosphorylation at Ser519 by Hog1 resulted in an increase of Rck2 kinase activity. Overexpression of Rck2 partially suppressed the osmosensitive phenotype of hog1 Delta and pbs2 Delta cells, suggesting that Rck2 is acting downstream of Hog1. Consistently, growth arrest caused by hyperactivation of the Hog1 MAPK was abolished by deletion of the RCK2 gene. Furthermore, overexpression of a catalytically impaired (presumably dominant inhibitory) Rck2 kinase resulted in a decrease of osmotolerance in wild-type cells but not in hog1 Delta cells. Taken together, our data suggest that Rck2 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK upon osmotic stress.