Phosphorylated Ssk1 prevents unphosphorylated Ssk1 from activating the Ssk2 mitogen-activated protein kinase kinase kinase in the yeast high-osmolarity glycerol osmoregulatory pathway
Phosphorylated Ssk1 prevents unphosphorylated Ssk1 from activating the Ssk2 mitogen-activated protein kinase kinase kinase in the yeast high-osmolarity glycerol osmoregulatory pathway
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DOI：
10.1128/mcb.00589-08
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发表时间：
2008-09-01
影响因子：
5.3
通讯作者：
Saito, Haruo
中科院分区：
文献类型：
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作者：
Horie, Tetsuro;Tatebayashi, Kazuo;Saito, Haruo
在酿酒酵母中，外部高渗透压激活Hog 1丝裂原活化蛋白激酶（MAPK），它控制着酿酒酵母适应的各个方面。Ssk 1是细菌双组分反应调节剂的同源物，并激活Hog 1上游的Ssk 2 MAPK激酶激酶。已经提出，未磷酸化的Ssk 1（Ssk 1-OH）是活性形式，并且通过Sln 1-Ypd 1-Ssk 1多步磷酸化机制在Asp 554处磷酸化的Ssk 1（Ssk 1类似于P）是非活性形式。在这项研究中，我们表明，组成性激活SSK 2发生时，SSK 1磷酸化被阻断的SSK 1突变的磷酸化位点或SSK 1突变，抑制其与Ypd 1，磷酸盐的供体SSK 1的相互作用。因此，Ssk 1-OH确实是Ssk 2激活所必需的。然而，野生型Ssk 1或不能结合Ssk 2的Ssk 1突变体的过表达阻止了组成型活性Ssk 1突变体激活Ssk 2。因此，Ssk 1具有作为Ssk 2的激活剂和Ssk 1自身的抑制剂的双重功能。我们还发现Ssk 1主要以二聚体的形式存在于细胞内。从突变体表型，我们推断，只有Ssk 1-OH/Ssk 1-OH二聚体可以有效地激活Ssk 2。因此，由于Ssk 1类似于P结合并抑制Ssk 1-OH，因此Ssk 1-OH水平的适度波动不会导致Hog 1的非生理性和有害的活化。
In Saccharomyces cerevisiae, external high osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK), which controls various aspects of osmoadaptation. Ssk1 is a homolog of bacterial two-component response regulators and activates the Ssk2 MAPK kinase kinase upstream of Hog1. It has been proposed that unphosphorylated Ssk1 (Ssk1-OH) is the active form and that Ssk1 phosphorylated (Ssk1 similar to P) at Asp554 by the Sln1-Ypd1-Ssk1 multistep phosphorelay mechanism is the inactive form. In this study, we show that constitutive activation of Ssk2 occurs when Ssk1 phosphorylation is blocked by either an Ssk1 mutation at the phosphorylation site or an Ssk1 mutation that inhibits its interaction with Ypd1, the donor of phosphate to Ssk1. Thus, Ssk1-OH is indeed necessary for Ssk2 activation. However, overexpression of wild-type Ssk1 or of an Ssk1 mutant that cannot bind Ssk2 prevents constitutively active Ssk1 mutants from activating Ssk2. Therefore, Ssk1 has a dual function as both an activator of Ssk2 and an inhibitor of Ssk1 itself. We also found that Ssk1 exists mostly as a dimer within cells. From mutant phenotypes, we deduce that only the Ssk1-OH/Ssk1-OH dimer can activate Ssk2 efficiently. Hence, because Ssk1 similar to P binds to and inhibits Ssk1-OH, moderate fluctuation of the level of Ssk1-OH does not lead to nonphysiological and detrimental activation of Hog1.