Isolation of hyperactive mutants of the MAPK p38/Hog1 that are independent of MAPK kinase activation
Isolation of hyperactive mutants of the MAPK p38/Hog1 that are independent of MAPK kinase activation
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DOI：
10.1074/jbc.m101818200
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发表时间：
2001-07-06
影响因子：
4.8
通讯作者：
Engelberg, D
中科院分区：
文献类型：
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作者：
Bell, M;Capone, R;Engelberg, D
丝裂原活化蛋白激酶（MAPK）在生长、发育、分化和凋亡过程中起着关键作用，但在这些过程中特定MAPK的确切作用尚不完全清楚。这个问题可以用这些酶的活性形式来解决，这些酶独立于外部刺激和上游调节，然而，这样的分子是不可用的。MAPK激活需要双磷酸化，在邻近的Tyr和Thr残基上，由MAPK激酶（MAPKKs）催化。目前尚不清楚如何迫使MAPK激活独立于MAPKK磷酸化，这里我们描述了一系列的9个过度活跃（催化和生物学），MAPK Hog1的非MAPKK变体，每个活性分子只包含一个单点突变。6个突变位于该蛋白的保守L16结构域。通过一种新的遗传筛选获得了活性Hog1突变体，该突变体可用于分离其他家族的活性MAPKs。将等效突变引入人类p38 α，即使在大肠杆菌中也能使该酶具有活性，这表明突变增加了p38的内在催化活性，这意味着激活突变可直接用于从酵母到人类产生活性形式的mapk，并为揭示其生物学功能开辟了道路。
Mitogen-activated protein kinases (MAPKs) play pivotal roles in growth, development, differentiation, and apoptosis, The exact role of a given MAPK in these processes is not fully understood. This question could be addressed using active forms of these enzymes that are independent of external stimulation and upstream regulation, Yet, such molecules are not available. MAPK activation requires dual phosphorylation, on neighboring Tyr and Thr residues, catalyzed by MAPK kinases (MAPKKs). It is not known how to force MAPK activation independent of MAPKK phosphorylation, Here we describe a series of nine hyperactive (catalytically and biologically), MAPKK-independent variants of the MAPK Hog1, Each of the active molecules contains just a single point mutation. Six mutations are in the conserved L16 domain of the protein. The active Hog1 mutants were obtained through a novel genetic screen that could be applied for isolation of active MAPKs of other families. Equivalent mutations, introduced to the human p38 alpha, rendered the enzyme active even when produced in Escherichia coli, showing that the mutations increased the intrinsic catalytic activity of p38, It implies that the activating mutations could be directly used for production of active forms of MAPKs from yeasts to humans and could open the way to revealing their biological functions.