MNK1, a new MAP kinase-activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates
MNK1, a new MAP kinase-activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates
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DOI：
10.1093/emboj/16.8.1921
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发表时间：
1997-04-15
期刊：
影响因子：
11.4
通讯作者：
Hunter, T
中科院分区：
文献类型：
--
作者：
Fukunaga, R;Hunter, T
我们建立了一种新的蛋白激酶底物的表达筛选方法。在该方法中，用纯化的蛋白激酶和[γ-P-32]ATP通过原位固相磷酸化筛选λ噬菌体cDNA表达文库，用ERK 1 MAP激酶筛选HeLa cDNA文库，获得先前表征的ERK底物c-Myc和p90（RSK）的cDNA，在该筛选中分离的命名为MNK 1的新克隆编码蛋白丝氨酸/苏氨酸激酶，其与MAP激酶活化的蛋白激酶2（MAPKAP-K2）、3 pK/MAPKAP-K3和p90（RSK）最相似。细菌表达的MNK 1在体外被ERK 1和p38 MAP激酶磷酸化和激活，但不被JNK/SAPK激活。此外，MNK 1在用12-O-十四酰基佛波醇-13-乙酸酯、胎牛血清、茴香霉素、UV照射、肿瘤坏死因子-α、白细胞介素-1 β或渗透压休克刺激HeLa细胞时被激活，并且由这些刺激引起的激活被MEK抑制剂PD 098059或p38 MAP激酶抑制剂SB 202190差异性地抑制。总之，这些结果表明，MNK 1是一类新的蛋白激酶，通过ERK和p38 MAP激酶信号通路激活。
We have developed a novel expression screening method for identifying protein kinase substrates, In this method, a lambda phage cDNA expression library is screened by in situ, solid-phase phosphorylation using purified protein kinase and [gamma-P-32]ATP, Screening a HeLa cDNA library with ERK1 MAP kinase yielded cDNAs of previously characterized ERK substrates, c-Myc and p90(RSK), demonstrating the utility of this method for identifying physiological protein kinase substrates, A novel clone isolated in this screen, designated MNK1, encodes a protein-serine/threonine kinase, which is most similar to MAP kinase-activated protein kinase 2 (MAPKAP-K2), 3pK/MAPKAP-K3 and p90(RSK). Bacterially expressed MNK1 was phosphorylated and activated in vitro by ERK1 and p38 MAP kinases but not by JNK/SAPK. Further, MNK1 was activated upon stimulation of HeLa cells with 12-O-tetradecanoylphorbol-13-acetate, fetal calf serum, anisomycin, UV irradiation, tumor necrosis factor-alpha, interleukin-1 beta or osmotic shock, and the activation by these stimuli was differentially inhibited by the MEK inhibitor PD098059 or the p38 MAP kinase inhibitor SB202190. Together, these results indicate that MNK1 is a novel class of protein kinase that is activated through both the ERK and p38 MAP kinase signaling pathways.