Hirai S, Noda K, Moriguchi T, Nishida E, Yamashita A, Deyama T, Fukuyama K, Ohno S
Department of Molecular Biology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan.
J Biol Chem. 1998 Mar 27;273(13):7406-12. doi: 10.1074/jbc.273.13.7406.
MKN28-derived nonreceptor type of serine/threonine kinase/mixed lineage kinase 2 (MST/MLK2) directly phosphorylates and activates SEK1/MKK4/JNKK1/SKK1 in vitro, thereby acting as a mitogen-activated protein (MAP) kinase kinase kinase in the JNK/SAPK pathway (Hirai, S. -i., Katoh, M., Terada, M., Kyriakis, J. M., Zon, L. I., Rana, A., Avruch, J., and Ohno, S. (1997) J. Biol. Chem. 272, 15167-15173). The in vitro reconstitution system for the kinase cascade allowed us now to identify JNK/SAPK activators involved in the MST/MLK2-dependent activation of JNK/SAPK in vivo. We show that at least two distinct MST/MLK2-dependent JNK/SAPK activators are present in the fractionated COS-1 cell lysate, and that they appear to be SEK1/MKK4/JNKK1/SKK1 and MKK7/JNKK2/SKK4 by Western blot analysis. Notably, a majority of the MST/MLK2-dependent JNK/SAPK-activating activity is found in MKK7-containing fractions, whereas the MEKK1-dependent activity is comparably distributed in SEK1- and MKK7-containing fractions. Moreover, MST/MLK2 activates recombinant MKK7 more effectively than recombinant SEK1, whereas MEKK1 activates both to a similar extent. In addition, the deletion analysis on MST/MLK2 showed that the kinase domain is responsible for the determination of substrate specificity. These results provide a molecular aspect to the differential regulation of the two JNK activators by a variety of cellular stimuli.
源自MKN28的非受体型丝氨酸/苏氨酸激酶/混合谱系激酶2（MST/MLK2）在体外直接磷酸化并激活SEK1/MKK4/JNKK1/SKK1，从而在JNK/SAPK途径中作为丝裂原活化蛋白（MAP）激酶激酶激酶发挥作用（平井，S.-i.，加藤，M.，寺田，M.，基里亚基斯，J.M.，佐恩，L.I.，拉纳，A.，阿夫鲁奇，J.，及大野，S.（1997年）《生物化学杂志》272，15167 - 15173）。激酶级联反应的体外重组系统使我们现在能够鉴定体内参与MST/MLK2依赖性JNK/SAPK激活的JNK/SAPK激活剂。我们发现，在分级分离的COS - 1细胞裂解物中存在至少两种不同的MST/MLK2依赖性JNK/SAPK激活剂，通过蛋白质印迹分析显示它们似乎是SEK1/MKK4/JNKK1/SKK1和MKK7/JNKK2/SKK4。值得注意的是，大部分MST/MLK2依赖性JNK/SAPK激活活性存在于含MKK7的组分中，而MEKK1依赖性活性在含SEK1和含MKK7的组分中分布相当。此外，MST/MLK2比重组SEK1更有效地激活重组MKK7，而MEKK1对两者的激活程度相似。另外，对MST/MLK2的缺失分析表明激酶结构域负责底物特异性的确定。这些结果为多种细胞刺激对两种JNK激活剂的差异调节提供了分子层面的依据。
