Identification of a specific domain responsible for JNK2α2 autophosphorylation
Identification of a specific domain responsible for JNK2α2 autophosphorylation
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DOI：
10.1074/jbc.m412165200
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发表时间：
2005-03-18
影响因子：
4.8
通讯作者：
Wong, AJ
中科院分区：
文献类型：
--
作者：
Cui, J;Holgado-Madruga, M;Wong, AJ
c-Jun n-末端激酶（JNKs）是一组丝裂原活化蛋白激酶家族成员，在调节细胞生长、增殖和凋亡中起重要作用。JNK通路的激活与几种人类肿瘤的形成有关。我们之前已经证明，在86%的人类脑肿瘤中，55-kDa JNK异构体是组成性激活的，最近又证明该异构体是JNK2 α 2或JNK2 β 2。重要的是，我们还发现在已知的10种JNK异构体中，JNK2异构体在体外和体内的自磷酸化能力是独一无二的。这不需要任何上游激酶的参与，也导致底物激酶活性在体外和体内。为了阐明JNK2 α 2自激活的机制，我们生成了一系列嵌合cdna，将JNK1 α 2的部分（无法检测到的自磷酸化活性）与JNK2 α 2的部分（具有最强的自磷酸化活性）连接起来。通过体内和体外激酶测定，我们能够在JNK2 α 2中定义一个氨基酸范围从218到226的区域，这是其自磷酸化所必需的。在体内和体外，JNK2 α 2在该区域突变为JNK1 α 2的对应物时，其自磷酸化活性和c-Jun底物激酶活性均被破坏。值得注意的是，JNK1 α 2在这9个氨基酸位点转换为JNK2 α 2，使JNK1 α 2在体内和体外获得自磷酸化活性。我们还发现了另外两个参与JNK2 α 2活性的功能位点。JNK2 α 2的363 - 382个氨基酸位点是体外c-Jun有效结合所必需的，而383 - 424个氨基酸位点增强了自磷酸化强度，尽管它不是体外触发自磷酸化所必需的。这些发现揭示了JNK2 α 2自磷酸化所需的区域，这些信息可以用作阻止JNK2 α 2激活的潜在靶点。
c-Jun N-terminal kinases (JNKs) are a group of mitogen-activated protein kinase family members that are important in regulating cell growth, proliferation, and apoptosis. Activation of the JNK pathway has been implicated in the formation of several human tumors. We have previously demonstrated that a 55-kDa JNK isoform is constitutively activated in 86% of human brain tumors and more recently demonstrated that this isoform is either JNK2 alpha 2 or JNK2 beta 2. Importantly, we have also found that among the 10 known JNK isoforms, the JNK2 isoforms are unique in their ability to autophosphorylate in vitro and in vivo. This does not require the participation of any upstream kinases and also leads to substrate kinase activity in vitro and in vivo. To clarify the mechanism of JNK2 alpha 2 autoactivation, we have generated a series of chimeric cDNAs joining portions of JNK1 alpha 2, which does not have detectable autophosphorylation activity, with portions of JNK2 alpha 2, which has the strongest autophosphorylation activity. Through in vivo and in vitro kinase assays, we were able to define a domain ranging from amino acids 218 to 226 within JNK2 alpha 2 that is required for its autophosphorylation. Mutation of JNK2 alpha 2 to its counterpart of JNK1 alpha 2 in this region abrogated the autophosphorylation activity and c-Jun substrate kinase activity in vivo and in vitro. Notably, switching of JNK1 alpha 2 to JNK2 alpha 2 at this 9-amino acid site enabled JNK1 alpha 2 to gain the autophosphorylation activity in vivo and in vitro. We also found two other functional sites that participate in JNK2 alpha 2 activity. One site ranging from amino acids 363 to 382 of JNK2 alpha 2 is required for efficient c-Jun binding in vitro, and a site ranging from amino acids 383 to 424 enhances autophosphorylation intensity, although it is not required for triggering the autophosphorylation in vitro. These findings have uncovered the regions required for JNK2 alpha 2 autophosphorylation, and this information could be used as potential targets to block JNK2 alpha 2 activation.