Identification of a Specific Domain Responsible for JNK2α2 Autophosphorylation

Jian Cui,Marina Holgado-Madruga,Wanwen Su,Hiromasa Tsuiki,Philip Wedegaertner,Albert J Wong

doi:10.1074/jbc.m412165200

Jian Cui, Marina Holgado-Madruga + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m412165200

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Abstract

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 JNK2alpha2 or JNK2beta2. 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 JNK2alpha2 autoactivation, we have generated a series of chimeric cDNAs joining portions of JNK1alpha2, which does not have detectable autophosphorylation activity, with portions of JNK2alpha2, 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 JNK2alpha2 that is required for its autophosphorylation. Mutation of JNK2alpha2 to its counterpart of JNK1alpha2 in this region abrogated the autophosphorylation activity and c-Jun substrate kinase activity in vivo and in vitro. Notably, switching of JNK1alpha2 to JNK2alpha2 at this 9-amino acid site enabled JNK1alpha2 to gain the autophosphorylation activity in vivo and in vitro. We also found two other functional sites that participate in JNK2alpha2 activity. One site ranging from amino acids 363 to 382 of JNK2alpha2 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 JNK2alpha2 autophosphorylation, and this information could be used as potential targets to block JNK2alpha2 activation.

Highlights

The c-Jun N-terminal kinases (JNK),1 together with ERK and p38, constitute the mitogen-activated protein kinase family
Domains I and IV are located in the N terminus, which has been shown to be critical for both catalytic function and ATP binding for all of the JNK isoforms
We have demonstrated for the first time that amino acids 218 –226 of JNK2␣2 (LVKGCVIFQ) are crucial for its autophosphorylation

Summary

Introduction

The c-Jun N-terminal kinases (JNK), together with ERK and p38, constitute the mitogen-activated protein kinase family. We have found that all of the known JNK2 isoforms, but not the JNK1 or JNK3 isoforms, have autophosphorylation activity and constitutive substrate kinase activity in vitro and in vivo in the absence of upstream kinases [18]. These data support the notion that JNK2 may play a more crucial role in tumor formation than JNK1 or JNK3. We asked whether there were a specific domain(s) within JNK2 that leads to its constitutive activation. Identifying such a domain(s) will reveal a novel mechanism for how JNKs are activated but will assist in the design of inhibitors that could be used to block the activation of this tumorigenic JNK isoform

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