Identification of a specific domain responsible for JNK2α2 autophosphorylation

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 JNK2α2 or JNK2β2. 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 JNK2α2 autoactivation, we have generated a series of chimeric cDNAs joining portions of JNK1α2, which does not have detectable autophosphorylation activity, with portions of JNK2α2, 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 JNK2α2 that is required for its autophosphorylation. Mutation of JNK2α2 to its counterpart of JNK1α2 in this region abrogated the autophosphorylation activity and c-Jun substrate kinase activity in vivo and in vitro. Notably, switching of JNK1α2 to JNK2α2 at this 9-amino acid site enabled JNK1α2 to gain the autophosphorylation activity in vivo and in vitro. We also found two other functional sites that participate in JNK2α2 activity. One site ranging from amino acids 363 to 382 of JNK2α2 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 JNK2α2 autophosphorylation, and this information could be used as potential targets to block JNK2α2 activation.