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.