Constitutive Activity of JNK2α2 Is Dependent on a Unique Mechanism of MAPK Activation

c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase ( MAPK) family and are important regulators of cell growth, proliferation, and apoptosis. Typically, a sequential series of events are necessary for MAPK activation: phosphorylation, dimerization, and then subsequent translocation to the nucleus. Interestingly, a constitutively active JNK isoform, JNK2 alpha 2, possesses the ability to autophosphorylate and has been implicated in several human tumors, including glioblastoma multiforme. Because overexpression of JNK2 alpha 2 enhances several tumorigenic phenotypes, including cell growth and tumor formation in mice, we studied the mechanisms of JNK2 alpha 2 autophosphorylation and autoactivation. We find that JNK2 alpha 2 dimerization in vitro and in vivo occurs independently of its autophosphorylation but is dependent on nine amino acids, known as the alpha-region. Alanine scanning mutagenesis of the alpha-region reveals that five specific mutants (L218A, K220A, G221A, I224A, and F225A) prevent JNK2 alpha 2 dimerization rendering JNK2 alpha 2 inactive and incapable of stimulating tumor formation. Previous studies coupled with additional mutagenesis of neighboring isoleucines and leucines (I208A, I214A, I231A, and I238A) suggest that a leucine zipper may play an important role in JNK2 alpha 2 homodimerization. We also show that a kinase-inactive JNK2 alpha 2 mutant can interact with and inhibit wild type JNK2 alpha 2 autophosphorylation, suggesting that JNK2 alpha 2 undergoes trans-autophosphorylation. Together, our results demonstrate that JNK2 alpha 2 differs from other MAPK proteins in two major ways; its autoactivation/autophosphorylation is dependent on dimerization, and dimerization most likely precedes autophosphorylation. In addition, we show that dimerization is essential for JNK2 alpha 2 activity and that prevention of dimerization may decrease JNK2 alpha 2 induced tumorigenic phenotypes.