Identification of structural and functional domains in mixed lineage kinase dual leucine zipper-bearing kinase required for complex formation and stress-activated protein kinase activation

Accumulating evidence suggests that mitogen-activated protein kinase signal ing pathways form modular signaling complexes. Because the mixed lineage ki nase dual leucine zipper-bearing kinase (DLK) is a large modular protein, s tructure-function analysis was undertaken to examine the role of DLK domain s in macromolecular complex formation. DLK mutants were used to demonstrate that a DLK leucine zipper-leucine zipper interaction is necessary for DLK dimerization and to show that DLK dimerization mediated by the leucine zipp er domain is prerequisite for DLK activity and subsequent activation of str ess-activated protein kinase (SAPK), Heterologous mixed lineage kinase fami ly members can he co-immunoprecipitated. However, the DLK leucine zipper do main interacted specifically only with the DLK leucine zipper domain; in co ntrast, DLK NH2-terminal region was sufficient to co-immunoprecipitate leuc ine zipper kinase and DLK, DLK has been shown to associate with the putativ e scaffold protein JIP1, This association occurred through the DLK NH2-term inal region and occurred independently of DLK catalytic activity. Although the DLK NH2-terminal region associated directly with JIP-1, this region did not interact directly with either DLK or leucine zipper kinase. Therefore, DLK may interact with heterologous mixed lineage kinase proteins via inter mediary proteins. The NH2-terminal region of overexpressed DLK was required for activation of SAPK, These results provide evidence that protein comple x formation is required for signal transduction from DLK to SAPK.