The mixed lineage kinase DLK is oligomerized by tissue transglutaminase during apoptosis

Current evidence suggests that the mixed lineage kinase family member dual leucine zipper-bearing kinase (DLK) might play a significant role in the regulation of cell growth and differentiation, particularly during the process of tissue remodeling. To further explore this working model, we have investigated the regulation of host and recombinant DLK in NIH3T3 and COS-1 cells undergoing apoptosis. Using calphostin C, a potent and selective inhibitor of protein kinase C and a recognized apoptosis inducer for various cell types, we demonstrate, by immunoblot analysis, that DLK protein levels are rapidly and dramatically down-regulated during the early phases of apoptosis. Down-regulation in calphostin C-treated cells was also accompanied by the appearance of SDS- and mercaptoethanol-resistant high molecular weight DLK immunoreactive oligomers. Experiments aimed at elucidating the mechanism(s) underlying DLK oligomerization revealed that the tissue transglutaminase (tTG) inhibitor monodansylcadaverine antagonized the effects of calphostin C almost completely, thereby suggesting the involvement of a tTG-catalyzed reaction as the root cause of DLK down-regulation and accumulation as high molecular weight species. In support of this notion, we also show that DLK can serve as a substrate for tTG-dependent cross-linking in vitro and that this covalent post-translational modification leads to the functional inactivation of DLK. Taken together, these observations suggest that transglutamination and oligomerization may constitute a relevant physiological mechanism for the regulation of DLK activity.