Distinct functions of the dual leucine zipper kinase depending on its subcellular localization.

The dual leucine zipper kinase DLK induces ß-cell apoptosis by inhibiting the transcriptional activity conferred by the ß-cell protective transcription factor cAMP response element binding protein CREB. This action might contribute to ß-cell loss and ultimately diabetes. Within its kinase domain DLK shares high homology with the mixed lineage kinase (MLK) 3, which is activated by tumor necrosis factor (TNF) α and interleukin (IL)-1ß, known prediabetic signals. In the present study, the regulation of DLK in ß-cells by these cytokines was investigated. Both, TNFα and IL-1ß induced the nuclear translocation of DLK. Mutations within a putative nuclear localization signal (NLS) prevented basal and cytokine-induced nuclear localization of DLK and binding to the importin receptor importin α, thereby demonstrating a functional NLS within DLK. DLK NLS mutants were catalytically active as they phosphorylated their down-stream kinase c-Jun N-terminal kinase to the same extent as DLK wild-type but did neither inhibit CREB-dependent gene transcription nor transcription conferred by the promoter of the anti-apoptotic protein BCL-xL. In addition, the ß-cell apoptosis-inducing effect of DLK was severely diminished by mutation of its NLS. In a murine model of prediabetes, enhanced nuclear DLK was found. These data demonstrate that DLK exerts distinct functions, depending on its subcellular localization and thus provide a novel level of regulating DLK action. Furthermore, the prevention of the nuclear localization of DLK as induced by prediabetic signals with consecutive suppression of ß-cell apoptosis might constitute a novel target in the therapy of diabetes mellitus.

Inhibition of human insulin gene transcription and MafA transcriptional activity by the dual leucine zipper kinase.

Insulin biosynthesis is an essential ß-cell function and inappropriate insulin secretion and biosynthesis contribute to the pathogenesis of diabetes mellitus type 2. Previous studies showed that the dual leucine zipper kinase (DLK) induces ß-cell apoptosis. Since ß-cell dysfunction precedes ß-cell loss, in the present study the effect of DLK on insulin gene transcription was investigated in the HIT-T15 ß-cell line. Downregulation of endogenous DLK increased whereas overexpression of DLK decreased human insulin gene transcription. 5'- and 3'-deletion human insulin promoter analyses resulted in the identification of a DLK responsive element that mapped to the DNA binding-site for the ß-cell specific transcription factor MafA. Overexpression of DLK wild-type but not its kinase-dead mutant inhibited MafA transcriptional activity conferred by its transactivation domain. Furthermore, in the non-ß-cell line JEG DLK inhibited MafA overexpression-induced human insulin promoter activity. Overexpression of MafA and DLK or its kinase-dead mutant into JEG cells revealed that DLK but not its mutant reduced MafA protein content. Inhibition of the down-stream DLK kinase c-Jun N-terminal kinase (JNK) by SP600125 attenuated DLK-induced MafA loss. Furthermore, mutation of the serine 65 to alanine, shown to confer MafA protein stability, increased MafA-dependent insulin gene transcription and prevented DLK-induced MafA loss in JEG cells. These data suggest that DLK by activating JNK triggers the phosphorylation and degradation of MafA thereby attenuating insulin gene transcription. Given the importance of MafA for ß-cell function, the inhibition of DLK might preserve ß-cell function and ultimately retard the development of diabetes mellitus type 2.