Regulation of Dual Leucine Zipper Kinase (DLK) by Prediabetic Signalsdissertation
Аннотация: Diabetes mellitus type 2 is the most increasing metabolic disease worldwide. The disease is recognized by the disability of endocrine pancreas to afford the high metabolic demand which has been raised due to insulin resistance followed by hyperglycemia. This failure might be addressed to both β-cell dysfunction and β-cell mass decline. CRE- (cAMP response element) binding protein (CREB) is a transcriptional factor which plays an outstanding role in the survival and maintenance of β-cells. The mitogen activated protein kinase kinase kinase (MAPKKK) DLK (dual leucine zipper kinase) has been shown to inhibit the membrane depolarization-induced transcriptional activity of CREB and its coactivator CRTC. DLK phosphorylates and activates the stress activated protein kinase JNK (C-Jun N-terminal kinase). In this study the regulation of DLK by prediabetic signals was investigated in two ways. First the regulation of DLK kinase activity by prediabetic signals, and second the subcellular translocation of DLK induced by pro-inflammatory cytokines. Two point mutations were generated in the putative phosphorylation sites of DLK (Serin-298 and Serin-302) and the participation of these phosphorylation sites in the activity of DLK was investigated. Mutation in Serin-302 residue abolished the inhibitory effect of DLK on CRE- and CBP-dependent gene transcription. The Serin-302 mutant was also unable to phosphorylate and activate JNK. These data suggest that Serin-302 residue is an important residue in the kinase activity of DLK. An antibody against DLK was produced which recognizes the phosphorylation of DLK specifically at Serin-302 residue. This antibody provides a valuable tool for investigating the activity of DLK. By use of this antibody we showed that pro-inflammatory cytokine TNFα alone and in combination with IFNγ activate DLK by phosphorylation of DLK at Serin-302 residue. Previous study showed that pro-inflammatory cytokines TNFα and IL-1β induce nuclear localization of DLK in β-cell line HIT. In this study the interaction of DLK protein with nuclear receptors (α-importin, β-importin, and transportin) was investigated. The results showed that DLK interacts with α-importin but neither with β-importin nor with transportin. The mutation in DLK bipartite NLS destroyed the interaction with α-importin. These data show that DLK contains a functional bipartite NLS which interacts with the classic nuclear localization component α-importin which transports DLK into the nucleus. The mutation in leucine zipper sequence of DLK (DLK-PP) which prevents the homodimerization of DLK had no effect on the interaction of DLK with α-importin but decreased the nuclear localization induced by TNFα compared to DLKwt. This data suggest that the homodimerization of DLK is not necessary for the interaction with nuclear receptor, but has an obvious effect on the stimulated nuclear translocation of the protein. The role of DLK in the establishment of diet-induced diabetes was investigated using DLK heterozygote mice and their wild-type littermates. The data showed no establishment of diabetes in neither in wild-type nor in heterozygote mice. DLK knock-out mice die perinatally. To investigate the role of DLK in function and survival of β-cells in an animal model, a targeting knock-out vector was designed and generated in this study. In this vector the exons 3 to 6 of DLK are flanked by two loxP sites which are recognized by Cre recombinase. The mice generated using this targeting vector, will be mated with the mice expressing Cre recombinase under the control of rat insulin promoter. The next generation express Cre recombinase in β-cells where it can cut out the loxP flanked DLK, resulting in the β-cell-specific DLK knock-out mice.
Год издания: 2013
Авторы: Rohollah Babaeikelishomi
Ключевые слова: FOXO transcription factor regulation, Pancreatic function and diabetes, Metabolism, Diabetes, and Cancer
Открытый доступ: hybrid