Department of Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany; DZHK Standort Hamburg, Kiel, Lübeck, Germany.
Department of Pharmacology, University of Göttingen, Robert-Koch-Str. 40, 37099 Göttingen, Germany.
Cell Signal. 2021 Jun;82:109953. doi: 10.1016/j.cellsig.2021.109953. Epub 2021 Feb 16.
Hyperglycemia enhancing the intracellular levels of reactive oxygen species (ROS) contributes to dysfunction and progressive loss of beta cells and thereby to diabetes mellitus. The oxidation sensitive calcium/calmodulin dependent phosphatase calcineurin promotes pancreatic beta cell function and survival whereas the dual leucine zipper kinase (DLK) induces apoptosis. Therefore, it was studied whether calcineurin interferes with DLK action. In a beta cell line similar concentrations of HO decreased calcineurin activity and activated DLK. DLK interacted via its φLxVP motif (aa 362-365) with the interface of the calcineurin subunits A and B. Mutation of the Val prevented this protein protein interaction, hinting at a distinct φLxVP motif. Indeed, mutational analysis revealed an ordered structure of DLK's φLxVP motif whereby Val mediates the interaction with calcineurin and Leu maintains an enzymatically active conformation. Overexpression of DLK wild-type but not the DLK mutant unable to bind calcineurin diminished calcineurin-induced nuclear localisation of the nuclear factor of activated T-cells (NFAT), suggesting that both, DLK and NFAT compete for the substrate binding site of calcineurin. The calcineurin binding-deficient DLK mutant exhibited increased DLK activity measured as phosphorylation of the downstream c-Jun N-terminal kinase, inhibition of CRE-dependent gene transcription and induction of apoptosis. These findings show that calcineurin interacts with DLK; and inhibition of calcineurin increases DLK activity. Hence, this study demonstrates a novel mechanism regulating DLK action. These findings suggest that ROS through inhibition of calcineurin enhance DLK activity and thereby lead to beta cell dysfunction and loss and ultimately diabetes mellitus.
高血糖会增加细胞内活性氧（ROS）的水平，导致β细胞功能障碍和进行性丧失，从而引发糖尿病。氧化敏感的钙/钙调蛋白依赖性磷酸酶钙调神经磷酸酶促进胰岛β细胞的功能和存活，而双亮氨酸拉链激酶（DLK）则诱导细胞凋亡。因此，研究人员研究了钙调神经磷酸酶是否会干扰 DLK 的作用。在胰岛β细胞系中，相似浓度的 H2O2 降低了钙调神经磷酸酶的活性并激活了 DLK。DLK 通过其 φLxVP 基序（aa 362-365）与钙调神经磷酸酶 A 和 B 亚基的界面相互作用。突变 Val 阻止了这种蛋白质-蛋白质相互作用，提示存在一个独特的 φLxVP 基序。事实上，突变分析揭示了 DLK 的 φLxVP 基序的有序结构，其中 Val 介导与钙调神经磷酸酶的相互作用，而 Leu 则维持酶活性构象。DLK 野生型的过表达而不是不能与钙调神经磷酸酶结合的 DLK 突变体减少了钙调神经磷酸酶诱导的核因子活化 T 细胞（NFAT）的核定位，表明 DLK 和 NFAT 都竞争钙调神经磷酸酶的底物结合位点。钙调神经磷酸酶结合缺陷的 DLK 突变体表现出增加的 DLK 活性，如磷酸化下游的 c-Jun N 末端激酶、抑制 CRE 依赖性基因转录和诱导细胞凋亡。这些发现表明钙调神经磷酸酶与 DLK 相互作用；抑制钙调神经磷酸酶增加了 DLK 的活性。因此，本研究证明了一种调节 DLK 作用的新机制。这些发现表明，ROS 通过抑制钙调神经磷酸酶增强了 DLK 的活性，从而导致β细胞功能障碍和丧失，并最终导致糖尿病。
