Mata M, Merritt S E, Fan G, Yu G G, Holzman L B
Department of Neurology, University of Pittsburgh Medical School, Pittsburgh, Pennsylvania 15261, USA.
J Biol Chem. 1996 Jul 12;271(28):16888-96. doi: 10.1074/jbc.271.28.16888.
The biochemistry and regulation of dual leucine zipper bearing kinase (DLK), a member of the mixed lineage kinase or MLK subfamily of protein kinases, was examined in the nervous system. DLK transcript expression in the nervous system was predominantly neuronal. DLK protein was present in synaptic terminals where it was associated with both plasma membrane and cytosol fractions. Within these two fractions, DLK had differing characteristics. Cytosolic DLK existed in both a phosphorylated and dephosphorylated state; DLK associated with plasma membrane existed in the dephosphorylated state only. On nonreducing SDS-polyacrylamide gel electrophoresis, cytosolic DLK migrated at 130 kDa, while membrane associated DLK migrated with an apparent Mr >/= 260,000. Similarly, DLK transiently expressed in COS 7 cells autophosphorylated in vivo and migrated at approximately 260 kDa when separated by nonreducing SDS-polyacrylamide gel electrophoresis. In cotransfection experiments, FLAG-tagged DLK or a FLAG-tagged truncated DLK mutant (F-Delta520) was coimmunoprecipitated with Myc-tagged DLK and formed complexes under nonreducing conditions consistent with the conclusion that DLK formed covalently associated homodimers in overexpressing COS 7 cells. In aggregating neuronal-glial cultures, depolarization of plasma membrane lead to dephosphorylation of DLK. Treatment of aggregates with 5 nM or 200 nM okadaic acid lead to a shift in electrophoretic mobility consistent with phosphorylation of DLK. Treatment with cyclosporin A, a specific inhibitor of the calcium/calmodulin-dependent protein phosphatase 2B (calcineurin), had no effect on DLK phosphorylation under basal conditions. However, cyclosporin A completely inhibited DLK dephosphorylation upon membrane depolarization.
在神经系统中研究了双亮氨酸拉链激酶（DLK）的生物化学性质和调控机制，DLK是蛋白激酶混合谱系激酶或MLK亚家族的成员。DLK转录本在神经系统中的表达主要集中在神经元。DLK蛋白存在于突触末端，与质膜和胞质部分均有关联。在这两个部分中，DLK具有不同的特性。胞质中的DLK以磷酸化和去磷酸化两种状态存在；与质膜相关的DLK仅以去磷酸化状态存在。在非还原SDS聚丙烯酰胺凝胶电泳中，胞质中的DLK迁移率为130 kDa，而与膜相关的DLK迁移率的表观分子量≥260,000。同样，在COS 7细胞中瞬时表达的DLK在体内发生自磷酸化，经非还原SDS聚丙烯酰胺凝胶电泳分离时迁移率约为260 kDa。在共转染实验中，带有FLAG标签的DLK或带有FLAG标签的截短DLK突变体（F-Delta520）与带有Myc标签的DLK共同免疫沉淀，并在非还原条件下形成复合物，这一结果支持了在过表达的COS 7细胞中DLK形成共价结合的同二聚体的结论。在神经元-神经胶质细胞聚集培养物中，质膜去极化导致DLK去磷酸化。用5 nM或200 nM冈田酸处理聚集物会导致电泳迁移率发生变化，这与DLK的磷酸化一致。用环孢素A（钙/钙调蛋白依赖性蛋白磷酸酶2B（钙调神经磷酸酶）的特异性抑制剂）处理，在基础条件下对DLK磷酸化没有影响。然而，环孢素A完全抑制了膜去极化时DLK的去磷酸化。
