Shuang R, Zhang L, Fletcher A, Groblewski G E, Pevsner J, Stuenkel E L
Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622, USA.
J Biol Chem. 1998 Feb 27;273(9):4957-66. doi: 10.1074/jbc.273.9.4957.
The Munc-18-syntaxin 1A complex has been postulated to act as a negative control on the regulated exocytotic process because its formation blocks the interaction of syntaxin with vesicle SNARE proteins. However, the formation of this complex is simultaneously essential for the final stages of secretion as evidenced by the necessity of Munc-18's homologues in Saccharomyces cerevisiae (Sec1p), Drosophila (ROP), and Caenorhabditis elegans (Unc-18) for proper secretion in these organisms. As such, any event that regulates the interaction of these two proteins is important for the control of secretion. One candidate for such regulation is cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division cycle kinases that has recently been copurified with Munc-18 from rat brain. The present study shows that Cdk5 bound to its neural specific activator p35 not only binds to Munc-18 but utilizes it as a substrate for phosphorylation. Furthermore, it is demonstrated that Munc-18 that has been phosphorylated by Cdk5 has a significantly reduced affinity for syntaxin 1A. Finally, it is shown that Cdk5 can also bind to syntaxin 1A and that a complex of Cdk5, p35, Munc-18, and syntaxin 1A can be fashioned in the absence of ATP and promptly disassembled upon the addition of ATP. These results suggest a model in which p35-activated Cdk5 becomes localized to the Munc-18-syntaxin 1A complex by its affinity for both proteins so that it may phosphorylate Munc-18 and thus permit the positive interaction of syntaxin 1A with upstream protein effectors of the secretory mechanism.
Munc-18- syntaxin 1A复合物被认为对调节性胞吐过程起负调控作用，因为其形成会阻断syntaxin与囊泡SNARE蛋白的相互作用。然而，该复合物的形成对于分泌的最后阶段同时也是必不可少的，酿酒酵母（Sec1p）、果蝇（ROP）和秀丽隐杆线虫（Unc-18）中Munc-18的同源物对这些生物体的正常分泌具有必要性，这一事实证明了这一点。因此，任何调节这两种蛋白质相互作用的事件对于分泌的控制都很重要。这种调节的一个候选者是细胞周期蛋白依赖性激酶5（Cdk5），它是细胞分裂周期激酶Cdc2家族的成员，最近已从大鼠脑中与Munc-18一起共纯化出来。本研究表明，与神经特异性激活剂p35结合的Cdk5不仅与Munc-18结合，还将其用作磷酸化的底物。此外，还证明了被Cdk5磷酸化的Munc-18对syntaxin 1A的亲和力显著降低。最后，研究表明Cdk5也可以与syntaxin 1A结合，并且在没有ATP的情况下可以形成Cdk5、p35、Munc-18和syntaxin 1A的复合物，加入ATP后该复合物会迅速分解。这些结果提示了一个模型，其中p35激活的Cdk5通过其对两种蛋白质的亲和力而定位于Munc-18- syntaxin 1A复合物，从而使其可以磷酸化Munc-18，进而允许syntaxin 1A与分泌机制的上游蛋白质效应器进行正向相互作用。
