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首页> 外文期刊>The European Journal of Neuroscience >Dynamics of munc18-1 phosphorylation/dephosphorylation in rat brain nerve terminals.
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Dynamics of munc18-1 phosphorylation/dephosphorylation in rat brain nerve terminals.

机译：大鼠脑神经末梢中munc18-1磷酸化/去磷酸化的动力学。

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摘要

Munc18-1 is a mammalian member of the SEC1 protein family implicated in neuronal secretion. Its sequence contains several consensus sites for phosphorylation by protein kinase C (PKC), a kinase known to enhance secretion. We have characterized the phosphorylation of the synaptic munc18-1 pool by endogenous, presynaptic PKC-isoforms. In isolated rat brain nerve terminals, munc18-1 was almost completely nonphosphorylated. Its phosphorylation state increased by 250% on inhibition of endogenous phosphatases and by 1500% on additional, direct PKC activation using phorbol esters. K+-evoked depolarization also increased munc18-1 phosphorylation, by 50% within 5 s in a Ca2+-dependent manner. Munc18-1 phosphorylation in nerve terminals was blocked by PKC inhibitors. Activation of endogenous PKC in nerve terminals inhibited the interaction of synaptic munc18-1 with its binding partner syntaxin-1A by 50%. Munc18-1 antisera precipitated 80% of native, brain-derived munc18-1 from salt solutions, but only 12% from synaptosomal lysates, together with 6% synaptic syntaxin-1A/B; these amounts were not changed by PKC activation. In this 12%, the phosphate incorporation per mole of munc18 was four-fold lower than the total pool. We conclude that the synaptic munc18-1 pool can be readily and rapidly phosphorylated by endogenous presynaptic PKC isoforms. A high constitutive phosphatase activity keeps its basal phosphorylation state low so that PKC activation can increase the phosphorylation state dramatically. These phosphorylation dynamics and the effects on the interaction with syntaxin-1A make munc18-1 a prominent candidate to account for PKC-dependent enhancement of secretion.
机译：Munc18-1是SEC1蛋白家族的哺乳动物成员，与神经元分泌有关。其序列包含几个被蛋白激酶C（PKC）磷酸化的共有位点，PKC是一种已知会增强分泌的激酶。我们已经表征了内源性，突触前PKC亚型的突触munc18-1库的磷酸化。在孤立的大鼠脑神经末梢中，munc18-1几乎完全未被磷酸化。在抑制内源性磷酸酶的作用下，其磷酸化状态增加了250％，在使用佛波醇酯进行的直接PKC激活后，其磷酸化状态增加了1500％。 K +引起的去极化也以5 Ca依赖于Ca 2+的方式使munc18-1磷酸化增加了50％。神经末梢中的Munc18-1磷酸化被PKC抑制剂阻断。神经末梢内源性PKC的激活抑制了突触munc18-1及其结合伴侣syntaxin-1A的相互作用50％。 Munc18-1抗血清从盐溶液中沉淀出80％的天然脑源性munc18-1，而从突触体裂解物中仅沉淀了12％，以及6％突触语法Synthemin-1A / B。这些数量不会因PKC激活而改变。在这12％中，每摩尔munc18的磷酸盐掺入量比总溶液量低四倍。我们得出的结论是，内源性突触前PKC异构体可以很容易地将突触munc18-1磷酸化。较高的组成型磷酸酶活性使其基础磷酸化状态保持较低，因此PKC活化可显着增加磷酸化状态。这些磷酸化动力学以及对与syntaxin-1A相互作用的影响使munc18-1成为解决PKC依赖性分泌增加的重要候选者。

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