Dynamics of munc18-1 phosphorylation/dephosphorylation in rat brain nerve terminals
Dynamics of munc18-1 phosphorylation/dephosphorylation in rat brain nerve terminals
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DOI：
10.1046/j.1460-9568.2000.00931.x
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发表时间：
2000-01-01
影响因子：
3.4
通讯作者：
Verhage, M
中科院分区：
文献类型：
--
作者：
de Vries, KJ;Geijtenbeek, A;Verhage, M
Munc 18 -1是参与神经元分泌的SEC 1蛋白家族的哺乳动物成员。它的序列包含几个由蛋白激酶C（PKC）磷酸化的共有位点，蛋白激酶C是一种已知的增强分泌的激酶。我们的特点是磷酸化的突触munc 18 -1池的内源性，突触前PKC亚型。在离体大鼠脑神经末梢，munc 18 -1几乎完全非磷酸化。其磷酸化状态增加了250%的内源性磷酸酶的抑制和1500%的额外的，直接PKC激活使用佛波醇酯。K+-诱发的去极化也增加munc 18 -1磷酸化，在5秒内以Ca 2+依赖的方式增加50%。PKC抑制剂可阻断神经末梢中Munc 18 -1的磷酸化。神经末梢内源性PKC的激活抑制突触munc 18 -1与其结合伙伴syntaxin-1A的相互作用50%。munc 18 -1抗血清从盐溶液中沉淀出80%的天然脑源性munc 18 -1，但从突触体裂解物中仅沉淀出12%，以及6%的突触突触融合蛋白-1A/B;这些量不受PKC激活的影响。在这12%中，每摩尔munc 18的磷酸盐掺入量比总库低四倍。我们的结论是，突触munc 18 -1池可以很容易和迅速磷酸化的内源性突触前PKC亚型。高组成性磷酸酶活性保持其基础磷酸化状态低，使得PKC活化可显著增加磷酸化状态。这些磷酸化动力学和对与syntaxin-1A相互作用的影响使得munc 18 -1成为解释PKC依赖性分泌增强的突出候选者。
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.