Nagy Gábor, Reim Kerstin, Matti Ulf, Brose Nils, Binz Thomas, Rettig Jens, Neher Erwin, Sørensen Jakob B
Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Neuron. 2004 Feb 5;41(3):417-29. doi: 10.1016/s0896-6273(04)00038-8.
Protein kinase A (PKA) is a key regulator of neurosecretion, but the molecular targets remain elusive. We combined pharmacological manipulations of kinase and phosphatase activities with mutational studies on the exocytotic machinery driving fusion of catecholamine-containing vesicles from chromaffin cells. We found that constitutive PKA activity was necessary to maintain a large number of vesicles in the release-ready, so-called primed, state, whereas calcineurin (protein phosphatase 2B) activity antagonized this effect. Overexpression of the SNARE protein SNAP-25a mutated in a PKA phosphorylation site (Thr-138) eliminated the effect of PKA inhibitors on the vesicle priming process. Another, unidentified, PKA target regulated the relative size of two different primed vesicle pools that are distinguished by their release kinetics. Overexpression of the SNAP-25b isoform increased the size of both primed vesicle pools by a factor of two, and mutations in the conserved Thr-138 site had similar effects as in the a isoform.
蛋白激酶A（PKA）是神经分泌的关键调节因子，但其分子靶点仍不清楚。我们将激酶和磷酸酶活性的药理学操作与对驱动嗜铬细胞中含儿茶酚胺囊泡融合的胞吐机制的突变研究相结合。我们发现，组成型PKA活性对于维持大量囊泡处于释放就绪状态（即所谓的引发状态）是必要的，而钙调神经磷酸酶（蛋白磷酸酶2B）活性则拮抗这种作用。在PKA磷酸化位点（Thr-138）发生突变的SNARE蛋白SNAP-25a的过表达消除了PKA抑制剂对囊泡引发过程的影响。另一个未确定的PKA靶点调节了两个不同引发囊泡池的相对大小，这两个池通过其释放动力学来区分。SNAP-25b亚型的过表达使两个引发囊泡池的大小增加了两倍，并且保守的Thr-138位点的突变与a亚型具有相似的效果。
