Rubenstein J L, Greengard P, Czernik A J
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021-6399.
Synapse. 1993 Feb;13(2):161-72. doi: 10.1002/syn.890130207.
The phosphorylation of synaptophysin, a major integral membrane protein of small synaptic vesicles, was found to be regulated in a Ca(2+)-dependent manner in rat cerebrocortical slices, synaptosome preparations, and highly purified synaptic vesicles isolated from rat forebrain. K(+)-induced depolarization of slices and synaptosomes prelabeled with 32P-orthophosphate produced a rapid, transient increase in serine phosphorylation of synaptophysin. In synaptosomes, the depolarization-dependent increase in synaptophysin phosphorylation required the presence of external Ca2+ in the incubation medium. The addition of Ca2+ plus calmodulin to purified synaptic vesicles resulted in a 4-fold increase in serine phosphorylation of synaptophysin, and this phosphorylation was antagonized by a peptide inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II(. Purified rat forebrain CaM kinase II phosphorylated both purified synaptophysin and endogenous, vesicle-associated synaptophysin, and the resulting 2-dimensional chymotryptic phosphopeptide maps were similar to those derived from synaptophysin phosphorylated in cerebrocortical slices. These data demonstrate that Ca(2+)-dependent phosphorylation of synaptophysin, mediated by CaM kinase II, occurs under physiological conditions.
在大鼠脑皮质切片、突触体标本以及从大鼠前脑分离出的高度纯化的突触小泡中，发现小突触小泡的主要整合膜蛋白突触素的磷酸化是以钙（Ca²⁺）依赖的方式调节的。用³²P - 正磷酸盐预标记的切片和突触体经钾（K⁺）诱导去极化后，突触素的丝氨酸磷酸化迅速短暂增加。在突触体中，突触素磷酸化的去极化依赖性增加需要孵育培养基中存在细胞外Ca²⁺。向纯化的突触小泡中添加Ca²⁺加钙调蛋白导致突触素的丝氨酸磷酸化增加4倍，并且这种磷酸化被钙/钙调蛋白依赖性蛋白激酶II（CaM激酶II）的肽抑制剂所拮抗。纯化的大鼠前脑CaM激酶II使纯化的突触素和内源性的、与小泡相关的突触素磷酸化，所得的二维胰凝乳蛋白酶磷酸肽图谱与在脑皮质切片中磷酸化的突触素的图谱相似。这些数据表明，由CaM激酶II介导的突触素的钙（Ca²⁺）依赖性磷酸化在生理条件下发生。
