Osawa S, Hall P F
Endocrinology. 1985 Dec;117(6):2347-56. doi: 10.1210/endo-117-6-2347.
Preparations of cytoskeleton from Y-1 cells were found to phosphorylate various cytoskeletal proteins when incubated with [gamma-32P]ATP. When cAMP was added to the cytoskeleton, a rapid increase in phosphorylation of cytoskeletal protein was observed, and changes were seen in the phosphorylation of individual proteins; four additional proteins were phosphorylated (mol wt, 165,000, 92,000, 45,000, and 24,000) and three proteins were more intensely phosphorylated than without cAMP (mol wt, 125,000, 51,000, and 38,000). In addition, one protein (mol wt, 96,000) that was intensely phosphorylated without cAMP was not phosphorylated with the cyclic nucleotide, and a second (mol wt, 48,000) was less phosphorylated. The increased level of total phosphorylation returned to the unstimulated level within 10 min. The increased phosphorylation of proteins produced by cAMP was inhibited by protein kinase inhibitor. cAMP-dependent protein kinase activity was closely associated with the cytoskeleton, since it was not removed by Triton X-100 (1%, wt/vol), although some activity could be extracted with buffer containing high concentrations of salt. When the cytoskeleton of Y-1 cells was subjected to treatments that disrupt the cytoskeleton before the cells were extracted (cytochalasin B, colchicine, and sonication), no change was seen in cAMP-dependent protein kinase activity. However, cytochalasin B increased phosphorylation of two proteins that were not phosphorylated by cAMP-dependent kinase (mol wt, 63,000 and 43,000). Sonication of the cytoskeleton before addition of [gamma-32P]ATP caused a number of changes in cAMP-independent phosphorylation, but did not affect cAMP-dependent phosphorylation. cAMP-dependent phosphorylation required Mg2+ and was inhibited by Ca2+. It is concluded that the cytoskeleton of Y-1 cells contains bound cAMP-dependent protein kinase that phosphorylates certain cytoskeleton proteins. The cytoskeleton also contains one or more cAMP-independent kinase systems. It is suggested that the cAMP-dependent protein kinase described here may be important in the cytoskeletal responses to ACTH.
当用[γ-32P]ATP孵育时，发现从Y-1细胞制备的细胞骨架能使多种细胞骨架蛋白磷酸化。当向细胞骨架中加入cAMP时，观察到细胞骨架蛋白的磷酸化迅速增加，并且单个蛋白的磷酸化发生了变化；另外四种蛋白被磷酸化（分子量分别为165,000、92,000、45,000和24,000），还有三种蛋白比未加cAMP时磷酸化程度更高（分子量分别为125,000、51,000和38,000）。此外，一种在未加cAMP时被强烈磷酸化的蛋白（分子量96,000）在加入环核苷酸后未被磷酸化，另一种蛋白（分子量48,000）的磷酸化程度降低。总磷酸化水平的增加在10分钟内恢复到未刺激时的水平。cAMP诱导的蛋白磷酸化增加被蛋白激酶抑制剂抑制。cAMP依赖性蛋白激酶活性与细胞骨架紧密相关，因为它不能被Triton X-100（1%，重量/体积）去除，尽管一些活性可以用含有高浓度盐的缓冲液提取。当在提取细胞之前对Y-1细胞的细胞骨架进行破坏细胞骨架的处理（细胞松弛素B、秋水仙碱和超声处理）时，cAMP依赖性蛋白激酶活性没有变化。然而，细胞松弛素B增加了两种未被cAMP依赖性激酶磷酸化的蛋白（分子量63,000和43,000）的磷酸化。在加入[γ-32P]ATP之前对细胞骨架进行超声处理导致了许多非cAMP依赖性磷酸化的变化，但不影响cAMP依赖性磷酸化。cAMP依赖性磷酸化需要Mg2+，并被Ca2+抑制。得出的结论是，Y-1细胞的细胞骨架含有结合的cAMP依赖性蛋白激酶，该激酶能使某些细胞骨架蛋白磷酸化。细胞骨架还含有一个或多个非cAMP依赖性激酶系统。有人提出，这里描述的cAMP依赖性蛋白激酶可能在细胞骨架对促肾上腺皮质激素的反应中起重要作用。
