Pryzwansky K B, Wyatt T A, Lincoln T M
Department of Pathology, University of North Carolina, Chapel Hill 27599-7525.
Blood. 1995 Jan 1;85(1):222-30.
The effects of the calcium ionophore, A23187, on human neutrophil activation were studied in relation to the signaling mechanism of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G-kinase). Immunocytochemistry demonstrated that G-kinase translocated from a diffuse localization in the cytoplasm to the cytoskeleton after stimulation with A23187. Over a period of 5 minutes, G-kinase was transiently colocalized with the intermediate filament protein, vimentin. At 3 minutes' stimulation with A23187, colocalization of G-kinase and vimentin was predominantly confined to filaments that extended into the uropod. The time of colocalization of G-kinase and vimentin was reduced in the A23187-stimulated cell from 3 minutes to 1 minute by 8-Br-cGMP. Coincident with colocalization was an increase in cGMP levels and transient phosphorylation of vimentin in adhered A23187-stimulated cells. Phosphorylation of vimentin was maximal after 3 minutes with A23187, and was essentially over at 5 minutes. The time of phosphorylation of vimentin was also reduced from 3 minutes to 1 minute when cells were preincubated with 8-Br-cGMP and then stimulated with A23187, which suggests that cyclic adenosine monophosphate (cAMP)-dependent protein kinase does not phosphorylate vimentin in A23187-treated neutrophils. Phosphorylation of vimentin was not observed in nonactivated cells treated only with 8-Br-cGMP. The presence of the protein kinase C inhibitors, staurosporine or H-7, did not inhibit vimentin phosphorylation in A23187-treated cells, which provides supportive data that protein kinase C is not the phosphorylating enzyme. These results suggest that vimentin and G-kinase are colocalized in a Ca(2+)-dependent manner in neutrophils, and that vimentin is transiently phosphorylated by G-kinase in response to the colocalization of the two proteins. The transient redistribution of compartmentalized G-kinase represents one type of neutrophil activation mechanism.
研究了钙离子载体A23187对人中性粒细胞激活的影响，并探讨其与环磷酸鸟苷（cGMP）依赖性蛋白激酶（G激酶）信号转导机制的关系。免疫细胞化学显示，用A23187刺激后，G激酶从细胞质中的弥散定位转移至细胞骨架。在5分钟内，G激酶与中间丝蛋白波形蛋白短暂共定位。用A23187刺激3分钟时，G激酶与波形蛋白的共定位主要局限于延伸至尾足的细丝。8 - 溴 - cGMP可使A23187刺激细胞中G激酶与波形蛋白的共定位时间从3分钟缩短至1分钟。在黏附的A23187刺激细胞中，与共定位同时发生的是cGMP水平升高以及波形蛋白的短暂磷酸化。用A23187刺激3分钟后波形蛋白磷酸化达到最大值，5分钟时基本结束。当细胞先用8 - 溴 - cGMP预孵育然后用A23187刺激时，波形蛋白的磷酸化时间也从3分钟缩短至1分钟，这表明环磷酸腺苷（cAMP）依赖性蛋白激酶不会使A23187处理的中性粒细胞中的波形蛋白磷酸化。仅用8 - 溴 - cGMP处理的未激活细胞中未观察到波形蛋白的磷酸化。蛋白激酶C抑制剂星形孢菌素或H - 7的存在并不抑制A23187处理细胞中波形蛋白的磷酸化，这提供了支持性数据，表明蛋白激酶C不是磷酸化酶。这些结果表明，波形蛋白和G激酶在中性粒细胞中以钙（2 +）依赖性方式共定位，并且响应于两种蛋白的共定位，波形蛋白被G激酶短暂磷酸化。分隔的G激酶的短暂重新分布代表了中性粒细胞激活机制的一种类型。
