Vimentin is transiently co-localized with and phosphorylated by cyclic GMP-dependent protein kinase in formyl-peptide-stimulated neutrophils.
Vimentin is transiently co-localized with and phosphorylated by cyclic GMP-dependent protein kinase in formyl-peptide-stimulated neutrophils.
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DOI：
10.1016/s0021-9258(18)54851-1
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发表时间：
1991-11
期刊：
影响因子：
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通讯作者：
Todd A. Wyatt;Thomas M. Lincoln;K. Pryzwansky
中科院分区：
文献类型：
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作者：
Todd A. Wyatt;Thomas M. Lincoln;K. Pryzwansky
在活化的人中性粒细胞中研究了cGMP依赖性蛋白激酶（G-激酶）（cGMP的主要细胞受体）的作用。免疫细胞化学结果显示，G激酶在fMLP刺激后从弥漫性的胞浆定位转移到细胞骨架和细胞核，并与中间丝蛋白波形蛋白短暂共定位。在此期间，在用fMLP刺激1-2.5分钟之间观察到G-激酶和波形蛋白的最显著的共定位。当时，G-激酶和波形蛋白的共定位主要局限于从邻近细胞核的区域延伸到尾足的细丝。在微管组织中心和细胞核的常染色质中观察到G-激酶的独特定位。在8-Br-cGMP存在下，G-激酶和波形蛋白的丝状染色模式增强。与粘附中性粒细胞中G-激酶和波形蛋白的共定位一致的是cGMP水平的瞬时增加和fMLP刺激的细胞中波形蛋白磷酸化的增加。cGMP水平的增加是依赖于细胞粘附，增强预孵育的中性粒细胞与L-精氨酸（一氧化氮合成的前体），并减弱与一氧化氮合酶抑制剂，NG-单甲基-L-精氨酸。在存在或不存在外源性cGMP的情况下观察到fMLP刺激的中性粒细胞中波形蛋白的磷酸化，尽管在低浓度8-Br-cGMP的存在下观察到波形蛋白的更快速磷酸化，其与G-激酶和波形蛋白的增强的共定位相关。在用8-Br-cGMP处理的非活化细胞中未观察到波形蛋白的磷酸化，表明磷酸化仅在G-激酶与波形蛋白共定位时发生。蛋白激酶C抑制剂staurosporine或H-7的存在下，不抑制波形蛋白磷酸化过程中fMLP刺激，而8-Br-cGMP增强磷酸化在fMLP处理的细胞。这表明蛋白激酶C和cAMP依赖性蛋白激酶都不催化fMLP激活的中性粒细胞中波形蛋白的磷酸化。这些结果表明，波形蛋白和G-激酶共定位于中性粒细胞和波形蛋白磷酸化的G-激酶在响应于这两种蛋白质的共定位。G-激酶和波形蛋白的靶向模型提出假设，G-激酶的瞬时再分布可以调节中性粒细胞活化。
The effects of cGMP-dependent protein kinase (G-kinase), a major cellular receptor of cGMP, were investigated in activated human neutrophils. Immunocytochemistry demonstrated that G-kinase translocated from a diffuse localization in the cytoplasm to the cytoskeleton and nucleus after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP), and transiently co-localized with the intermediate filament protein, vimentin. During this time period, the most remarkable co-localization of G-kinase and vimentin was observed between 1-2.5 min stimulation with fMLP. At that time co-localization of G-kinase and vimentin was predominantly confined to filaments which extended from regions adjacent to the nucleus into the uropod. Distinctive localization for only G-kinase was observed at the microtubule organizing center and euchromatin of the nucleus. The filamentous staining pattern for G-kinase and vimentin was enhanced in the presence of 8-Br-cGMP. Coincident with co-localization of G-kinase and vimentin in adherent neutrophils was a transient increase in cGMP levels and an increase in the phosphorylation of vimentin in fMLP-stimulated cells. The increase in cGMP levels was dependent upon cell adherence, was enhanced by preincubating neutrophils with L-arginine (the precursor for nitric oxide synthesis), and attenuated with the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine. Phosphorylation of vimentin in the fMLP-stimulated neutrophil was observed in the presence or absence of exogenous cGMP, although in the presence of low concentrations of 8-Br-cGMP a more rapid phosphorylation of vimentin was observed that correlated with the enhanced co-localization of G-kinase and vimentin. Phosphorylation of vimentin was not observed in non-activated cells treated with 8-Br-cGMP, suggesting that phosphorylation only occurs when G-kinase is co-localized with vimentin. The presence of the protein kinase C inhibitors, staurosporine or H-7, did not inhibit vimentin phosphorylation during fMLP stimulation, while 8-Br-cGMP enhanced phosphorylation in fMLP-treated cells. This suggests that neither protein kinase C nor cAMP-dependent protein kinase catalyze the phosphorylation of vimentin in neutrophils activated by fMLP. These results indicate that vimentin and G-kinase are co-localized in neutrophils and that vimentin is phosphorylated by G-kinase in response to the co-localization of the two proteins. A model for the targeting of G-kinase and vimentin is presented which hypothesizes that the transient redistribution of G-kinase may regulate neutrophil activation.