Wyatt T A, Lincoln T M, Pryzwansky K B
Department of Pathology, University of North Carolina, Chapel Hill 27599.
J Biol Chem. 1991 Nov 5;266(31):21274-80.
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.
环磷酸鸟苷（cGMP）依赖性蛋白激酶（G激酶）是cGMP的主要细胞受体，本研究对其在活化的人中性粒细胞中的作用进行了探究。免疫细胞化学结果显示，在用N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）刺激后，G激酶从细胞质中的弥散定位转位至细胞骨架和细胞核，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分钟时观察到G激酶与波形蛋白的共定位最为显著。此时，G激酶与波形蛋白的共定位主要局限于从细胞核附近区域延伸至尾足的细丝。仅在细胞核的微管组织中心和常染色质处观察到G激酶的独特定位。在8-溴环磷酸鸟苷（8-Br-cGMP）存在的情况下，G激酶和波形蛋白的丝状染色模式增强。在贴壁中性粒细胞中，G激酶与波形蛋白的共定位同时伴随着cGMP水平的短暂升高以及fMLP刺激细胞中波形蛋白磷酸化的增加。cGMP水平的升高依赖于细胞黏附，用L-精氨酸（一氧化氮合成的前体）预孵育中性粒细胞可增强该升高，而用一氧化氮合酶抑制剂NG-单甲基-L-精氨酸可使其减弱。无论是否存在外源性cGMP，均可观察到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存在的情况下，观察到波形蛋白的磷酸化更快，这与G激酶和波形蛋白共定位的增强相关。在用8-Br-cGMP处理的未活化细胞中未观察到波形蛋白的磷酸化，这表明磷酸化仅在G激酶与波形蛋白共定位时发生。蛋白激酶C抑制剂星形孢菌素或H-7的存在在fMLP刺激期间并不抑制波形蛋白的磷酸化，而8-Br-cGMP可增强fMLP处理细胞中的磷酸化。这表明蛋白激酶C和cAMP依赖性蛋白激酶均不催化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激酶在中性粒细胞中共定位，并且波形蛋白在两种蛋白共定位时被G激酶磷酸化。本文提出了一个G激酶和波形蛋白靶向作用的模型，该模型假设G激酶的短暂重新分布可能调节中性粒细胞的活化。
