CYCLIC GUANOSINE MONOPHOSPHATE-DEPENDENT PROTEIN-KINASE IS TARGETED TO INTERMEDIATE FILAMENTS AND PHOSPHORYLATES VIMENTIN IN A23187-STIMULATED HUMAN NEUTROPHILS

The effects of the calcium ionophore, A23187, on human neutrophil acti vation were studied in relation to the signaling mechanism of cyclic g uanosine monophosphate (cGMP)-dependent protein kinase (G-kinase). Imm unocytochemistry demonstrated that G-kinase translocated from a diffus e localization in the cytoplasm to the cytoskeleton after stimulation with A23187. Over a period of 5 minutes, G-kinase was transiently colo calized 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. Th e time of colocalization of G-kinase and vimentin was reduced in the A 23187-stimulated cell from 3 minutes to 1 minute by 8-Br-cGMP. Coincid ent with colocalization was an increase in cGMP levels and transient p hosphorylation of vimentin in adhered A23187-stimulated cells. Phospho rylation 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 preincuba ted with 8-Br-cGMP and then stimulated with A23187, which suggests tha t cyclic adenosine monophosphate (cAMP)-dependent protein kinase does not phosphorylate vimentin in A23187-treated neutrophils. Phosphorylat ion of vimentin was not observed in nonactivated cells treated only wi th 8-Br-cGMP. The presence of the protein kinase C inhibitors, stauros porine or H-7, did not inhibit vimentin phosphorylation in A23187-trea ted 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 Ca2+-dependent manner in neutrophils, and that vimentin is transiently phosphorylated by G-kinase in response t o the colocalization of the two proteins. The transient redistribution of compartmentalized G-kinase represents one type of neutrophil activ ation mechanism. (C) 1995 by The American Society of Hematology.