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 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.
Pryzwansky
K B
KB
Department of Pathology, University of North Carolina, Chapel Hill 27599-7525.
Wyatt
T A
TA
Lincoln
T M
TM
D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000032:analysis / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D006801:Humans; D007382:Intermediate Filaments / Q000201:enzymology*; D009504:Neutrophils / Q000737:chemistry / Q000201:enzymology* / Q000648:ultrastructure; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D014746:Vimentin / Q000032:analysis / Q000378:metabolism*
D014746:Vimentin; C016276:8-bromocyclic GMP; D000001:Calcimycin; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569718:PRKG1 protein, human; D011493:Protein Kinase C; D006152:Cyclic GMP; D002118:Calcium