Cyclic guanosine monophosphate-dependent protein kinase is targeted to intermediate filaments and phosphorylates vimentin in A23187-stimulated human neutrophils

K. B. Pryzwansky, T. A. Wyatt, T. M. Lincoln

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Abstract

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 Ca2+-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.

Original languageEnglish (US)
Pages (from-to)222-230
Number of pages9
JournalBlood
Volume85
Issue number1
StatePublished - Jan 1 1995

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ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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