Localization of cyclic GMP-dependent protein kinase in human mononuclear phagocytes.
The presence and physiological role of cGMP-dependent protein kinase (G-kinase) was investigated in human mononuclear phagocytes. Western blots of monocyte extracts revealed a single polypeptide band that comigrated with purified bovine lung G-kinase. G-kinase was localized by immunofluorescence microscopy in freshly isolated adherent human monocytes, monocyte-derived macrophages cultured from 4 to 14 days, and alveolar macrophages. In monocytes, G-kinase was localized in granules or vesicles in the cytoplasm, at the microtubule organizing center, on filaments, and in the nucleus. In monocyte-derived macrophages, intense staining for G-kinase was found in the vicinity of the Golgi, in vesicles throughout the cytoplasm, and diffusely in the nucleus. Dual-label confocal laser scanning microscopy demonstrated that G-kinase was colocalized with the endoplasmic reticulum. For comparison, G-kinase was localized in alveolar macrophages that were adhered from 3 to 30 min. In these cells, G-kinase was prominent within the organelle-rich area pericortical to the nucleus. However, a well-defined area of intense staining was also observed at the cell periphery at early time points during adherence and spreading. Rhodamine-labeled phalloidin showed that this peripheral area was rich in F-actin. Cytochalasin D, but not nocodazole, inhibited G-kinase targeting to the cell margin. Furthermore, the guanylate cyclase inhibitor LY83583 inhibited alveolar macrophage spreading and staining for G-kinase at the cell periphery. These data suggest that G-kinase may play an important role in cGMP-mediated regulation involved in protein processing and cell motility.
Pryzwansky
K B
KB
Department of Pathology, University of North Carolina, Chapel Hill 27599-7525, USA.
Kidao
S
S
Wyatt
T A
TA
Reed
W
W
Lincoln
T M
TM
D000634:Aminoquinolines / Q000494:pharmacology; D015153:Blotting, Western; D017869:Cyclic GMP-Dependent Protein Kinases / Q000032:analysis* / Q000502:physiology; D003599:Cytoskeleton / Q000187:drug effects / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors; D006801:Humans; D016676:Macrophages, Alveolar / Q000201:enzymology; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000201:enzymology; D010586:Phagocytes / Q000201:enzymology*
D000634:Aminoquinolines; C041715:6-anilino-5,8-quinolinedione; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase