Cyclic adenosine 3':5'-monophosphate-dependent protein kinase on the external surface of LS-174T human colon carcinoma cells.
The analysis of purified plasma membranes and the surface of intact cells revealed the presence of cyclic adenosine 3':5'-monophosphate-(cAMP) dependent protein kinase (PKA) on the external surface of LS-174T human colon carcinoma cells. Photoaffinity labeling of intact cells at confluence with 8-azido-[32P]cAMP identified the cAMP-binding proteins on the surface. Immunoprecipitation identified the photoaffinity-labeled cAMP-binding proteins as the RIIalpha regulatory subunit of PKA. During the logarithmic stage of growth, both the RIalpha and RIIalpha subunits of PKA were localized on the cell surface. Intact LS-174T cells catalyzed the phosphorylation of Kemptide in a cAMP-dependent manner; upon substitution of cAMP in the medium with 8-chloroadenosine, which did not compete with cAMP for the binding on intact cells, the ecto-PKA was no longer activated. The specific inhibitory protein for PKA, PKI, abolished the stimulation of phosphorylation by cAMP. Forskolin, which elevates intracellular levels of cAMP, activated ecto-PKA. Moreover, probenecid, which blocks the export of cAMP, inhibited the forskolin-mediated activation of ecto-PKA. These results demonstrate that LS-174T colon carcinoma cells possess an ecto-PKA on the external surface. This ecto-PKA is similar, if not identical, to the soluble intracellular PKA.
Kondrashin
A
A
Cellular Biochemistry Section, Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, Maryland 20892-1750, USA.
Nesterova
M
M
Cho-Chung
Y S
YS
D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D019920:Photoaffinity Labels / Q000378:metabolism; D011339:Probenecid / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D001386:Azides; D009842:Oligopeptides; D019920:Photoaffinity Labels; D005576:Colforsin; C017042:8-azidoadenosine-3',5'-monophosphate; C037642:kemptide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011339:Probenecid