Cytochemical identification of the regulatory subunit of the cAMP-dependent protein kinase by use of fluorescently labeled catalytic subunit. Examination of protein kinase dissociation in hepatoma cells responding to 8-Br-cAMP stimulation.
Homogeneous catalytic subunit from the cAMP-dependent protein kinase, when derivatized with a fluorophore, was used as a cytochemical probe to locate intracellular sites of the protein kinase regulatory subunit. After conjugation, the fluoresceinated catalytic subunit (F:C), derivatized to a stoichiometry of approximately 1 mol/mol, retained near full activity as judged by specific activity and by titration against either regulatory subunit or Inhibitor Protein of the protein kinase. With this molecular probe the dissociated regulatory subunit was localized by direct cytochemistry in Reuber H-35 hepatoma cells that had been exposed, while intact, for 0-120 min to 10(-4) M 8-Br-cAMP. After stimulation, cultures were fixed and washed and then incubated for 16 h with F:C. Following 8-Br-cAMP stimulation, extensive binding of the probe to both cytoplasmic and nucleolar sites was observed. This binding was diminished but not eliminated when 50 microM cAMP was present during the incubation of the fixed cells with F:C that was eliminated by a 40-fold molar excess of underivatized catalytic subunit but not by heat-denatured catalytic subunit, and was not reduced by a 20-fold molar excess of cGMP-dependent protein kinase, examined plus or minus cGMP. Collectively, the results allow the conclusion that the F:C probe binds free regulatory subunit. The time course of its change with 8-Br-cAMP (measured as the difference between binding in the presence or absence of cAMP during the postfixation treatment) mirrors that previously reported for changes in the catalytic subunit in these cells, also identified cytochemically (Byus, C. V., and Fletcher, W.H. (1982) J. Cell Biol. 93, 727-734). The binding of the F:C probe, detected when cAMP is present during postfixation treatment, may possibly represent binding to free Inhibitor Protein of the cAMP-dependent protein kinase. If so, it was at a level of approximately 20% of the maximal level of detectable regulatory subunit, and it also showed cytosolic and nucleolar localization.
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism; D006651:Histocytochemistry; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence; D011494:Protein Kinases / Q000032:analysis*; D051381:Rats; D013861:Thiocyanates / Q000378:metabolism
D005452:Fluoresceins; D046911:Macromolecular Substances; D013861:Thiocyanates; D015124:8-Bromo Cyclic Adenosine Monophosphate; D011494:Protein Kinases; D006152:Cyclic GMP; D016650:Fluorescein-5-isothiocyanate