Intracellular kinetics of free catalytic units dissociated from adenosine 3',5'-monophosphate-dependent protein kinase in adrenocortical tumor cells (Y-1).
To define the role of cAMP in the actions of ACTH, the dissociation of cAMP-dependent protein kinase and the subsequent intracellular location of its free catalytic units were monitored after exposure of Y-1 cells to ACTH, FSH, or cyclic nucleotide analog. To accomplish this, a fluorescinated cytochemical probe was used that complexes specifically with free catalytic units from cAMP-dependent protein kinase. Also, the effects of hormone or nucleotide on secretion of fluorogenic steroids and DNA synthesis were examined. Y-1 cells dissociated protein kinase in a dose-dependent fashion when exposed to ACTH or cAMP analog, but did not respond to FSH, which was one of the control agents used. After 30 min of treatment with 1.5 X 10(-10) M ACTH, free catalytic units were observed only in the cytoplasm of Y-1 cells, whereas a similar time of exposure to 3 X 10(-10) M ACTH led to the appearance of catalytic units in nucleolus as well as in cytoplasm. ACTH (6 X 10(-10) M) caused a rise in cytoplasmic and nucleolar protein kinase dissociation proportionally greater than that seen in cultures exposed to 3 X 10(-10) M ACTH. Upon treatment with 6 X 10(-10) M ACTH, the amount of free catalytic units in cytoplasm and nucleolus was detectably greater than that in controls within 1 min of stimulation and continued to rise with increasing time of exposure to hormone. The nuclear, mostly nucleolar, content of free catalytic unit appeared to peak after 15 min of stimulation, while cytoplasmic enzyme levels continued to rise up to 60 min. Exposure of Y-1 cells to nucleotide analog caused cAMP-dependent protein kinase dissociation with temporal kinetics and a subcellular distribution similar to that seen after ACTH stimulation. We conclude that actions of ACTH are mediated by cAMP-dependent protein kinases. Further, there appear to be two intracellular pools of protein kinase, one nucleolar, the other cytoplasmic, and these may be independently regulated, with the nucleolar enzyme requiring higher concentrations of ACTH for dissociation than those needed for cytoplasm protein kinase. These observations may be relevant to the fact that more ACTH is required to inhibit DNA synthesis than is necessary to enhance steroid production.
Murray
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Byus
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Fletcher
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D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000306:Adrenal Cortex Neoplasms / Q000201:enzymology*; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins; D002460:Cell Line; D002466:Cell Nucleolus / Q000201:enzymology; D000242:Cyclic AMP / Q000494:pharmacology*; D003593:Cytoplasm / Q000201:enzymology; D004247:DNA / Q000096:biosynthesis; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006651:Histocytochemistry; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013256:Steroids / Q000096:biosynthesis; D013861:Thiocyanates
D002352:Carrier Proteins; D005452:Fluoresceins; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D013256:Steroids; D013861:Thiocyanates; C015482:protein kinase modulator; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000324:Adrenocorticotropic Hormone; D005640:Follicle Stimulating Hormone; D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases; D016650:Fluorescein-5-isothiocyanate