Post-transcriptional regulation of cAMP-dependent protein kinase activity by cAMP in GH3 pituitary tumor cells: Evidence for increased degradation of catalytic subunit in the presence of cAMP

Jeanne M. Richardson, Paul Howard, John S. Massa, Richard Maurer

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Abstract

The effects of cyclic AMP treatment on total cAMP-dependent protein kinase activity in GH3 pituitary tumor cells have been studied. Incubation of cells for 24 h with 1 μM forskolin resulted in a 50% decrease in total cAMP-dependent protein kinase activity which was reversible upon removal of forskolin from culture media. A similar response was observed in GH3 cells treated with 5 ng/ml cholera toxin and 0.5 mM dibutyryl cAMP but not 0.5 mM dibutyryl cGMP. Northern blot analysis demonstrated that the steady-state level of the mRNA for each of the six kinase subunit isoforms studied was not detectably altered after treatment with 1 μM forskolin for 24 h. The concentration of catalytic subunit was also assessed by binding studies using a radiolabeled heat-stable protein kinase inhibitor. Treatment of GH3 cells with 1 μM forskolin for 24 h reduced protein kinase inhibitor binding activity by 50%, consistent with the observed forskolin-induced decrease in total kinase activity. Analysis of endogenous heat-stable protein kinase inhibitor activity in GH3 cell extracts showed no significant difference between forskolin-treated cells and cells maintained under control conditions. To assess possible effects on catalytic subunit degradation, pulse-chase experiments were performed and radiolabeled catalytic subunit was isolated by affinity chromatography. The results demonstrated that treatment of cells with chlorophenylthio-cAMP detectably increased the apparent degradation of radiolabeled catalytic subunit. The increased degradation of the catalytic subunit was sufficient to account for the observed decreases in kinase activity. These results suggest that relatively long term cAMP treatment can alter total cAMP-dependent protein kinase activity through effects to alter the degradation of the catalytic subunit of the enzyme.

Original languageEnglish (US)
Pages (from-to)13635-13640
Number of pages6
JournalJournal of Biological Chemistry
Volume265
Issue number23
StatePublished - Aug 15 1990
Externally publishedYes

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Pituitary Neoplasms
Colforsin
Cyclic AMP-Dependent Protein Kinases
Tumors
Catalytic Domain
Cells
Degradation
Protein Kinase Inhibitors
Phosphotransferases
Hot Temperature
Affinity chromatography
Cholera Toxin
Cyclic AMP
Culture Media
Cell Extracts
Affinity Chromatography
Protein Isoforms
Northern Blotting
Messenger RNA
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Post-transcriptional regulation of cAMP-dependent protein kinase activity by cAMP in GH3 pituitary tumor cells: Evidence for increased degradation of catalytic subunit in the presence of cAMP",
abstract = "The effects of cyclic AMP treatment on total cAMP-dependent protein kinase activity in GH3 pituitary tumor cells have been studied. Incubation of cells for 24 h with 1 μM forskolin resulted in a 50{\%} decrease in total cAMP-dependent protein kinase activity which was reversible upon removal of forskolin from culture media. A similar response was observed in GH3 cells treated with 5 ng/ml cholera toxin and 0.5 mM dibutyryl cAMP but not 0.5 mM dibutyryl cGMP. Northern blot analysis demonstrated that the steady-state level of the mRNA for each of the six kinase subunit isoforms studied was not detectably altered after treatment with 1 μM forskolin for 24 h. The concentration of catalytic subunit was also assessed by binding studies using a radiolabeled heat-stable protein kinase inhibitor. Treatment of GH3 cells with 1 μM forskolin for 24 h reduced protein kinase inhibitor binding activity by 50{\%}, consistent with the observed forskolin-induced decrease in total kinase activity. Analysis of endogenous heat-stable protein kinase inhibitor activity in GH3 cell extracts showed no significant difference between forskolin-treated cells and cells maintained under control conditions. To assess possible effects on catalytic subunit degradation, pulse-chase experiments were performed and radiolabeled catalytic subunit was isolated by affinity chromatography. The results demonstrated that treatment of cells with chlorophenylthio-cAMP detectably increased the apparent degradation of radiolabeled catalytic subunit. The increased degradation of the catalytic subunit was sufficient to account for the observed decreases in kinase activity. These results suggest that relatively long term cAMP treatment can alter total cAMP-dependent protein kinase activity through effects to alter the degradation of the catalytic subunit of the enzyme.",
author = "Richardson, {Jeanne M.} and Paul Howard and Massa, {John S.} and Richard Maurer",
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TY - JOUR

T1 - Post-transcriptional regulation of cAMP-dependent protein kinase activity by cAMP in GH3 pituitary tumor cells

T2 - Evidence for increased degradation of catalytic subunit in the presence of cAMP

AU - Richardson, Jeanne M.

AU - Howard, Paul

AU - Massa, John S.

AU - Maurer, Richard

PY - 1990/8/15

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N2 - The effects of cyclic AMP treatment on total cAMP-dependent protein kinase activity in GH3 pituitary tumor cells have been studied. Incubation of cells for 24 h with 1 μM forskolin resulted in a 50% decrease in total cAMP-dependent protein kinase activity which was reversible upon removal of forskolin from culture media. A similar response was observed in GH3 cells treated with 5 ng/ml cholera toxin and 0.5 mM dibutyryl cAMP but not 0.5 mM dibutyryl cGMP. Northern blot analysis demonstrated that the steady-state level of the mRNA for each of the six kinase subunit isoforms studied was not detectably altered after treatment with 1 μM forskolin for 24 h. The concentration of catalytic subunit was also assessed by binding studies using a radiolabeled heat-stable protein kinase inhibitor. Treatment of GH3 cells with 1 μM forskolin for 24 h reduced protein kinase inhibitor binding activity by 50%, consistent with the observed forskolin-induced decrease in total kinase activity. Analysis of endogenous heat-stable protein kinase inhibitor activity in GH3 cell extracts showed no significant difference between forskolin-treated cells and cells maintained under control conditions. To assess possible effects on catalytic subunit degradation, pulse-chase experiments were performed and radiolabeled catalytic subunit was isolated by affinity chromatography. The results demonstrated that treatment of cells with chlorophenylthio-cAMP detectably increased the apparent degradation of radiolabeled catalytic subunit. The increased degradation of the catalytic subunit was sufficient to account for the observed decreases in kinase activity. These results suggest that relatively long term cAMP treatment can alter total cAMP-dependent protein kinase activity through effects to alter the degradation of the catalytic subunit of the enzyme.

AB - The effects of cyclic AMP treatment on total cAMP-dependent protein kinase activity in GH3 pituitary tumor cells have been studied. Incubation of cells for 24 h with 1 μM forskolin resulted in a 50% decrease in total cAMP-dependent protein kinase activity which was reversible upon removal of forskolin from culture media. A similar response was observed in GH3 cells treated with 5 ng/ml cholera toxin and 0.5 mM dibutyryl cAMP but not 0.5 mM dibutyryl cGMP. Northern blot analysis demonstrated that the steady-state level of the mRNA for each of the six kinase subunit isoforms studied was not detectably altered after treatment with 1 μM forskolin for 24 h. The concentration of catalytic subunit was also assessed by binding studies using a radiolabeled heat-stable protein kinase inhibitor. Treatment of GH3 cells with 1 μM forskolin for 24 h reduced protein kinase inhibitor binding activity by 50%, consistent with the observed forskolin-induced decrease in total kinase activity. Analysis of endogenous heat-stable protein kinase inhibitor activity in GH3 cell extracts showed no significant difference between forskolin-treated cells and cells maintained under control conditions. To assess possible effects on catalytic subunit degradation, pulse-chase experiments were performed and radiolabeled catalytic subunit was isolated by affinity chromatography. The results demonstrated that treatment of cells with chlorophenylthio-cAMP detectably increased the apparent degradation of radiolabeled catalytic subunit. The increased degradation of the catalytic subunit was sufficient to account for the observed decreases in kinase activity. These results suggest that relatively long term cAMP treatment can alter total cAMP-dependent protein kinase activity through effects to alter the degradation of the catalytic subunit of the enzyme.

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