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Recombinant fragment of protein kinase inhibitor blocks cyclic AMP-dependent gene transcription
J R Grove1, D J Price, H M Goodman
1Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02114.
Abstract:
Transcriptional regulation by cyclic adenosine monophosphate (cAMP) in mammalian cells could be mediated by a phosphoprotein substrate of the cAMP-dependent protein kinase or, as in prokaryotes, by a cAMP-binding protein. Two synthetic genes that code for an active fragment of the protein inhibitor of this kinase and a mutant inactive fragment were constructed and used to distinguish these alternatives. Transient expression of the active peptide product specifically inhibited the cAMP-stimulated expression of a cotransfected reporter gene by more than 90 percent, whereas the expression of the inactive peptide did not alter cAMP-stimulated gene expression. The results indicate that an active kinase catalytic subunit is a necessary intermediate in the cAMP stimulation of gene transcription.
Insights
Cyclic adenosine monophosphate (cAMP) regulates gene transcription in mammals via a protein kinase pathway. Active kinase intermediates are essential for cAMP-stimulated gene expression, as demonstrated by inhibiting reporter gene activity.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- Cyclic adenosine monophosphate (cAMP) is a crucial second messenger involved in cellular signaling.
- Mechanisms of cAMP-mediated transcriptional regulation in mammalian cells were unclear, with possibilities including protein kinase substrates or direct cAMP-binding proteins.
Purpose of the Study:
- To elucidate the precise mechanism of cAMP-mediated transcriptional regulation in mammalian cells.
- To differentiate between a protein kinase-dependent pathway and a direct cAMP-binding protein pathway.
Main Methods:
- Construction of synthetic genes encoding an active and an inactive fragment of a cAMP-dependent protein kinase inhibitor.
- Transient expression of these synthetic gene products in mammalian cells.
- Co-transfection with a reporter gene to quantify cAMP-stimulated gene expression.
Main Results:
- The active peptide inhibitor significantly suppressed cAMP-stimulated reporter gene expression by over 90%.
- The inactive peptide inhibitor had no effect on cAMP-stimulated gene expression.
- This strongly supports a role for the cAMP-dependent protein kinase pathway.
Conclusions:
- The findings indicate that an active catalytic subunit of the cAMP-dependent protein kinase is a necessary intermediate in cAMP-stimulated gene transcription.
- This study clarifies a key aspect of intracellular signal transduction and gene regulation by cAMP.