A protein kinase inhibitor gene reduces both basal and multihormone-stimulated prolactin gene transcription.
The possible role of the catalytic subunit of the cAMP-dependent protein kinase in mediating the regulation of prolactin gene transcription has been investigated through the use of a synthetic gene encoding the heat-stable inhibitor of the cAMP-dependent protein kinase. To assess the effects of protein kinase inhibitor expression on cAMP induction of prolactin gene transcription, a marker gene containing the rat prolactin promoter and adjacent 5'-flanking sequences linked to the bacterial chloramphenicol acetyltransferase gene was cotransfected with a protein kinase inhibitor-expression vector. The results demonstrate that the protein kinase inhibitor-expression vector reduced both basal and cAMP-stimulated expression of the cotransfected prolactin-chloramphenicol acetyltransferase gene. A mutant protein kinase inhibitor-expression vector, coding for an inactive inhibitor protein, did not inhibit basal or cAMP-stimulated prolactin gene transcription. Furthermore, the protein kinase inhibitor-expression vector did not inhibit zinc induction of the metallothionein promoter. Analysis of protein kinase activity in transfected cells demonstrated that the protein kinase inhibitor expression vector reduced cAMP-dependent protein kinase activity but did not reduce protein kinase C activity. Nuclease protection experiments confirmed that the effects of the inhibitor vector involved changes in correctly initiated transcripts produced from the prolactin promoter. Surprisingly, the protein kinase inhibitor-expression vector reduced the effects of several different agents including epidermal growth factor, thyrotropin-releasing hormone, phorbol esters, and estrogen on prolactin gene expression to the same extent as it altered cAMP effects.
Day
R N
RN
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
Walder
J A
JA
Maurer
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000502:physiology; D004791:Enzyme Inhibitors / Q000235:genetics*; D005796:Genes / Q000187:drug effects; D005813:Genes, Synthetic; D006728:Hormones / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D010911:Pituitary Neoplasms; D011388:Prolactin / Q000235:genetics*; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D006728:Hormones; D047908:Intracellular Signaling Peptides and Proteins; C015482:protein kinase modulator; D011388:Prolactin; D000242:Cyclic AMP; D011494:Protein Kinases