title: Cyclic AMP-dependent protein kinase regulates basal and cyclic AMP-stimulated but not phorbol ester-stimulated transcription of the tyrosine hydroxylase gene.
abstract: To define the precise role of cyclic AMP (cAMP)-dependent protein kinase (PKA) in transcriptional regulation of the tyrosine hydroxylase (TH) gene, we performed transient cotransfection analyses of a reporter construct containing the upstream 2,400 bp sequence of the rat TH gene with expression plasmids encoding a heat-stable specific inhibitor of PKA (PKI), a mutant regulatory subunit of PKA, or the catalytic subunit of PKA. Inhibition of PKA activity by expression of either PKI or mutant regulatory subunit blocked cAMP-stimulated induction and reduced basal transcription of the TH-reporter construct. Expression of the catalytic subunit of PKA induced the expression of the TH-reporter construct up to 50-fold in a dose-dependent manner. Primer extension analysis confirmed that PKA-mediated induction of TH-reporter expression occurred at the correct transcription initiation site. Expression of PKI did not affect induction following phorbol ester treatment, suggesting that PKA and protein kinase C (PKC) induce TH transcription by independent mechanisms. Finally, a double mutation within the cAMP response element (CRE) of TH2400-CAT diminished its basal and forskolin-stimulated transcription to the level of the promoterless plasmid, pBLCAT3, but did not alter the induction following treatment with phorbol ester, indicating that the CRE is not required for PKC-mediated transcriptional induction. Our results indicate that PKA, via the CRE, plays a crucial role for basal and cAMP-inducible transcription of the TH gene.
authors.affiliation: Laboratory of Molecular Neurobiology, W. M. Burke Medical Research Institute, Cornell University Medical College, White Plains, New York 10605.
authors.forename: K S
authors.initials: KS
authors.lastname: Kim
authors.forename: C
authors.initials: C
authors.lastname: Tinti
authors.forename: B
authors.initials: B
authors.lastname: Song
authors.forename: J F
authors.initials: JF
authors.lastname: Cubells
authors.forename: T H
authors.initials: TH
authors.lastname: Joh
mesh_terms: D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D011493:Protein Kinase C / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*
chemical_list: D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; C015482:protein kinase modulator; D005576:Colforsin; D004247:DNA; D000242:Cyclic AMP; D014446:Tyrosine 3-Monooxygenase; D015500:Chloramphenicol O-Acetyltransferase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate