CYCLIC-AMP-DEPENDENT PROTEIN-KINASE REGULATES BASAL AND CYCLIC AMP-STIMULATED BUT NOT PHORBOL ESTER-STIMULATED TRANSCRIPTION OF THE TYROSINE-HYDROXYLASE GENE

To define the precise role of cyclic AMP (cAMP)-dependent protein kina se (PKA) in transcriptional regulation of the tyrosine hydroxylase (TH ) gene, we performed transient cotransfection analyses of a reporter c onstruct 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 subuni t of PKA. Inhibition of PKA activity by expression of either PKI or mu tant regulatory subunit blocked cAMP-stimulated induction and reduced basal transcription of the TH-reporter construct. Expression of the ca talytic subunit of PKA induced the expression of the TH-reporter const ruct up to 50-fold in a dose-dependent manner. Primer extension analys is confirmed that PKA-mediated induction of TH-reporter expression occ urred 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 indepe ndent mechanisms. Finally, a double mutation within the cAMP response element (CRE) of TH2400-CAT diminished its basal and forskolin-stimula ted transcription to the level of the promoterless plasmid, pBLCAT3, b ut did not alter the induction following treatment with phorbol ester, indicating that the CRE is not required for PKC-mediated transcriptio nal induction. Our results indicate that PKA, via the CRE, plays a cru cial role for basal and cAMP-inducible transcription of the TH gene.