No abstract

Tyrosine hydroxylase (TH) catalyzes the conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine, the first and rate-limiting step in catecholamine biosynthesis. The cAMP-dependent protein kinase (PKA) phosphorylates and activates the TH enzyme and is thought to mediate transcriptional induction of the TH gene. To better understand the functional role of PKA in TH gene regulation, we studied TH gene expression at the transcriptional, translational, and posttranslational levels in several PKA-deficient cell lines derived from rat PC12 pheochromocytoma cells. Strikingly, all PKAdeficient cell lines analyzed in this study showed substantial deficits in basal TH expression as measured by TH enzymatic activity, level of TH immunoreactivity, TH protein level, and steady-state mRNA level. Interestingly, the steady-state level of mRNA correlated well with levels of TH activity, immunoreactivity, and protein. In addition, PKA-deficient cell lines lacked transcriptional induction of the TH gene following treatment with dibutyryl cAMP. Cotransfection of PKAdeficient cells with an expression plasmid for the catalytic subunit of PKA fully reversed transcriptional defect, as indicated by robust transcriptional induction of a reporter construct containing 2400 bp of TH upstream sequence in all PC12 cells tested. These data indicate that the PKA system regulates both the basal and the cAMP-inducible expression of the TH gene primarily at the transcriptional level in PC12 cells.The nervous system performs important adaptive processes through activation of various postsynaptic receptors and their associated signal transduction pathways. These processes can elicit either acute changes in the function of proteins-for instance, enzyme activities-by covalent modification ofpreexisting molecules or delayed changes in levels of protein by altering gene expression. The regulation of neurotransmitter-biosynthesizing-enzyme genes in particular is thought to play a pivotal role, since the resultant change in the amount of available neurotransmitter molecules can trigger substantial variations in neuronal activity.Tyrosine hydroxylase [TH; L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] catalyzes the conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine (L-dopa), which is the first and rate-limiting step in the biosynthesis of the catecholamine neurotransmitters (1). A variety of trans-synaptic and hormonal stimuli are known to produce either acute increases in TH activity via phosphorylation of the preexisting enzyme proteins (refs. 2 and 3; for review see ref. 4) or delayed increases in TH (5-7). The latter responses correlate with increases in TH mRNA and enzyme molecules and are blocked by inhibitors of RNA and protein synthesis, indicating that transcriptional regulation of the TH gene is directly involved (8,9).

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.