Regulation of tyrosine hydroxylase by protein phosphatase 2A

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation on its regulatory domain and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A comprising of an AC core dimer composed of catalytic (C) and scaffolding (A) subunit is complexed to a variable regulatory subunit derived from three gene families (B, B’, B”). My thesis work was focused on studying the regulation of TH by PP2A. I found out that in catecholaminesecreting PC12 cells, inducible expression of PP2A/B’β decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B’β had the opposite effect. Furthermore, PP2A/ B’β directly dephosphorylated TH in vitro. B’β was highly expressed in dopaminergic cell bodies in the rat substantia nigra, however it was excluded from TH-positive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. I detected higher TH phosphorylation in processes than in somata of dopaminergic neurons. This is consistent with a model in which B’β enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals. I further investigated the mechanism of substrate specificity by B’β holoenzyme. Using the PP2A/ B’γ crystal structure as a guide, I identified Glu of B’β as a critical residue for dephosphorylation of TH in PC12 cells. PC12 cell lines expressing B’β/ E153R mutant were unable to dephosphorylate TH and this mutant demonstrated reduced pSer-TH phosphatase activity in vitro. By site directed mutagenesis, I demonstrated that Arg and Arg within the PKA-Ser {part of a PKA consensus sequence (-RRXS-)} sequence of TH was critical for dephosphorylation by B’β. I also showed that PC12 cell line expressing B’β/E153R mutant exhibited reduced