Document Type

PhD diss.

Date of Degree

2008

Degree Name

PhD (Doctor of Philosophy)

Department

Pharmacology

First Advisor

Stefan Strack

Abstract

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 catecholamine-secreting 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 Glu153 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 pSer40-TH phosphatase activity in vitro. By site directed mutagenesis, I demonstrated that Arg37 and Arg38 within the PKA-Ser40 {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 ERK phosphorylation in response to NGF treatment as compared to increase observed in wild type B'β expressing cells. Taken together, these results show that B'β recruits PP2A to modulate TH activity in a tissue- and cell compartment specific fashion and that the Glu153 residue of B'β is crucial for dephosphorylation of TH.

Pages

ix, 88

Bibliography

77-88

Copyright

Copyright 2008 Amit Saraf

Included in

Pharmacology Commons

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