Differential Expression of the B′β Regulatory Subunit of Protein Phosphatase 2A Modulates Tyrosine Hydroxylase Phosphorylation and Catecholamine Synthesis

Amit Saraf,David M Virshup,Stefan Strack

doi:10.1074/jbc.m607407200

Abstract

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation by several kinases and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A is a family of heterotrimeric holoenzymes containing one of more than a dozen different regulatory subunits. In comparison with rat forebrain extracts, adrenal gland extracts exhibited TH hyperphosphorylation at Ser(19), Ser(31), and Ser(40), as well as reduced phosphatase activity selectively toward phosphorylated TH. Because the B'beta regulatory subunit of PP2A is expressed in brain but not in adrenal glands, we tested the hypothesis that PP2A/B'beta is a specific TH phosphatase. In catecholamine-secreting PC12 cells, inducible expression of B'beta decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B'beta had the opposite effect. Furthermore, PP2A/B'beta directly dephosphorylated TH in vitro. As to specificity, other PP2A regulatory subunits had negligible effects on TH activity and phosphorylation in situ and in vitro. Whereas B'beta was highly expressed in dopaminergic cell bodies in the substantia nigra, the PP2A regulatory subunit was excluded from TH-positive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. Consistent with a model in which B'beta enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals, TH phosphorylation was higher in processes than in somata of dopaminergic neurons. In summary, we show that B'beta recruits PP2A to modulate TH activity in a tissue- and cell compartment specific fashion.

Highlights

Tyrosine hydroxylase (TH)2 catalyzes the rate-limiting step in the biosynthesis of catecholamines from the amino acid precursor L-tyro
phosphatase 2A (PP2A) regulatory subunit could be responsible for the comparative hypophosphorylation of TH in this tissue, we explored the expression pattern of different PP2A subunits by immunoblotting tissue extract after affinity purification with microcystin-agarose
TH activity is subject to multiple levels of regulation, involving both long term and short term mechanisms

Summary

Introduction

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamines (dopamine, norepinephrine, and epinephrine) from the amino acid precursor L-tyro-. Several kinases regulate TH activity by phosphorylating key serines in the regulatory domain (Ser, Ser, Ser, and Ser). Best characterized is Ser phosphorylation by cyclic AMP-dependent protein kinase (PKA), which markedly enhances TH catalytic activity both in vitro and in vivo by relieving feedback inhibition by the catecholamines (4 –9). The kinases that regulate catecholamine synthesis have been studied extensively, little is know about inactivation of TH by protein Ser/Thr phosphatases. Previous studies used the phosphatase inhibitor okadaic acid to implicate PP2A as a major negative regulator of TH [12,13,14]. As one of four major groups of serine/threonine phosphatases, PP2A exists predominantly as a heterotrimer of a 36-kDa catalytic or C subunit, a 65-kDa scaffolding or A subunit, and a variable regulatory subunit [17, 18]. We provide evidence that tissue- and cell compartment-specific expression of BЈ␤ modulates TH phosphorylation locally, thereby defining sites of elevated catecholamine synthesis

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