Beyond the dopamine receptor: regulation and roles of serine/threonine protein phosphatases

Abstract

Dopamine plays an important modulatory role in the central nervous system, helping to control critical aspects of motor function and reward learning. Alteration in normal dopaminergic neurotransmission underlies multiple neurological diseases including schizophrenia, Huntington's disease, and Parkinson's disease. Modulation of dopamine-regulated signaling pathways is also important in the addictive actions of most drugs of abuse. Our studies over the last 30 years have focused on the molecular actions of dopamine acting on medium spiny neurons, the predominant neurons of the neostriatum. Striatum-enriched phosphoproteins, particularly dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein of 32 kDa (DARPP-32), regulator of calmodulin signaling (RCS), and ARPP-16, mediate pleiotropic actions of dopamine. Notably, each of these proteins, either directly or indirectly, regulates the activity of one of the three major subclasses of serine/threonine protein phosphatases, PP1, PP2B, and PP2A, respectively. For example, phosphorylation of DARPP-32 at Thr34 by protein kinase A results in potent inhibition of PP1, leading to potentiation of dopaminergic signaling at multiple steps from the dopamine receptor to the nucleus. The discovery of DARPP-32 and its emergence as a critical molecular integrator of striatal signaling will be discussed, as will more recent studies that highlight novel roles for RCS and ARPP-16 in dopamine-regulated striatal signaling pathways.

Keywords: ARPP-16; ARPP-21; DARPP-32; RCS; calcineurin; phosphorylation; protein kinase A; protein phosphatase.

Figure 1

Localization of DARPP-32, RCS, and ARPP-16 in rat brain. DARPP-32 (left, sagittal section, positive immunoreactivity black), RCS (middle, coronal section, positive immunoreactivity white; caudate/putamen (CP), and nucleus accumbens (A); inset at right shows RCS enrichment in nucleus accumbens (left of dashed line) in more rostral section), ARPP-16 (right, sagittal section, immunoreactivity white). Simple domain diagrams of each protein with their amino acid number and site of PKA phosphorylation are shown below the respective immunolocalization panels.

Figure 2

Domain organization of DARPP-32. DARPP-32 is phosphorylated at Thr34 by PKA (and PKG, not shown), at Thr75 by Cdk5, at Ser97 by CK2, and at Ser130 by CK1. Thr34 is preferentially dephosphorylated by PP2B (calcineurin); Thr75 is preferentially dephosphorylated by PP2A; Ser97 is also preferentially dephosphorylated by PP2A (not shown); Ser130 is dephosphorylated by PP2C. Phosphorylation of Thr34 converts DARPP-32 into a potent inhibitor of PP1. A PP1 docking motif and phosphorylation of Thr34 are required for binding and inhibition of PP1. Phosphorylation of Thr75 converts DARPP-32 into an inhibitor of PKA, reducing its ability to phosphorylate DARPP-32 and other substrates. Phosphorylation of Ser130 increases phosphorylation of Thr34 through inhibition of PP2B and potentiates dopaminergic signaling via the cAMP/PKA/DARPP-32/PP-1 pathway. In contrast, phosphorylation of Thr75 acts to inhibit dopaminergic signaling via this pathway. Phosphorylation of Ser97 in conjunction with a nuclear export signal (NES) act to export DARPP-32 from the nucleus and maintain the cytoplasmic localization of the protein observed under basal conditions.

Figure 3

Interactive roles of DARPP-32, RCS, and ARPP-16 in regulation of signal transduction in striatal MSNs. The efficacy of phosphorylation by PKA of numerous PKA/PP1 substrates is increased by PKA phosphorylation of DARPP-32, which inhibits PP1. In an analogous manner, the efficacy of phosphorylation by PKA of numerous PKA/PP2B substrates is increased by PKA phosphorylation of RCS, which inhibits PP2B. In contrast, ARPP-16 appears to be basally phosphorylated by MAST3 kinase, leading to inhibition of the action of PP2A on selective substrates including Thr75 of DARPP-32 (a site that acts basally to attenuate PKA’s ability to phosphorylate Thr34 of DARPP-32; not shown, see Figure 2). PKA may modulate the ability of MAST to phosphorylate ARPP-16 or influence the effect of ARPP-16 on PP2A. RCS and ARPP-16 in different ways may act to control DARPP-32’s ability to inhibit PP1.

Figure 4

Domain organization of ARPP-16/19/ENSA family members. ARPP-16 and ARPP-19 are generated by alternative splicing with ARPP-19 containing an additional 16 amino acids at the N-terminus. ENSA is generated from a distinct gene, and contains a 20-amino acid N-terminal region distinct from ARPP-19. Within the conserved domains of the three proteins (blue), ARPP-16 and ARPP-19 are identical and ENSA is highly homologous. MAST kinases (or Gwl in non-mammalian systems) phosphorylate a common serine residue in a conserved central domain, while PKA phosphorylates a conserved site at the C-terminus.