Molecular Determinants for PP2A Substrate Specificity: Charged Residues Mediate Dephosphorylation of Tyrosine Hydroxylase by the PP2A/B′ Regulatory Subunit

摘要

Together with protein phosphatase 1, protein phosphatase 2A (PP2A) contributes the bulk ofnSer/Thr phosphatase activity in most cell types. The predominant form of PP2A is a heterotrimer of catalyticn(C), scaffolding (A), and diverse regulatory subunits (B, B0n, and B00n). We have previously shown thatnN-terminal phosphorylation sites in tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholaminensynthesis, are specifically dephosphorylated by PP2A holoenzymes containing the B0nβ regulatory subunit.nHere, we identify a Glu residue conserved in B0nregulatory subunits that is critical for dephosphorylation andninactivation of tyrosine hydroxylase in vitro and in PC12 cells. According to the PP2A heterotrimer crystalnstructure, Glu153 (B0nβ numbering) abuts the catalytic site on the C subunit, and we demonstrate that Glu153nsubstitution inhibitsmultisite THdephosphorylationwithout compromising PP2A/B0nβ holoenzyme assemblynor in vitro dephosphorylation of model substrates. Apart from its role in modulating TH activity, Glu153 isnalso necessary for PP2A/B0nβ-mediated enhancement of nerve growth factor signaling. Furthermore, globalnphosphoproteome analysis suggests that Glu153 mediates dephosphorylation of most B0nβ substrates in PC12ncells.With regard to selectivity determinants in the substrate, we show that B0nβ Glu153 recognizes Arg37 andnArg38 in TH to direct dephosphorylation of both upstream (Ser31) and downstream (Ser40) sites. Thesenresults provide evidence of a subunit-spanning substrate docking site on the PP2A/B0nholoenzyme, in whichnnegatively charged side chains in the regulatory subunit interact with positive charges proximal tonphosphorylated residues to mediate site-specific dephosphorylation.