Together with protein phosphatase 1, protein phosphatase 2A (PP2A) contributes the bulk of Ser/Thr phosphatase activity in most cell types. The predominant form of PP2A is a heterotrimer of catalytic (C), scaffolding (A), and diverse regulatory subunits (B, B′, and B″). We have previously shown that N-terminal phosphorylation sites in tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, are specifically dephosphorylated by PP2A holoenzymes containing the B′β regulatory subunit. Here, we identify a Glu residue conserved in B′ regulatory subunits that is critical for dephosphorylation and inactivation of tyrosine hydroxylase in vitro and in PC12 cells. According to the PP2A heterotrimer crystal structure, Glu153 (B′β numbering) abuts the catalytic site on the C subunit, and we demonstrate that Glu153 substitution inhibits multisite TH dephosphorylation without compromising PP2A/B′β holoenzyme assembly or in vitro dephosphorylation of model substrates. Apart from its role in modulating TH activity, Glu153 is also necessary for PP2A/B′β-mediated enhancement of nerve growth factor signaling. Furthermore, global phosphoproteome analysis suggests that Glu153 mediates dephosphorylation of most B′β substrates in PC12 cells. With regard to selectivity determinants in the substrate, we show that B′β Glu153 recognizes Arg37 and Arg38 in TH to direct dephosphorylation of both upstream (Ser31) and downstream (Ser40) sites. These results provide evidence of a subunit-spanning substrate docking site on the PP2A/B′ holoenzyme, in which negatively charged side chains in the regulatory subunit interact with positive charges proximal to phosphorylated residues to mediate site-specific dephosphorylation.

Background: Proteasomal degradation of PP2A regulatory subunits has been described, but responsible E3 ubiquitin ligases have remained elusive. Results: KLHL15 is an E3 ubiquitin ligase adaptor targeting the BЈ/B56␤ regulatory subunit for proteasomal degradation, promoting formation of alternative PP2A holoenzymes. Conclusion: KLHL15 contributes to brain-specific expression of BЈ␤ and modifies PP2A holoenzyme composition. Significance: E3 ligase-mediated B subunit degradation is a novel mechanism to remodel the PP2A heterotrimer.

Protein phosphatase 2A (PP2A), a ubiquitous and pleiotropic regulator of intracellular signaling, is composed of a core dimer (A scaffolding and C catalytic subunits) bound to a variable (B) regulatory subunit of either the B, B' or B'' families. Further genetic expansion and alternative splicing within each B subunit family affords the enzyme tremendous functional heterogeneity as PP2A contributes dozens of heterotrimers with varying subcellular locations and cellular substrates dictated by the variable B subunit. B'! is a brain-specific PP2A regulatory subunit that mediates dephosphorylation of Ca 2+ /calmodulin-dependent protein kinase II and tyrosine hydroxylase. Unbiased proteomic screens for B'! interactors identified Cullin3 (Cul3), a scaffolding component of E3 ubiquitin ligase complexes, and the previously uncharacterized Kelch-like 15 (KLHL15). KLHL15 is one of more than 40 Kelch-like proteins, many of which have been defined as adaptors for the recruitment of substrates to Cul3-based E3 ubiquitin ligases. KLHL15/Cul3 specifically targets B'! to promote protein turnover via ubiquitination and proteasomal degradation. Comparison of KLHL15 and B'! expression profiles suggest that the E3 ligase adaptor contributes to selective expression of the PP2A/B'! holoenzyme in the brain. Mapping of KLHL15 residues critical for Cul3 binding and protein dimerization indicate two distinct and independent functions of KLHL15's N-terminal BTB domain while similar analysis of the C-terminal kelch domain identifies a B'!-specific binding core. While B' regulatory subunit association with the AC dimer is mediated by a highly conserved inner core of roughly 400 amino acids, the divergent N-terminus of B'! is found to be both necessary and sufficient for KLHL15-mediated degradation, with Tyr52 having an obligatory role, underlying the selective association of KLHL15 with the B'! regulatory subunit only. KLHL15 can interact with not only the monomeric version of B'! but also the more stable, PP2A/B'! heterotrimer. However, proteasomal targeting is reserved for the B subunit only. The loss of B'! promotes an exchange of B subunits and a reciprocal ! "! upregulation of alternative B subunit-containing heterotrimers. That is, excess KLHL15 may not only downregulate B'!-guided PP2A dephosphorylation activity, but moreover, may upregulate PP2A activity dictated by alternative B subunits. Taken together, these data suggest regulatory subunit-specific ubiquitination and proteasomal degradation as a novel mechanism for controlling total cellular PP2A activity.