Molecular determinants for PP2A substrate specificity: charged residues mediate dephosphorylation of tyrosine hydroxylase by the PP2A/B' regulatory subunit

Abstract

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'beta 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'beta numbering) abuts the catalytic site on the C subunit, and we demonstrate that Glu153 substitution inhibits multisite TH dephosphorylation without compromising PP2A/B'beta holoenzyme assembly or in vitro dephosphorylation of model substrates. Apart from its role in modulating TH activity, Glu153 is also necessary for PP2A/B'beta-mediated enhancement of nerve growth factor signaling. Furthermore, global phosphoproteome analysis suggests that Glu153 mediates dephosphorylation of most B'beta substrates in PC12 cells. With regard to selectivity determinants in the substrate, we show that B'beta 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.

Figure 1

The divergent N-terminus of B′β is dispensable for TH dephosphorylation. (A) Domain diagram of B′β truncation mutants and summary of PP2A heterotrimer incorporation experiments. Light gray areas are not required for A and C subunit association. (B) The indicated FLAG-tagged B′β constructs (WT, wild-type) were transiently expressed in COS cells and assessed for holoenzyme incorporation by co-immunoprecipitation (IP) of FLAG-B′β with endogenous A (arrow) and C subunits. (C and D) Total lysates of PC12 cells transiently transfected with the indicated plasmids were analyzed for TH phosphorylation at Ser40 (pTH S40), total TH, and B′β expression. Shown are a representative blot (C) and a quantification of phospho-Ser40 TH over total TH relative to empty vector-transfected cells (D, mean±SE from three experiments). By a Student's t test compared to vector control, one asterisk indicates p < 0.05 and two asterisks indicate p < 0.01.

FIGURE 2

Glu152 and Glu153 mutations do not affect incorporation of B′β into the PP2A heterotrimer. (A) Structure of the PP2A/B′ heterotrimer (A and C subunits in space filling and B′ in ribbon representation) highlighting Glu153 (B′β numbering) at the tip of a loop that connects to the active site (inset). The carboxyl group of Glu153 contacts microcystin, while its carbonyl oxygen contacts Arg268 on the C subunit (stippled lines). (B) Phylogenetic alignment of loop residues surrounding Glu152 and Glu153 in B′β (Hs, Homo sapiens;Dm, Drosophila melanogaster; wid, widerborst; Ce, Caenorhabditis elegans;Sc, Saccharomyces cerevisiae; a, A subunit contacts; c, C subunit contacts). (C) The indicated plasmids were transiently expressed in COS cells, and lysates were analyzed for co-immunoprecipitation of FLAG-B′β with the endogenous PP2A catalytic (C) subunit. (D) Stable PC12 cell lines were treated for 2 days with either vehicle (−) or doxycycline (Dox) to induce expression of wild-type or mutant FLAG-B′β. FLAG immunoprecipitates probed for B′β and the C subunits demonstrate equivalent PP2A holoenzyme association of wild-type and mutant B′β.

FIGURE 3

B′β Glu153 is critical for TH dephosphorylation and inactivation in PC12 cells. (A and B) PC12 cell lines were treated with or without doxycycline (Dox) to induce wild-type or mutant B′β expression. Total cell lysates were then analyzed for TH phosphorylation with the indicated antibodies. Shown are representative blots (A) and quantification of phospho (p)TH over TH signals normalized to uninduced (no Dox) cultures (mean ± SE of three to six independent experiments). (C and D) Stable PC12 cell lines were induced with or without Dox and incubated with L-[1-¹⁴C]tyrosine, and ¹⁴CO₂ release was assessed by scintillation counting as a measure of dopamine synthesis. Panel C depicts the assay principle, and panel D shows TH activity relative to uninduced (no Dox) controls (mean ± SE of three to seven experiments). One asterisk indicates p<0.05. Two asterisks indicate p<5 × 10⁻⁴. Three asterisks indicate p<5× 10⁻⁵.

FIGURE 4

B′β Glu153 recognizes selectivity determinants close to the TH Ser40 phosphorylation site. (A) Diagram of glutathione S-transferase (GST) TH fusion proteins used in this figure. (B–F) Wild-type and mutant B′β-containing PP2A holoenzymes were isolated via FLAG immunoprecipitation (IP) from stable PC12 cell lines (B, C, E, and F) or from transiently transfected COS-1 cells (D). Enzyme aliquots containing equivalent C subunit (insets of panels B, D, and E) were assayed toward the indicated ³²P-labeled GST fusion proteins (in vitro phosphorylated by PKA on TH Ser40 and Mfn2 Ser442). Representative phosphatase assays are shown in panels B, D, and E (mean ± standard deviation of triplicate reactions), while data summaries are shown in panels C and F (mean ± SE of three assays). An asterisk indicates p< 0.05.

FIGURE 5

Truncation of Mfn2 to the PKA phosphorylation site renders dephosphorylation dependent on Glu153 of B′β. (A) Diagram of GST–Mfn2 fusion proteins used in this figure and alignment of the PKA phosphorylation sites (arrow) in Mfn2 and TH. (B and C) PKA-phosphorylated Mfn2 fusion proteins were assayed for dephosphorylation by wild-type or mutant PP2A/B′β. A representative assay is shown in panel B (mean ± standard deviation of triplicate experiments) and a summary in panel C (ratio of wild-type B′β and E153R B′β activities; mean ± SE of three to seven assays). An asterisk indicates p< 0.05.

FIGURE 6

Arg37 and Arg38 are required for efficient dephosphorylation of TH by wild-type but not Glu153 mutant PP2A/B′β. (A) Sequence of the wild-type and Arg37 and Arg38 mutant TH–GST fusion protein and peptide, indicating ERK (Ser31) and PKA (Ser40) phosphorylation sites. (B and C) The indicated proteins were phosphorylated in vitro with ERK2 and assayed for dephosphorylation by wild-type and mutant PP2A/B′β: (B) representative assay showing the mean ± standard deviation of triplicate experiments and (C) summary of specific activities normalized to reactions with wild-type proteins (mean ± SE of three to five assays). (D) Phospho-Ser40 containing wild-type and Arg mutant TH peptides were assayed for dephosphorylation by wild-type and mutant PP2A/B′β using a colorimetric assay. Phosphatase activities are shown as means ± SE of three to five independent assays normalized to wild-type TH peptide. An asterisk indicates p< 0.05.

FIGURE 7

Glu153 is required for B′β-mediated stimulation of NGF signaling. (A) Schematic of NGF signal transduction to ERK. PP2A/B′β sustains NGF signaling by enhancing Tyr autophosphorylation of the NGF receptor TrkA (22). (B and C) PC12 cells inducibly expressing wild-type or E153R mutant B′β were cultured with or without Dox and stimulated with 20 ng/mL NGF for the indicated times, and total extracts were probed for phosphorylated (pERK) and total ERK. Blots from a representative experiment are shown in panel B, while panel C quantifies ERK phosphosphorylation from three experiments (mean ± SE of pERK1 divided by total ERK1 normalized to the highest value for vehicle-treated cells). One asterisk indicates p< 0.05; two asterisks indicate p< 0.01.

FIGURE 8

Glu153 is required for dephosphorylation of multiple B′β substrates in PC12 cells. Lysates of stable PC12 cell lines cultured for 36–42 h either in the absence (−) or in the presence (+) of doxycycline (Dox) to induce expression of wild-type (A and B) or E153R (B) PP2A/B′β were fractionated into Triton X-100 soluble and insoluble fractions. Soluble proteins were subjected to isoelectric focusing over pH 5.0 to 8.0 gradients, further separated by SDS–PAGE, and then stained with Pro Q Diamond to detect phosphoproteins. In panel B, the same gels were restained for total protein with Coomassie Blue R250. In panel A, gels from uninduced (−Dox) and B′β-expressing (+Dox) cells are shown individually and as a pseudocolored overlay subsequent to digital warping to bring spot patterns into registration. Red and blue circles highlight increases and decreases in spot intensity, respectively, upon Dox addition. Results are representative of four or more experiments. (C) Model of PP2A/B′β substrate interactions (see the text for details).