Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

Abstract

Protein phosphatase 2A (PP2A) is a large enzyme family responsible for most cellular Ser/Thr dephosphorylation events. PP2A substrate specificity, localization, and regulation by second messengers rely on more than a dozen regulatory subunits (including B/R2, B′/R5, and B″/R3), which form the PP2A heterotrimeric holoenzyme by associating with a dimer comprising scaffolding (A) and catalytic (C) subunits. Because of partial redundancy and high endogenous expression of PP2A holoenzymes, traditional approaches of overexpressing, knocking down, or knocking out PP2A regulatory subunits have yielded only limited insights into their biological roles and substrates. To this end, here we sought to reduce the complexity of cellular PP2A holoenzymes. We used tetracycline-inducible expression of pairs of scaffolding and regulatory subunits with complementary charge-reversal substitutions in their interaction interfaces. For each of the three regulatory subunit families, we engineered A/B charge–swap variants that could bind to one another, but not to endogenous A and B subunits. Because endogenous Aα was targeted by a co-induced shRNA, endogenous B subunits were rapidly degraded, resulting in expression of predominantly a single PP2A heterotrimer composed of the A/B charge–swap pair and the endogenous catalytic subunit. Using B′δ/PPP2R5D, we show that PP2A complexity reduction, but not PP2A overexpression, reveals a role of this holoenzyme in suppression of extracellular signal–regulated kinase signaling and protein kinase A substrate dephosphorylation. When combined with global phosphoproteomics, the PP2A/B′δ reduction approach identified consensus dephosphorylation motifs in its substrates and suggested that residues surrounding the phosphorylation site play roles in PP2A substrate specificity.

A disorder-related variant (E420K) of a PP2A-regulatory subunit (PPP2R5D) causes constitutively active AKT-mTOR signaling and uncoordinated cell growth

“…A major hurdle to determining how the E420K mutation alters the biological functions of PPP2R5D is that only a few studies have explored the "normal" action(s) of PPP2R5D at a cellular level (31,32). Therefore, we chose a global unbiased approach, employing TMT labeling for relative and absolute quantitation with LC-MS 3 , to obtain proteomic (Fig.…”

Endosomal cAMP production broadly impacts the cellular phosphoproteome

“…Of these 232 phosphopeptides, 168 peptides increased and 64 decreased in abundance relative to matched untreated controls reflecting increase or decrease in phosphorylation status, respectively (Dataset S1). Comparison with available phosphoproteomic datasets derived from studies of GPCR/cAMP activation revealed extensive overlap (65/218 sites = 30%) [7][8][9][10][11][12][13][14] . Of particular significance, we confirmed 1/3 of GPCR/cAMP target phosphosites previously reported in the same cell type, HEK293 cells 14 .…”

“…In this subset, 20 of these 22 peptides (>90%) had the motif (p < 1.2x10 -4 by Fisher's exact test). Two protein phosphatases, PP1A and PP2A, are known to modify prolinedirected sequences 11,24 . Hence, we searched the mass spectrometry data and found a peptide corresponding to a known regulatory site within the PP2A-B56δ subunit, S573.…”

Endosomal cAMP production broadly impacts the cellular phosphoproteome

“…In this subset, 20 of these 22 peptides (>90%) had the motif ( p < 1.2×10 −4 by Fisher’s exact test). Two protein phosphatases, PP1A and PP2A, are known to modify proline-directed sequences 11,24 . Hence, we searched the mass spectrometry data and found a peptide corresponding to a known regulatory site within the PP2A-B56δ subunit, S573.…”