Abstract
Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine protein phosphatase 2 (PP2A) holoenzyme, PP2A/B55α, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55α recruits p107, a pRB-related tumor suppressor and B55α substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an 'HxRVxxV619-625' short linear motif (SLiM) in p107 as necessary for B55α binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55α/PP2A substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, 'p[ST]-P-x(4,10)-[RK]-V-x-x-[VI]-R.' Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55α, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55α groove, and phosphosite presentation. Altogether, these data provide key insights into PP2A/B55α's mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55α's role in multiple cellular processes.
Highlights
Protein phosphorylation is a reversible post-translational modification that is critical for the regulation of signaling and other cellular processes
Members of the phosphoprotein phosphatase (PPP) family of serine/threonine phosphatases are responsible for the majority of dephosphorylation in eukaryotic cells, with protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) accounting for more than 90% of the total phosphatase activity
Within the PPP family, the molecular details of substrate recognition have been best studied for PP1 and calcineurin, and involve the recognition of defined short linear motifs or SLiMs including RVxF, φφ, SILK, among others for PP1 (Choy et al, 2014; Kumar et al, 2018); and LxVP and PxIxIT for phosphatase 2B (PP2B)
Summary
Protein phosphorylation is a reversible post-translational modification that is critical for the regulation of signaling and other cellular processes. Within the PPP family, the molecular details of substrate recognition have been best studied for PP1 and calcineurin (or PP2B), and involve the recognition of defined short linear motifs or SLiMs including RVxF, φφ (where φ refers to a hydrophobic residue), SILK, among others for PP1 (Choy et al, 2014; Kumar et al, 2018); and LxVP and PxIxIT for PP2B. These motifs are characterized by the presence of three or four core interacting amino acids that are part of a 4-10 amino acid stretch within intrinsically disordered regions of regulator and/or substrate proteins. Since modulation of PP2A activity is actively being explored in many cancer types, our data will further help in turning PP2A in a key cancer drug target
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