PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107

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 mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55α role in multiple cellular processes.

VPA mediates bidirectional regulation of cell cycle progression through the PPP2R2A-Chk1 signaling axis in response to HU

“…To further investigate which sites in PPP2R2A would be essential for the regulation of Chk1 dephosphorylation and contribute to VPA-bidirectional effects, we used 9 Myc-tagged PPP2R2A variants (Fig. 6A ) which lie in the top of the β-propeller [ 42 , 43 ]. By co-immunoprecipitation, we found PPP2R2A D197K and N181A variants showed the most profound impact on the binding of Chk1 when directly compared to wild-type (WT) PPP2R2A (Fig.…”

Orchestrating serine/threonine phosphorylation and elucidating downstream effects by short linear motifs

“…Compared with the well-defined docking motifs of PP1, the information about the motif-based interactions that contribute to PP2A activity remains scarce. Recently, a [RK]Vxx[VI]R docking motif was described for the B55 regulatory subunit [ 191 ]. This motif has been found closely downstream the site of dephosphorylation in substrates such as the retinoblastoma-like protein 1 (RBL1, also called p107) and the microtubule-associated protein tau.…”

The role of serine/threonine phosphatases in human development: Evidence from congenital disorders

“…Furthermore, dimers of PP2A between the C- and A-subunit have been described, although to a lesser extent than trimers ( Janssens and Goris, 2001 ). The B-subunits define substrate specificity of PP2A, in part by the presence of specific Short Linear Interaction Motifs (SLIMs) that function as substrate docking motifs ( Cundell et al, 2016 ; Hertz et al, 2016 ; Kruse et al, 2020 ; Fowle et al, 2021 ). The PP2A-related phosphatases PP4 and PP6 also form trimers and dimers, alike to PP2A ( Figure 1B ), but the overall number of holoenzyme complexes is significantly smaller ( Ohama, 2019 ; Park and Lee, 2020 ).…”