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

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/B55 alpha, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55 alpha recruits p107, a pRB-related tumor suppressor and B55 alpha 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 'HxRVxxV(619-625)' short linear motif (SLiM) in p107 as necessary for B55 alpha binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55 alpha/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/B55 alpha, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55 alpha groove, and phosphosite presentation. Altogether, these data provide key insights into PP2A/B55 alpha's mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55 alpha's role in multiple cellular processes.

Automated summary

The study elucidates the mechanism by which PP2A/B55 alpha recognizes substrates and dephosphorylates them, identifying a key binding site in p107 and validating the necessity of related SLiM motif sequences for substrate binding.