Mechanisms of site‐specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits

Abstract

PP2A is an essential protein phosphatase that regulates most cellular processes through the formation of holoenzymes containing distinct regulatory B‐subunits. Only a limited number of PP2A‐regulated phosphorylation sites are known. This hampers our understanding of the mechanisms of site‐specific dephosphorylation and of its tumor suppressor functions. Here, we develop phosphoproteomic strategies for global substrate identification of PP2A‐B56 and PP2A‐B55 holoenzymes. Strikingly, we find that B‐subunits directly affect the dephosphorylation site preference of the PP2A catalytic subunit, resulting in unique patterns of kinase opposition. For PP2A‐B56, these patterns are further modulated by affinity and position of B56 binding motifs. Our screens identify phosphorylation sites in the cancer target ADAM17 that are regulated through a conserved B56 binding site. Binding of PP2A‐B56 to ADAM17 protease decreases growth factor signaling and tumor development in mice. This work provides a roadmap for the identification of phosphatase substrates and reveals unexpected mechanisms governing PP2A dephosphorylation site specificity and tumor suppressor function.

Selective dephosphorylation by PP2A-B55 directs the meiosis I - meiosis II transition in oocytes

“…4C). This phosphorylation site preference is consistent with the consensus motif dephosphorylated by PP2A-B55 that we and others recently reported (Cundell et al, 2016;Kruse et al, 2020;McCloy et al, 2015). In contrast, MI-specific phosphorylation sites are depleted for serine and for downstream acidic residues.…”

“…Our analysis reveals that upon decrease in Cdk activity at the MI/MII transition, selective dephosphorylation of TP versus SP sites by PP2A-B55 is essential for meiotic progression. Examination of the TP sites dephosphorylated after MI identified an enrichment for basic amino acids starting in the +2 position, matching the known consensus for PP2A-B55 substrates (Cundell et al, 2016;Kruse et al, 2020). Moreover, we find that PP2A-B55 is re-activated at the MI/MII transition, based on phosphorylation signatures of its inhibitory pathway, including Greatwall and ARPP19, as well as conserved PP2A-B55 substrates.…”

BUB-1 targets PP2A:B56 to regulate chromosome congression during meiosis I in C. elegans oocytes

“…Since PP2A:B56 is concentrated in the midbivalent during meiosis I, we hypothesise that it could be balancing AIR-2 activity during meiosis. In this respect, we found that serine 612 in BUB-1 is phosphorylated and this serine is embedded in a sequence (RRLSI) closely resembling the consensus for PP2A:B56 reported in (Kruse et al, 2020). Furthermore, the sequence also fits into the loosely defined Aurora B consensus RRxS (where  is a Bel Borja et al…”

Kinetochore phosphatases suppress autonomous Polo-like kinase 1 activity to control the mitotic checkpoint

“…S9, A–C ). The relative activity of PP2A-B56 is strongly influenced by its position with respect to its substrates, and the presence of Pro +1 relative to pSer/Thr is a negative determinant for PP2A-B56 activity ( Kruse et al, 2020 ). Therefore, one interpretation is that the KARD must be close enough to enable dephosphorylation of the PBMs, which are otherwise predicted to be “poor” substrates.…”

“…Therefore, one interpretation is that the KARD must be close enough to enable dephosphorylation of the PBMs, which are otherwise predicted to be “poor” substrates. In relation to this, it is important to point out that although PP2A-B56 has low activity against Cdk1 sites ( Kruse et al, 2020 ), this does not mean that this phosphatase does not regulate these sites during mitosis. In fact, quite the opposite; it may simply ensure that the bulk of Cdk1 phosphorylation sites are unaffected by constitutive PP2A-B56 activity, while then allowing a subset of these sites to be dephosphorylated in a regulated manner by SLiM-mediated colocalization.…”