Expanding the PP2A Interactome by Defining a B56-Specific SLiM

Abstract

SUMMARY Specific interactions between proteins govern essential physiological processes including signaling. Many enzymes, especially the family of serine/threonine phosphatases (PSPs: PP1, PP2A and PP2B/calcineurin/CN), recruit substrates and regulatory proteins by binding Short Linear Motifs (SLiMs), short sequences found within intrinsically disordered regions (IDRs) that mediate specific protein:protein interactions. While tremendous progress had been made in identifying where and how SLiMs bind PSPs, especially PP1 and CN, essentially nothing is known about how SLiMs bind PP2A, a validated cancer drug target. Here we describe three structures of PP2A:SLiM interaction (B56: pS-RepoMan, B56:pS-BubR1 and B56:pSpS-BubR1), show that this PP2A-specific SLiM is defined as LSPIxE and then use this data to discover scores of likely PP2A regulators and substrates. Together, these data not only provide a powerful approach for dissecting PP2A interaction networks in cells but also for targeting PP2A diseases, such as cancer.

PP2A‐B56 binds to Apc1 and promotes Cdc20 association with the APC/C ubiquitin ligase in mitosis

“…Crucially, the activity of PP2A-B55d fluctuates during the cell cycle, via the PP2A-B55d/ENSA/Greatwall pathway, and is high in interphase and low in mitosis [18][19][20]. More recent work suggests that PP2A-B56 achieves its functional specificity by binding to a LxxIxE short linear motif (SLiM) in which Glu (E) at position 6 is crucial, but position 1 (Leu) and position 4 (Ile) can be replaced by other hydrophobic residues, and phosphorylation or the enrichment of acidic residues within and downstream from the motif can increase PP2A-B56 binding [25][26][27]. In contrast, PP2A-B56 has been shown to be active during mitosis.…”

The M-phase regulatory phosphatase PP2A-B55δ opposes protein kinase A on Arpp19 to initiate meiotic division

“…These M-phase roles of PP2A-B56 are not directly connected to Cdk1 regulation and do not involve Arpp19. Accordingly, Arpp19 lacks the two known binding motifs for PP2A-B56 but includes bipartite recognition determinants for PP2A-B55 [41][42][43] .…”

PP1 and PP2A use opposite phospho-dependencies to control distinct processes at the kinetochore

“…In particular, Aurora B phosphorylates the Knl1-SLiMs to inhibit PP1 (Liu et al, 2010), but when microtubule attachments were rescued by the recruitment of PP2A-B56 to Knl1, we directly tethered B56 and lost these regulatory inputs ( fig.3a). In addition, Cdk1 and Plk1 phosphorylate the BubR1-SLiM to recruit PP2A-B56 (Elowe et al, 2007;Huang et al, 2008;Kruse et al, 2013;Suijkerbuijk et al, 2012;Wang et al, 2016a;Wang et al, 2016b), but when the SAC was rescued by recruiting PP1 to BubR1 we removed this phospho-dependence ( fig.3e). Therefore, we rationalised that it may be the unique forms of SLiM regulation that prevents PP1-Knl1 from stabilising microtubule attachments when PP2A-B56 is removed, and restricts PP2A-B56 from silencing the SAC when PP1-Knl1 is absent.…”

“…almost 700 unique proteins (supp.table.1). Motif analysis demonstrates that serines and threonines are statistically enriched at positions within each motif where phosphorylation is known to inhibit (RVxF) or enhance (LxxIxE) phosphatase interaction ( fig.6a,b) (Hertz et al, 2016;Kim et al, 2003;Kumar et al, 2016;Nasa et al, 2018;Wang et al, 2016a;Wang et al, 2016b). Furthermore, up to 25% of the validated motifs are known to be phosphorylated in vivo, and 50% of the RVxF and 100% of the LxxIxE motifs contain phosphorylatable residues at the key positions ( fig.6c-e); which is a statistically significant enrichment (see amino acid matrices in supp.table.1).…”

“…In these cases, the regulation can occur directly on the SLiMs within these pathways that are needed to direct the phosphatases towards specific substrates. Interestingly, in this respect, PP1 and PP2A-B56 behave in opposite ways: PP1 binding to the RVxF motif can be repressed by phosphorylation (Kim et al, 2003;Nasa et al, 2018), whereas PP2A-B56 interaction with the LxxIxE motif can be enhanced by phosphorylation (Hertz et al, 2016;Wang et al, 2016a;Wang et al, 2016b). These unique modes of phospho-regulation could allow PP1 and PP2A-B56 to perform very different signalling roles, however, it is difficult to dissociate whether it is these properties or others, such as catalytic preferences, that are more important in any given situation.…”