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.

Serine/threonine phosphatases such as PP1 lack substrate specificity and associate with a large array of targeting subunits to achieve the requisite selectivity. The tumour suppressor ASPP (apoptosis-stimulating protein of p53) proteins associate with PP1 catalytic subunits and are implicated in multiple functions from transcriptional regulation to cell junction remodelling. Here we show that Drosophila ASPP is part of a multiprotein PP1 complex and that PP1 association is necessary for several in vivo functions of Drosophila ASPP. We solve the crystal structure of the human ASPP2/PP1 complex and show that ASPP2 recruits PP1 using both its canonical RVxF motif, which binds the PP1 catalytic domain, and its SH3 domain, which engages the PP1 C-terminal tail. The ASPP2 SH3 domain can discriminate between PP1 isoforms using an acidic specificity pocket in the n-Src domain, providing an exquisite mechanism where multiple motifs are used combinatorially to tune binding affinity to PP1.

The kinetochore scaffold 1 (KNL1) protein coordinates the spindle assembly checkpoint (SAC), a signaling pathway that delays chromosome segregation until all sister chromatids are properly attached to spindle microtubules. Recently, microtubules and protein phosphatase 1 (PP1), which both bind the N-terminal domain of KNL1, have emerged as regulators of the SAC; however, how these proteins interact to contribute to SAC signaling is unknown. Here, we use X-ray crystallography, nuclear magnetic resonance spectroscopy, and biochemical assays to show how KNL1 binds both PP1 and microtubules. Unexpectedly, we discovered that PP1 and microtubules bind KNL1 via overlapping binding sites. Further, we showed that Aurora B kinase phosphorylation results in distinct patterns of KNL1 complex disruption. Finally, combining this data with co-sedimentation assays unequivocally demonstrated that microtubules and PP1 binding to KNL1 is mutually exclusive, with preferential formation of the KNL1:PP1 holoenzyme in the presence of PP1.

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