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.

Very little is known about how Ser/Thr protein phosphatases specifically recruit and dephosphorylate substrates. Here, we identify how the Na + /H + -exchanger 1 (NHE1), a key regulator of cellular pH homeostasis, is regulated by the Ser/Thr phosphatase calcineurin (CN). NHE1 activity is increased by phosphorylation of NHE1 residue T779, which is specifically dephosphorylated by CN. While it is known that Ser/Thr protein phosphatases prefer p Thr over p Ser, we show that this preference is not key to this exquisite CN selectivity. Rather a combination of molecular mechanisms, including recognition motifs, dynamic charge-charge interactions and a substrate interaction pocket lead to selective dephosphorylation of p T779. Our data identify T779 as a site regulating NHE1-mediated cellular acid extrusion and provides a molecular understanding of NHE1 substrate selection by CN, specifically, and how phosphatases recruit specific substrates, generally.