Docking Motif-Guided Mapping of the Interactome of Protein Phosphatase-1

Abstract

The ubiquitous protein Ser/Thr phosphatase-1 (PP1) interacts with dozens of regulatory proteins that are structurally unrelated. However, most of them share a short, degenerate "RVxF"-type docking motif. Using a broad in silico screening based on a stringent definition of the RVxF motif, in combination with a multistep biochemical validation procedure, we have identified 78 novel mammalian PP1 interactors. A global analysis of the validated RVxF-based PP1 interactome not only provided insights into the conserved features of the RVxF motif but also led to the discovery of additional common PP1 binding elements, described as the "SILK" and "MyPhoNE" motifs. In addition to the doubling of the known mammalian PP1 interactome, our data contribute to the design of PP1 interaction networks. Notably, an interaction network linking PP1 interactors discloses a pleiotropic role of PP1 in cell polarity.

MiR-125b promotes cell migration and invasion by targeting PPP1CA-Rb signal pathways in gastric cancer, resulting in a poor prognosis

“…Protein serine/threonine (Ser/Thr) phosphatase-1 (PP1) is one of the most highly conserved enzymes known, and it plays a central role in a range of cellular processes, including protein synthesis, RNA splicing, cell-cycle progression, and glycogen metabolism [23,24]. A large array of regulatory subunits are associated with the PP1 catalytic subunit to determine its cellular localization and substrate specificity, mediating the control of these many physiological processes through PP1 holoenzymes [24][25][26]. There are 4 different isoforms of the PP1 catalytic subunit (a, b, c1, and c2) and each can bind to many non-substrate cellular proteins that function as regulatory subunits.…”

PPP1CC2 can form a kinase/phosphatase complex with the testis-specific proteins TSSK1 and TSKS in the mouse testis

“…In vitro experiments have previously demonstrated direct interactions between TSKS and PP1 fragments, as well as between TSKS and TSSK1 (Kueng et al 1997, Hendrickx et al 2009). While sedimentation assays indicated a reciprocal interaction between PPP1CC2 and TSSK1 in testis lysates, we sought to determine whether there was also a direct interaction between PPP1CC2 and TSSK1 in vitro.…”

“…In a previously published study, the TSSK1 substrate TSKS was bioinformatically predicted to be a PP1-interacting protein, and subsequent in vitro experiments showed that a TSKS fragment was capable of interacting with PPP1CA (Hendrickx et al 2009), the only PP1 isoform assayed. This prediction was based on the presence of a PP1 Table 1 Testis proteins identified in a SDS-PAGE gel band after sedimentation by GST-PPP1CC1 and GST-PPP1CC2.…”

“…These include both proteins involved in isoform-specific interactions such as SPZ1 and Endophilin B1t (SH3GLB1, testisspecific isoform) (Hrabchak & Varmuza 2004, Hrabchak et al 2007, and, more numerously, proteins that have the ability to interact with multiple PP1 isoforms, such as PPP1R11 (Cheng et al 2009). In addition, another testisspecific protein, TSSK substrate (TSKS), was bioinformatically predicted to interact with PP1 based on the presence of a high-affinity PP1 docking motif, and in vitro experiments have confirmed that a TSKS fragment is capable of interacting with PPP1CA (Hendrickx et al 2009). The ability of full-length TSKS to bind to other PP1 isoforms such as PPP1CC2 has not been tested.…”

Conserved Residues in the N Terminus of Lipin-1 Are Required for Binding to Protein Phosphatase-1c, Nuclear Translocation, and Phosphatidate Phosphatase Activity

“…PP-1c is a key regulator of protein dephosphorylation (23,24). Because PP-1c exhibits little substrate specificity, appropriate targeting of protein dephosphorylation is mediated by interaction with different regulatory subunits, which interact with PP-1c via a conserved RVXF motif as well as by secondary interactions (23-25, 32).…”