Degeneracy and Function of the Ubiquitous RVXF Motif That Mediates Binding to Protein Phosphatase-1

Abstract

Most interactors of protein phosphatase-1 (PP1) contain a variant of a so-called "RVXF" sequence that binds to a hydrophobic groove of the catalytic subunit. A combination of sequence alignments and site-directed mutagenesis has enabled us to further define the consensus sequence for this degenerate motif as [RK]-X 0 -1 -[VI]-{P}-[FW], where X denotes any residue and {P} any residue except Pro. Naturally occurring RVXF sequences differ in their affinity for PP1, and we show by swapping experiments that this binding affinity is an important determinant of the inhibitory potency of the regulators NIPP1 and inhibitor-1. Also, inhibition by NIPP1-(143-224) was retained when the RVXF motif (plus the preceding Ser) was swapped for either of two unrelated PP1-binding sequences from human inhibitor-2, i.e. KGILK or RKLHY. Conversely, the KGILK motif of inhibitor-2 could be functionally replaced by the RVXF motif of NIPP1. Our data provide additional evidence for the view that the RVXF and KGILK motifs function as anchors for PP1 and thereby promote the interaction of secondary binding sites that determine the activity and substrate specificity of the enzyme.The ubiquitous protein serine/threonine phosphatases of type 1 (PP1) 1 and type 2A (PP2A) interact with dozens of different polypeptides that function as substrates, inhibitors, chaperones, anchoring/scaffolding proteins, or substrate-specifiers and are often multifunctional (1-3). For example, the glycogen-associated G-subunits not only target PP1 to the glycogen particles but also increase the specific activity of PP1 toward the substrate glycogen synthase. Similarly, protein kinase Nek2 is a substrate for associated PP1 and targets the centrosomal protein C-Nap1 for dephosphorylation by PP1. In addition, Nek2 mediates cell cycle-dependent control of centrosomal PP1. The promiscuity of PP1 and PP2A in their interaction with other polypeptides accounts for the presence of these enzymes in a large variety of different holoenzymes. The sharing of catalytic subunits between holoenzymes also explains why higher eukaryotes can manage with 15 times fewer protein serine/threonine phosphatases than protein serine/threonine kinases (4).Mammalian genomes contain three genes that encode isoforms of PP1 (1, 3). These isoforms (35-38 kDa) are about 90% identical, and the differences are mainly concentrated in the extremities. Although some PP1 interactors such as the neurabins (5, 6) interact with PP1 in an isoform-specific manner, most interactors do not discriminate between PP1 isoforms, implying that the major interactor binding sites reside in the catalytic core, i.e. the central three-quarters of the protein. The surface of the catalytic core is too small to harbor specific binding sites for each of the 65 known mammalian interactors. The available evidence rather suggests that PP1 interactors compete for a limited number of common or overlapping binding sites (discussed in Ref.

Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex

“…PP1 proteins have indiscriminate phosphatase activity in vitro, and in vivo are directed by PP1 targeting proteins to physiologically relevant substrates or cellular locations. PP1 targeting proteins are diverse, generally showing no sequence similarity beyond the presence of at least one PP1 interaction (e.g., RVXF or SILK) motif (Wakula et al 2003). These PP1 interaction motifs are also found in other PP1-interacting proteins, including PP1 substrates and inhibitory regulators (Bollen et al 2010).…”

Molecular Characterization of Ypi1, a Novel Saccharomyces cerevisiae Type 1 Protein Phosphatase Inhibitor

“…To date, more than 45 bona fide or putative PP1c-regulating subunits have been defined in higher eukaryotes (15)(16)(17). These subunits are structurally quite different, but almost all of them present a consensus binding motif (R/K)(V/ I)X(F/W) necessary for PP1c regulation, which can also account for the mutually exclusive binding of the different subunits to PP1c (15)(16)(17)(18)(19)(20).…”

Interactor-mediated Nuclear Translocation and Retention of Protein Phosphatase-1

“…These PP1-binding motifs can be shared among PP1 interactors, accounting for the ability of PP1 to form stable complexes with a large number of structurally unrelated proteins. The best characterized and most common PP1-binding motif conforms to the consensus sequence RKX 0 -1 VI{P}FW in which X denotes any residue and {P} any residue except Pro; it is often referred to as the RVXF motif (6). The RVXF motif binds to a hydrophobic channel near the C terminus of PP1 (7).…”