Molecular Determinants of Nuclear Protein Phosphatase-1 Regulation by NIPP-1

Abstract

Reversible protein serine/threonine phosphorylation in higher eukaryotes is catalyzed by protein kinases and phosphatases (1). The functional diversity of protein phosphatases is increased by the presence of isoforms of the catalytic subunits and numerous noncatalytic or regulatory subunits. For example, there are four mammalian isoforms of the catalytic subunit of type-1 protein phosphatases (2, 3). These catalytic subunits (PP-1 C ) 1 are anchored at various subcellular locations by their association with targeting subunits that also define the substrate specificity and activity of the enzyme. Of the nearly 20 PP-1-targeting subunits thus far identified in mammalian cells, the best characterized is the G subunit which targets PP-1 C to glycogen and enhances its glycogen-synthase phosphatase activity (4 -6). Interaction of the skeletal muscle G M subunit with PP-1 C is in part mediated by an RVXF motif that binds in an extended conformation within a hydrophobic channel near the C terminus of PP-1 C (5, 6). Phosphorylation of G M by protein kinase A (PKA) at a threonine in the X position abolished PP-1 binding. Variants of the RVXF motif are present in many other PP-1 regulators, including the myosin-binding or M subunit, the p53 binding-protein-2, inhibitor-1, DARPP-32, and inhibitor-2 (5-10). Moreover, the regulation of PP-1 C by these proteins was impaired by mutations within the RVXF motif or by the presence of competing RVXF-containing peptides. However, these regulators also possessed other interaction sites with PP-1 C in addition to the RVXF motif (4,5,8). Biochemical (11) and immunofluorescence (12) studies showed that a large fraction of PP-1 is present in the nucleus. Nuclear PP-1 (PP-1N) has been implicated in the regulation of mRNA splicing (13), in the cell cycle-dependent control of the retinoblastoma protein (14) and lamin B (15), and in the dephosphorylation of the transcription factors CREB (16) and Sp1 (17). However, the PP-1 holoenzymes involved and their regulation remain poorly understood.

Structural basis of protein phosphatase 1 regulation

“…Because 34 amino acids N-terminal to the RVxF motif allow MYPT1 to reach the catalytic cleft of PP1, other regulatory subunits may follow a similar path, including the nuclear inhibitor of PP1 (NIPP1) 23 , spinophilin and neurabin 24 (Fig. 4).…”

“…4). Amino acids 143-217 of NIPP1 constitute a potent PP1 inhibitor 23 . This region contains regulatory phosphorylation sites at Ser 178, Ser 199, Thr 161 and Ser 204, and the consensus RVxF motif, which is preceded by a basic sequence as in MYPT1.…”

The Protein Phosphatase-1 (PP1) Regulator, Nuclear Inhibitor of PP1 (NIPP1), Interacts with the Polycomb Group Protein, Embryonic Ectoderm Development (EED), and Functions as a Transcriptional Repressor

“…However, mutation of this polybasic stretch as in NIPP1-(143-224)-K193A,R194A,K195A,R196A,K197A did not hamper the interaction with EED in two-hybrid assays (data not shown). A second PP1-binding site in the central domain of NIPP1 is represented by the 200 RVTF 203 sequence (2). This sequence is a variant of the so-called RVXF motif that is present in most interactors of PP1 (38).…”

“…NIPP1 1 (39 kDa, 351 residues) is a ubiquitously expressed nuclear protein that was originally discovered as a potent and specific inhibitor of protein Ser/Thr phosphatase-1 (PP1), hence its name nuclear inhibitor of PP1 (1). NIPP1 contains binding sites for PP1 in its central and COOH-terminal domains (2,3) and is complexed to 30 -50% of the nuclear pool of PP1 (4).…”

“…NIPP1 contains binding sites for PP1 in its central and COOH-terminal domains (2,3) and is complexed to 30 -50% of the nuclear pool of PP1 (4). The NIPP1-PP1 holoenzyme in nuclear extracts is inactive but can be activated by the phosphorylation of NIPP1 with protein kinase A (Ser-199), protein kinase CK2 (Ser-204), or protein tyrosine kinases of the Src family (Tyr-335) (3, 5-7).…”