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.

NIPP1 is a regulatory subunit of a species of protein phosphatase-1 (PP1) that co-localizes with splicing factors in nuclear speckles. We report that the N-terminal third of NIPP1 largely consists of a Forkhead-associated (FHA) protein interaction domain, a known phosphopeptide interaction module. A yeast two-hybrid screening revealed an interaction between this domain and a human homolog (CDC5L) of the fission yeast protein cdc5, which is required for G 2 /M progression and pre-mRNA splicing. CDC5L and NIPP1 co-localized in nuclear speckles in COS-1 cells. Furthermore, an interaction between CDC5L, NIPP1, and PP1 in rat liver nuclear extracts could be demonstrated by co-immunoprecipitation and/or co-purification experiments. The binding of the FHA domain of NIPP1 to CDC5L was dependent on the phosphorylation of CDC5L, e.g. by cyclin E-Cdk2. When expressed in COS-1 or HeLa cells, the FHA domain of NIPP1 did not affect the number of cells in the G 2 /M transition. However, the FHA domain blocked ␤-globin pre-mRNA splicing in nuclear extracts. A mutation in the FHA domain that abolished its interaction with CDC5L also canceled its anti-splicing effects. We suggest that NIPP1 either targets CDC5L or an associated protein for dephosphorylation by PP1 or serves as an anchor for both PP1 and CDC5L.Type 1 protein phosphatases (PP1) 1 belong to the PPP family of Ser/Thr protein phosphatases and regulate diverse cellular processes such as transcription, pre-mRNA splicing, intracellular transport, and metabolism (1-3). They consist of a single catalytic subunit (PP1 C ) and one or two regulatory subunits. The regulatory subunits act as substrate specifiers and anchor the holoenzymes in specific cell compartments in close vicinity to their substrates. In addition, the regulatory subunits mediate the control of the holoenzymes by hormones and growth factors through interaction with allosteric effectors or through phosphorylation by specific protein kinases. It has been estimated that mammalian cells contain tens of different regulatory proteins of PP1 (4). Altogether about 20 of these have already been characterized and cloned, including the glycogenbinding G-subunits, the myosin-binding M-subunits, the cytosolic regulator inhibitor-1, and the nuclear RNA-binding protein NIPP1 (1-3). Recent investigations have revealed that these regulatory proteins have multiple points of interaction with PP1 C , including a common phosphatase binding motif with the consensus sequence RVXF (5-10). In addition, most regulatory subunits contain domains that mediate the binding to substrates (e.g. myosin for the M-subunits) and/or a subcellular structure (e.g. glycogen for the G-subunits) to which the substrates are bound.In nuclear extracts, NIPP1 (39 kDa) is present as an inactive complex with PP1 C , termed PP1N NIPP1 (11). This heterodimer can be activated by phosphorylation of up to 4 Ser/Thr residues in the central domain of NIPP1 by protein kinases A and CK2 (12), which disrupts its interaction with PP1 C via the RVXF motif without dis...

Functional studies of the protein phosphatase-1 (PP1) regulator Sds22 suggest that it is indirectly and/or directly involved in one of the most ancient functions of PP1, i.e. reversing phosphorylation by the Aurora-related protein kinases. We predict that the conserved portion of Sds22 folds into a curved superhelix and demonstrate that mutation to alanine of any of eight residues (Asp 148 Among the protein phosphatases that occur in all studied eukaryotic lineages, the Ser/Thr-specific protein phosphatases of type-1 are the best conserved, with more than 70% of their residues nearly invariant (1). This conservation extends well beyond structurally and catalytically important residues to include exposed residues involved in the binding of regulatory proteins. As a catalytic subunit, PP1 1 depends on the interaction with one or two regulatory subunits for subcellular localization, substrate specificity, and activity regulation (1, 2). Eukaryotic cells contain a large variety of regulatory subunits of PP1, which account for the diversified action of this phosphatase. We have recently proposed that PP1 acquired an essential function during early eukaryotic evolution by the development of sites for interaction with a primordial regulatory subunit(s) (1). This essential primordial function and the sequential acquirement of additional interaction sites and functions would then have impeded further mutation of the corresponding portion(s) of the surface. The phylogenetic distribution of PP1 indicates that this primordial function must have been acquired before the divergence of the extant eukaryotic lineages. One of the most ancient functions of PP1 is to dephosphorylate substrates of the Aurora-related protein kinases, and this is essential for the completion of mitosis (3). The regulatory subunit(s) associated with this function of PP1 remain unknown, but the protein Sds22 (38 kDa) has emerged as a prime candidate. First, both yeast and mammalian Sds22 have been shown to interact with PP1 and to be part of a complex with PP1 that is enriched in the nucleus (4 -7). Second, the Sds22 encoding gene was identified independently in fission and in budding yeast as an extra-copy suppressor of the temperaturesensitive mitotic arrest phenotypes that are associated with certain mutations of PP1 (4,5,8). Deletion of the Sds22-encoding gene caused a similar mitotic arrest, and this phenotype could be complemented by the overexpression of PP1 (4,5,8). Third, the conditionally lethal phenotype in budding yeast that was conferred by a loss-of-function mutation of the Aurora-related kinase Ipl1 (Ipl1-2), was largely relieved by the expression of certain temperature-sensitive mutant versions of Sds22 or PP1 (9, 10). The mutant Sds22 version that rescued the Ipl1-2 phenotype showed a decreased ability to interact with PP1. The expression of this mutant Sds22 did not affect the cellular levels of PP1 or Sds22, but drastically reduced the nuclear level of PP1 and caused a redistribution of the nuclear pool of PP1 (9).Hitherto, little ...