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...

NIPP1 is a ubiquitously expressed nuclear protein that functions both as a regulator of protein Ser/Thr phosphatase-1 and as a splicing factor. The N-terminal part of NIPP1 consists of a phosphothreonine-interacting Forkhead-associated (FHA) domain. We show here that the FHA domain of NIPP1 interacts in vitro and in vivo with a TP dipeptide-rich fragment of the splicing factor SAP155/SF3b 155 , a component of the U2 small nuclear ribonucleoprotein particle. The NIPP1-SAP155 interaction was entirely dependent on the phosphorylation of specific TP motifs in SAP155. Mutagenesis and competition studies revealed that various phosphorylated TP motifs competed for binding to the same site in the FHA domain. The SAP155 kinases in cell lysates were blocked by the Ca 2؉ chelator EGTA and by the cyclin-dependent protein kinase inhibitor roscovitine. The phosphorylation level of SAP155 was dramatically increased during mitosis, and accordingly the activity of SAP155 kinases was augmented in mitotic lysates. We discuss how the interaction between NIPP1 and SAP155 could contribute to spliceosome (dis)assembly and the catalytic steps of splicing.Spliceosomes catalyze the removal of intronic sequences from primary transcripts (pre-mRNAs) in two consecutive transesterification reactions (1-3). Their major components are the U1, U2, U5, and U4/U6 small nuclear ribonucleoprotein particles (snRNPs), 1 which consist of small nuclear RNAs and a set of snRNP proteins. These snRNPs are recruited from nuclear storage/assembly sites known as the splicing factor compartments or speckles, and their assembly on pre-mRNAs occurs in a stepwise manner. According to a recent model of spliceosome assembly (2), U1 snRNP and U4/U6⅐U5 tri-snRNP first contact the 5Ј splice site. Subsequently U2 snRNP binds stably to the intronic branch site region. The creation of splicing-competent spliceosomes also depends on the recruitment of numerous nonsnRNP-associated splicing factors and requires multiple, ordered rearrangements between spliceosomal components.Substantial evidence implicates reversible protein phosphorylation in both spliceosome (dis)assembly and splicing catalysis (2, 4). For example, the SR family of splicing factors contain a domain that is rich in Arg/Ser dipeptides, and the phosphorylation of these motifs by specific "SR" kinases has been shown to modulate the interaction of SR proteins with other splicing factors and with RNA during spliceosome assembly (5). The shuttling of SR proteins between the speckles and the spliceosomes and the dispersion of SR proteins from the speckles during mitosis is also controlled by phosphorylation (6 -8). The splicing factor SF1/BBP, which interacts with U2AF65 in an early step of spliceosome assembly, is a substrate for phosphorylation by protein kinase G (9). The phosphorylation of SF1/ BBP inhibits its interaction with U2AF65 and blocks splicing complex formation. A role for protein phosphatases in pre-mRNA splicing has also been firmly established. The protein Ser/Thr phosphatase PP2C␥ is required ...

NIPP1 is a ubiquitous nuclear protein that is required for spliceosome assembly. We report here that the phosphothreonine-binding Forkhead-associated domain of NIPP1 interacts with the cell cycle-regulated protein Ser/Thr kinase MELK (maternal embryonic leucine zipper kinase). The NIPP1-MELK interaction was critically dependent on the phosphorylaton of Thr-478 of MELK and was increased in lysates from mitotically arrested cells. Recombinant MELK was a potent inhibitor of an early step of spliceosome assembly in nuclear extracts. This splicing defect was also seen with a kinase-dead mutant but was absent after mutation (T478A) of the NIPP1 binding site of MELK, indicating a mediatory role for NIPP1. Our data suggest that MELK has a role in the cell cycle-regulated control of pre-mRNA splicing.The nuclear protein NIPP1 1 (39 kDa) was originally discovered as a potent and specific inhibitor of protein Ser/Thr phosphatase-1 (PP1), hence its name, nuclear inhibitor of PP1 (1-7).More recently, we have demonstrated that NIPP1 is also implicated in transcription as well as in pre-mRNA splicing by mechanisms that do not involve PP1 (8, 9). In transient transfection experiments, NIPP1 acted as a transcriptional repressor, which may be accounted for by the binding of the central and C-terminal domains of NIPP1 to the Polycomb protein, EED (embryonic ectoderm development) (9). The latter promotes transcriptional repression by the recruitment of a histone methyltransferase and histone deacetylases. NIPP1 also appears to be required for the assembly of spliceosomes, the protein-RNA complexes that catalyze pre-mRNA splicing (8). The spliceosomal function of NIPP1 requires its C-terminal domain as well as its N-terminal Forkhead-associated (FHA) domain, an established phosphothreonine-binding module. The FHA domain of NIPP1 mediates targeting to both the spliceosomes and the nuclear storage sites for splicing factors, known as "speckles" (8, 10). The targeting function of the FHA domain of NIPP1 is likely explained by its ability to bind to phosphorylated forms of the essential splicing factors CDC5L (11) and SAP155 (12).Here we show that the protein kinase MELK, which is structurally related to the AMP-activated protein kinases, also interacts in a phosphorylation-dependent manner with the FHA domain of NIPP1 and that this interaction is increased during mitosis. Furthermore, we demonstrate that recombinant MELK blocks spliceosome assembly by a mechanism that involves NIPP1. Our data suggest a novel link between pre-mRNA processing and cell cycle progression. EXPERIMENTAL PROCEDURESYeast , cloned into the pEG202 vector in-frame with the LexA DNA-binding domain, was used as bait for the screening of a HeLa cell cDNA library (11). In this library, the cDNAs are subcloned behind a galactose-inducible promoter in the pJG4 -5 vector in-frame with the B42 activation domain. Interacting proteins were identified by growth of the yeast strain EGY188 in a Ϫleucine/ϩ galactose medium. The use of a plasmid-borne LacZ reporter gene (pSH18 ...

Various studies have provided evidence for the existence of spontaneously active cytosolic species of protein phosphatase 1, but these enzymes have never been purified and characterized. We have used chromatography on microcystinSepharose and Resource Q to purify cytosolic protein phosphatases from rat liver. Two of the isolated enzymes were identified by Western analysis and peptide sequencing as complexes of the catalytic subunit of protein phosphatase 1 and either the inhibitor NIPP1 or the myosin-binding subunit MYPT1, which reportedly is not present in chicken liver. In contrast, PCR cloning revealed the expression of two MYPT1 splice variants in rat liver.z 1999 Federation of European Biochemical Societies.

NIPP1 is a nuclear subunit of protein phosphatase-1 (PP1) that colocalizes with pre-mRNA splicing factors in speckles. We report here that the nuclear and subnuclear targeting of NIPP1, when expressed in HeLa cells or COS-1 cells as a fusion protein with the enhanced-green-fluorescent protein (EGFP), are mediated by distinct sequences. While NIPP1-EGFP can cross the nuclear membrane passively, the active transport to the nucleus is mediated by two independent nuclear localization signals in the central domain of NIPP1, which partially overlap with binding site(s) for PP1. Furthermore, the concentration of NIPP1-EGFP in the nuclear speckles requires the ‘ForkHead-Associated’ domain in the N terminus. This domain is also required for the nuclear retention of NIPP1 when active transport is blocked. Our data imply that the nuclear and subnuclear targeting of NIPP1 are controlled independently.