ABSTRACT
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.
Subject(s)
Carrier Proteins , Endoribonucleases , Intracellular Signaling Peptides and Proteins , Nuclear Localization Signals , RNA-Binding Proteins/metabolism , Subcellular Fractions/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Humans , Molecular Sequence Data , Phosphoprotein Phosphatases , Protein Phosphatase 1 , Protein Transport , RNA-Binding Proteins/chemistry , Sequence Homology, Amino AcidABSTRACT
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 beta-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.
Subject(s)
Carrier Proteins , Cell Cycle Proteins/metabolism , Endoribonucleases , Intracellular Signaling Peptides and Proteins , Mitosis , Phosphoprotein Phosphatases/metabolism , RNA Splicing , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Animals , COS Cells , Cattle , Cell Cycle Proteins/chemistry , Cell Nucleus/metabolism , Cell Separation , Flow Cytometry , Fluorescent Antibody Technique , Glutathione Transferase/metabolism , HeLa Cells , Humans , Liver/metabolism , Molecular Sequence Data , Muscle, Skeletal/chemistry , Mutation , Phosphoprotein Phosphatases/chemistry , Phosphorylation , Precipitin Tests , Protein Phosphatase 1 , Protein Structure, Tertiary , Rabbits , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Schizosaccharomyces/chemistry , Schizosaccharomyces pombe Proteins , Sequence Homology, Amino Acid , Two-Hybrid System TechniquesABSTRACT
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 microcystin-Sepharose 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.
