NIPP1-mediated interaction of protein phosphatase-1 with CDC5L, a regulator of pre-mRNA splicing and mitotic entry.
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.
Boudrez
A
A
Afdeling Biochemie and Center for Human Genetics (VIB), Faculteit Geneeskunde, Katholieke Universiteit Leuven, Belgium.
Beullens
M
M
Groenen
P
P
Van Eynde
A
A
Vulsteke
V
V
Jagiello
I
I
Murray
M
M
Krainer
A R
AR
Stalmans
W
W
Bollen
M
M
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins; D002417:Cattle; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D004722:Endoribonucleases; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000378:metabolism; D008938:Mitosis; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000737:chemistry; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D054645:Protein Phosphatase 1; D017434:Protein Structure, Tertiary; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012568:Schizosaccharomyces / Q000737:chemistry; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C085875:cdc5 protein, S pombe; C021042:protein phosphatase inhibitor-1; D005982:Glutathione Transferase; D004722:Endoribonucleases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C097081:PPP1R8 protein, human