NIPP1-mediated Interaction of Protein Phosphatase-1 with CDC5L, a Regulator of Pre-mRNA Splicing and Mitotic Entry

doi:10.1074/jbc.M001676200

NIPP1-mediated Interaction of Protein Phosphatase-1 with CDC5L, a Regulator of Pre-mRNA Splicing and Mitotic Entry*

From Afdeling Biochemie and ^§Center for Human Genetics (VIB), Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium and ^¶Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724

Abstract

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 G₂/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 G₂/M transition. However, the FHA domain blocked β-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.

Footnotes

↵* This work was supported by Fund for Scientific Research-Flanders Grant G.0179.97, a Flemish Concerted Research Action and the Prime Minister's Office IUAP P4/23.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Postdoctoral fellow of the National Fund for Scientific Research-Flanders.
↵‖ Supported by National Institutes of Health Grant GM-42699.
↵** To whom correspondence should be addressed: Afdeling Biochemie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32 16 34 57 01; Fax: 32 16 34 59 95; E-mail: Mathieu.Bollen@med.kuleuven.ac.be.
Published, JBC Papers in Press, May 24, 2000, DOI 10.1074/jbc.M001676200
↵2 ProfileScan is available on the World Wide Web.
Abbreviations:

PP1

protein phosphatase-1

EGFP

enhanced green fluorescent protein

GST

glutathione S-transferase

HA

hemagglutinin

PBS

phosphate-buffered saline

PP2A

protein phosphatase-2A

PP1_C

catalytic subunit of PP1

PP2A_C

catalytic subunit of PP2A

PP1N_NIPP1

heterodimeric complex of NIPP1 and PP1_C

TBS

Tris-buffered saline

FHA

Forkhead-associated

FACS

fluorescence-activated cell sorting

MES

4-morpholineethanesulfonic acid

Tricine

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
- Received February 29, 2000.
- Revision received May 15, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.