Molecular Cloning of NIPP-1, a Nuclear Inhibitor of Protein Phosphatase-1, Reveals Homology with Polypeptides Involved in RNA Processing

doi:10.1074/jbc.270.47.28068

Molecular Cloning of NIPP-1, a Nuclear Inhibitor of Protein Phosphatase-1, Reveals Homology with Polypeptides Involved in RNA Processing (*)

From the Afdeling Biochemie and
Center for Human Genetics, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium

^§ To whom correspondence should be addressed:
Afdeling Biochemie, Campus Gasthuisberg KULeuven, B-3000 Leuven, Belgium.
Tel.: 32-16-345700; Fax: 32-16-345995; mathieu.bollen{at}med.kuleuven.ac.be.

Abstract

NIPP-1 was originally isolated as a potent and specific nuclear inhibitory polypeptide (16-18 kDa) of protein phosphatase-1. We report here the cDNA cloning of NIPP-1 from bovine thymus and show that the native polypeptide consists of 351 residues and has a calculated mass of 38.5 kDa. The bacterially expressed central third of NIPP-1 completely inhibited the type-1 catalytic subunit, but displayed a reduced inhibitory potency after phosphorylation by protein kinase A and casein kinase 2. Translation of NIPP-1 mRNA in reticulocyte lysates resulted in the accumulation of both intact NIPP-1 and a smaller polypeptide generated by alternative initiation at the codon corresponding to Met. A data base search showed that the COOH terminus of NIPP-1 is nearly identical to the human ard-1 protein (13 kDa), which has been implicated in RNA processing (Wang, M., and Cohen, S. N.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10591-10595). Comparison of the cDNAs encoding ard-1 and NIPP-1 suggests that their mRNAs are generated by alternative splicing of the same pre-mRNA. Western blotting with antibodies against the COOH terminus of NIPP-1, however, showed a single polypeptide of 47 kDa, which was enriched in the nucleus. Northern analysis revealed a single transcript of 2.2 kilobases in bovine thymus and of 2.4 kilobases in various human tissues.

Footnotes

↵* This work was supported by the Algemene Spaar-en Lijfrentekas, by the Belgian Fund for Medical Scientific Research (Grant 3.0041.93) and by a Concerted Research Action of the “Vlaamse Executive.” The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z50748[GenBank].
↵⁽⁾ The abbreviations used are:

PP-1

protein phosphatase-1

PP-1

catalytic subunit of PP-1

PP-2A

catalytic subunit of PP-2A

PP-1N

nuclear PP-1

NIPP-1

nuclear inhibitor of protein phosphatase-1

Tricine

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine

PAGE

polyacrylamide gel electrophoresis

bp

base pair(s).
- Received July 10, 1995.
- Revision received August 30, 1995.