Properties and Phosphorylation Sites of Baculovirus-expressed Nuclear Inhibitor of Protein Phosphatase-1 (NIPP-1)

doi:10.1074/jbc.272.52.32972

Properties and Phosphorylation Sites of Baculovirus-expressed Nuclear Inhibitor of Protein Phosphatase-1 (NIPP-1)*

From the Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium

Abstract

NIPP-1 is the RNA-binding subunit of a major species of protein phosphatase-1 in the nucleus. We have expressed nuclear inhibitor of protein phosphatase-1 (NIPP-1) in Sf9 cells, using the baculovirus-expression system. The purified recombinant protein was a potent (K _i = 9.9 ± 0.3 pm) and specific inhibitor of protein phosphatase-1 and was stoichiometrically phosphorylated by protein kinases A and CK2. At physiological ionic strength, phosphorylation by these protein kinases drastically decreased the inhibitory potency of free NIPP-1. Phosphorylation of NIPP-1 in a heterodimeric complex with the catalytic subunit of protein phosphatase-1 resulted in an activation of the holoenzyme without a release of NIPP-1. Sequencing and phosphoamino acid analysis of tryptic phosphopeptides enabled us to identify Ser¹⁷⁸ and Ser¹⁹⁹ as the phosphorylation sites of protein kinase A, whereas Thr¹⁶¹ and Ser²⁰⁴were phosphorylated by protein kinase CK2. These residues all conform to consensus recognition sites for phosphorylation by protein kinases A or CK2 and are clustered near a RVXF sequence that has been identified as a motif that interacts with the catalytic subunit of protein phosphatase-1.

Footnotes

↵* This work was supported by the Algemene Spaar en Lijfrentekas, by Fund for Medical Scientific Research Grant G.0179.97, and by a Flemish Concerted Research Action.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.
↵‡ These authors contributed equally to this work.
↵§ Research associate of the Fund for Scientific Research-Flanders.
↵¶ Senior research associate of the Fund for Scientific Research-Flanders.
↵‖ 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.
↵1 The abbreviations used are: PP-1, protein phosphatase-1; NIPP-1, nuclear inhibitor of PP-1; PKA, protein kinase A; CK2, protein kinase CK2, previously called casein kinase-2; PP-1_C, catalytic subunit of PP-1; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis.
↵2 M. Beullens, Q. Jin, W. Stalmans, and M. Bollen, unpublished data.
- Received June 3, 1997.
- Revision received September 18, 1997.