Phosphorylation-dependent Inhibition of Protein Phosphatase-1 by G-substrate

doi:10.1074/jbc.274.6.3485

Phosphorylation-dependent Inhibition of Protein Phosphatase-1 by G-substrate

A PURKINJE CELL SUBSTRATE OF THE CYCLIC GMP-DEPENDENT PROTEIN KINASE*

From the ^‡Mental Health Research Institute, the ^§Department of Biological Chemistry, and the ^¶Neuroscience Program, University of Michigan, Ann Arbor, Michigan 48109 and the ^**Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202

Abstract

G-substrate, a specific substrate of the cGMP-dependent protein kinase, has previously been localized to the Purkinje cells of the cerebellum. We report here the isolation from mouse brain of a cDNA encoding G-substrate. This cDNA was used to localize G-substrate mRNA expression, as well as to produce recombinant protein for the characterization of G-substrate phosphatase inhibitory activity. Brain and eye were the only tissues in which a G-substrate transcript was detected. Within the brain, G-substrate transcripts were restricted almost entirely to the Purkinje cells of the cerebellum, although transcripts were also detected at low levels in the paraventricular region of the hypothalamus and the pons/medulla. Like the native protein, the recombinant protein was preferentially phosphorylated by cGMP-dependent protein kinase (K _m = 0.2 μm) over cAMP-dependent protein kinase (K _m = 2.0 μm). Phospho-G-substrate inhibited the catalytic subunit of native protein phosphatase-1 with an IC₅₀ of 131 ± 27 nm. Dephospho-G-substrate was not found to be inhibitory. Both dephospho- and phospho-G-substrate were weak inhibitors of native protein phosphatase-2A₁, which dephosphorylated G-substrate 20 times faster than the catalytic subunit of protein phosphatase-1. G-substrate potentiated the action of cAMP-dependent protein kinase on a cAMP-regulated luciferase reporter construct, consistent with an inhibition of cellular phosphatases in vivo. These results provide the first demonstration that G-substrate inhibits protein phosphatase-1 and suggest a novel mechanism by which cGMP-dependent protein kinase I can regulate the activity of the type 1 protein phosphatases.

Footnotes

↵* This work was supported by National Institutes of Health Grants GM50791 and DK36569.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF071562 (mouse) and AF071789 (human).
↵‖ Present address: Dept. of Biology, Texas A & M University, Kingsville, TX 78363.
↵‡ To whom correspondence should be addressed: Mental Health Research Institute, Neuroscience Laboratory Bldg., 1103 East Huron St., University of Michigan, Ann Arbor, MI 48109-1687. Tel.: 734-647-3172; Fax: 734-936-2690; E-mail: muhler{at}umich.edu.
Abbreviations:

cGK

cGMP-dependent protein kinase

cAK

cAMP-dependent protein kinase

DARPP-32

dopamine- and cAMP-regulated phosphoprotein of apparent M _r32,000 determined by SDS-PAGE

PAGE

polyacrylamide gel electrophoresis

PP1c

protein phosphatase-1 catalytic subunit

PCR

polymerase chain reaction

CMV

human cytomegalovirus

Cα

cAK catalytic subunit

PP2A₁

protein phosphatase type-2A₁

bp

base pairs

EST

expressed sequence tag

CPT-cAMP

chlorophenylthio-cAMP.
- Received June 18, 1998.
- Revision received November 4, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.