Phosphorylation-dependent inhibition of protein phosphatase-1 by G- substrate

A purkinje cell substrate of the cyclic GMP-dependent protein kinase

Kelly Umstott Hall, Sean P. Collins, David M. Gamm, Enrique Massa, Anna De Paoli-Roach, Michael D. Uhler

Research output: Contribution to journalArticle

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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 i protein phosphatases.

Original languageEnglish
Pages (from-to)3485-3495
Number of pages11
JournalJournal of Biological Chemistry
Volume274
Issue number6
DOIs
StatePublished - Feb 5 1999

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Glycogen-Synthase-D Phosphatase
Protein Phosphatase 1
Cyclic GMP-Dependent Protein Kinases
Phosphorylation
Purkinje Cells
Substrates
Brain
Phosphoprotein Phosphatases
Cyclic AMP-Dependent Protein Kinases
Phosphoric Monoester Hydrolases
Recombinant Proteins
Cerebellum
G-substrate
Cyclic GMP-Dependent Protein Kinase Type I
Complementary DNA
Pons

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphorylation-dependent inhibition of protein phosphatase-1 by G- substrate : A purkinje cell substrate of the cyclic GMP-dependent protein kinase. / Hall, Kelly Umstott; Collins, Sean P.; Gamm, David M.; Massa, Enrique; De Paoli-Roach, Anna; Uhler, Michael D.

In: Journal of Biological Chemistry, Vol. 274, No. 6, 05.02.1999, p. 3485-3495.

Research output: Contribution to journalArticle

Hall, Kelly Umstott ; Collins, Sean P. ; Gamm, David M. ; Massa, Enrique ; De Paoli-Roach, Anna ; Uhler, Michael D. / Phosphorylation-dependent inhibition of protein phosphatase-1 by G- substrate : A purkinje cell substrate of the cyclic GMP-dependent protein kinase. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 6. pp. 3485-3495.
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