ARPP-21, a cyclic AMP-regulated phosphoprotein (Mr = 21,000) enriched in dopamine-innervated brain regions. Amino acid sequence of the site phosphorylated by cyclic AMP in intact cells and kinetic studies of its phosphorylation in vitro

J Biol Chem. 1989 May 5;264(13):7726-33.

Abstract

ARPP-21 (cyclic AMP-regulated phosphoprotein, Mr = 21,000) is a cytosolic neuronal phosphoprotein that is highly enriched in regions of mammalian brain that receive dopaminergic innervation, in particular the striatum. The state of phosphorylation of ARPP-21 in brain slices prepared from rat striatum was shown to be regulated by 8-bromo-cyclic AMP. Phosphorylation occurred exclusively on seryl residues contained within a single tryptic phosphopeptide as analyzed by two-dimensional thin layer electrophoresis/chromatography. The tryptic phosphopeptide derived from ARPP-21 phosphorylated in intact cells comigrated with the tryptic phosphopeptide derived from purified ARPP-21 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase in vitro. Purified cyclic AMP-dependent protein kinase catalyzed the incorporation of 1.1 mol of [32P]phosphate/mol of ARPP-21 exclusively on seryl residues. The amino acid sequence surrounding the site in purified ARPP-21 phosphorylated by cyclic AMP-dependent protein kinase in vitro was determined by analyzing two overlapping chymotryptic peptides isolated from [32P]phospho-ARPP-21 by reverse phase high performance liquid chromatography. A combination of gas phase and solid phase amino acid sequencing yielded a phosphorylation site sequence of -Glu-Arg-Arg-Lys-Ser(P)-Lys-Ser-Gly-Ala-Gly-. Initial rate studies of the phosphorylation of purified ARPP-21 by the catalytic subunit of cyclic AMP-dependent protein kinase yielded an apparent Km of 0.78 microM and a kcat of 2.2 s-1. A synthetic peptide based on the phosphorylation site of ARPP-21 was phosphorylated on the corresponding seryl residue with an apparent Km of 40 microM and a kcat of 4.0 s-1. These results are compatible with a physiological role for the phosphorylation of ARPP-21 by cyclic AMP-dependent protein kinase in vivo, regulated by first messengers acting via cyclic AMP, e.g. dopamine and vasoactive intestinal peptide.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Caudate Nucleus / physiology*
  • Chromatography, High Pressure Liquid
  • Cyclic AMP / physiology*
  • Dopamine / physiology*
  • In Vitro Techniques
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • Nerve Tissue Proteins / metabolism*
  • Peptide Fragments / analysis
  • Peptide Fragments / metabolism
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Phosphoserine / metabolism
  • Protein Kinases / metabolism*
  • Rats
  • Rats, Inbred Strains
  • Time Factors

Substances

  • Nerve Tissue Proteins
  • Peptide Fragments
  • Phosphoproteins
  • Phosphoserine
  • Cyclic AMP
  • Protein Kinases
  • Dopamine