Title | 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. |
Publication Type | Journal Article |
Year of Publication | 1989 |
Authors | Hemmings HC, Girault JA, Williams KR, LoPresti MB, Greengard P |
Journal | J Biol Chem |
Volume | 264 |
Issue | 13 |
Pagination | 7726-33 |
Date Published | 1989 May 5 |
ISSN | 0021-9258 |
Keywords | Amino Acid Sequence, Animals, Caudate Nucleus, Chromatography, High Pressure Liquid, Cyclic AMP, Dopamine, Kinetics, Molecular Sequence Data, Molecular Weight, Nerve Tissue Proteins, Peptide Fragments, Phosphoproteins, Phosphorylation, Phosphoserine, Protein Kinases, Rats, Rats, Inbred Strains, Time Factors |
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. |
Alternate Journal | J. Biol. Chem. |
PubMed ID | 2540203 |
Grant List | MH-40899 / MH / NIMH NIH HHS / United States |