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.
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.
Hemmings
H C
HC
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.
Girault
J A
JA
Williams
K R
KR
LoPresti
M B
MB
Greengard
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002421:Caudate Nucleus / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000502:physiology*; D004298:Dopamine / Q000502:physiology*; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D010768:Phosphoserine; D000242:Cyclic AMP; D011494:Protein Kinases; D004298:Dopamine