ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Purification and characterization of the protein from bovine caudate nucleus.
ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by SDS/PAGE) is a major cytosolic substrate for cAMP-stimulated protein phosphorylation in dopamine-innervated regions of rat CNS (Walaas et al., 1983c). This acidic phosphoprotein has now been identified in bovine caudate nucleus cytosol and purified to homogeneity from this source. The purification procedure involved diethylaminoethyl-cellulose chromatography, ammonium sulfate fractionation, phenyl-Sepharose CL-4B chromatography, and fast protein liquid chromatography using Mono Q anion-exchange resin. Two isoforms of ARPP-21 (ARPP-21A and ARPP-21B) were obtained, which were present in approximately equal amounts in the starting material. ARPP-21A was purified 2610-fold with a final yield of 20% and ARPP-21B was purified 2940-fold with a final yield of 21%. The purified preparations of both isoforms were judged to be homogenous by SDS/PAGE. ARPP-21A and ARPP-21B yielded identical 2-dimensional thin-layer tryptic phosphopeptide maps, identical amino acid compositions and closely related, but distinct, reverse-phase high-pressure liquid chromatograms of tryptic digests. The amino acid composition of ARPP-21 showed a high content of glutamic acid/glutamine, and no methionine, tryptophan, tyrosine, phenylalanine, or histidine. ARPP-21 was stable to heat denaturation and to 50% (vol/vol) ethanol treatment and was partially soluble at pH 2. The Mr determined for ARPP-21 by SDS/PAGE was 21,000. The Stokes radius of ARPP-21 was 26.3 A, and the sedimentation coefficient of ARPP-21 was 1.3 S; these values yield a calculated molecular mass of 13,700 Da and a frictional ratio of 1.7, indicative of an elongated tertiary structure. ARPP-21 was an excellent substrate for cAMP-dependent protein kinase and was either not phosphorylated or only poorly phosphorylated by cGMP-dependent protein kinase, calcium/calmodulin-dependent protein kinase I, calcium/calmodulin-dependent protein kinase II, casein kinase II, or protein kinase C. The purified catalytic subunit of cAMP-dependent protein kinase catalyzed the incorporation of 1.2 mol phosphate/mol purified ARPP-21. Phosphorylation occurred exclusively on seryl residues. Phospho-ARPP-21 was dephosphorylated effectively by protein phosphatase-1 or -2A, but not by protein phosphatase-2B or -2C. Rabbit polyclonal and mouse monoclonal antibodies were prepared to purified ARPP-21. These antibodies specifically immunoprecipitated ARPP-21, which was found to be highly enriched in the caudate nucleus and putamen of monkey brain.(ABSTRACT TRUNCATED AT 400 WORDS)
Hemmings
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Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.
Greengard
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D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000502:physiology; D002417:Cattle; D002421:Caudate Nucleus / Q000032:analysis*; D002498:Centrifugation; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D000242:Cyclic AMP / Q000502:physiology*; D004298:Dopamine / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D007536:Isomerism; D008253:Macaca mulatta; D008297:Male; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000145:classification / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D014018:Tissue Distribution
D010750:Phosphoproteins; D000242:Cyclic AMP; D004298:Dopamine