DARPP-32, a dopamine- and adenosine 3‘:5‘-monophosphate-regulated neuronal phosphoprotein. I. Amino acid sequence around the phosphorylated threonine.

H C Hemmings,K R Williams,W H Konigsberg,P Greengard

doi:10.1016/s0021-9258(17)42625-1

H C Hemmings, K R Williams + Show 2 more

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https://doi.org/10.1016/s0021-9258(17)42625-1

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Journal: The Journal of biological chemistry	Publication Date: Dec 1, 1984
Citations: 100	License type: cc-by

Abstract

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000) is a major endogenous cytosolic substrate for dopamine- and cyclic AMP-stimulated protein phosphorylation in neurons of the basal ganglia of mammalian brain. It shares many properties with phosphatase inhibitor 1, a substrate for cyclic AMP-dependent protein kinase, and with G-substrate, a substrate for cyclic GMP-dependent protein kinase. We have, therefore, undertaken an analysis of the amino acid sequence around the site at which purified DARPP-32 is phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. The results indicate that DARPP-32 is phosphorylated at a single threonine residue contained in the sequence Arg-Arg-Arg-Pro-Thr(P)-Pro-Ala-Met-Leu-Phe-Arg. This sequence was obtained by automated solid phase sequencing of two overlapping tryptic phosphopeptides and one overlapping chymotryptic phosphopeptide which were purified by reverse-phase high-performance liquid chromatography. A 9-amino acid sequence containing the phosphorylatable threonine residue in DARPP-32 shares 8 identical residues with a sequence containing the phosphorylatable threonine residue in phosphatase inhibitor 1, and shares 5 identical residues with the two identical sequences surrounding the 2 phosphorylatable threonine residues in G-substrate. These observations support the view that DARPP-32, inhibitor 1, and G-substrate are members of a family of regulatory proteins which are involved in the control of protein phosphatase activity by both cyclic AMP and cyclic GMP, but which differ in their cellular and tissue distributions.

Highlights

= 32,000 which is highly enriched in brain regions known to receive dopaminergic innervation, including the basalganglia cytosolic substratefor dopamine- and cyclic AMP- (Walaas andGreengard, 1984;Ouimet et al, 1984).Dopamine, stimulated protein phosphorylation in neurons of the basalganglia of mammalian brain
Comparative Peptide Mapping of Dephospho-DARPP-32 and Phospho-DARPP-32after Tryptic Digestion--In order to determine the number of phosphorylated tryptic peptides in DARPP-32, tryptic digests of dephospho-DARPP-32 and phospho-DARPP-32 were subjected to reverse-phase HPLC on a C-18 column.Phosphorylation of DARPP-32 resulted in a 2 min decrease in the elution time for the two peaksthat eluted at 76-78 min in the dephosphoDARPP-32 digest (Fig. 1).Cerenkov counting of the fractions from the phospho-DARPP-32digest indicated that these were the only radioactivelylabeledpeaks.Based on amino acid
The overall recovery of radioactivity from this column was 84%, with 42% of the recovered radioactivity contained in the peak eluting at 53 min labeled C-11. This peptide was not completely pure (Table I),solid phase amino acid sequencing resulted in an unambiguous sequence (Table 11)that corresponded to an overlap of peptide T-10, and extended the sequence obtained for the phosphorylation site of DARPP-32 to 11amino acids (Table 111).The other major radioactive peak (C-11’, eluting at 56 min) contained about 15%of the totalrecovered radioactivity and had an amino acid composition identical with that of peptide C-11 except that it contained a phenylalanine residue

Summary

RESULTS

Comparative Peptide Mapping of Dephospho-DARPP-32 and Phospho-DARPP-32after Tryptic Digestion--In order to determine the number of phosphorylated tryptic peptides in DARPP-32, tryptic digests of dephospho-DARPP-32 and phospho-DARPP-32 were subjected to reverse-phase HPLC on a C-18 column.Phosphorylation of DARPP-32 resulted in a 2 min decrease in the elution time for the two peaks (labeled T-10and T-10')that eluted at 76-78 min in the dephosphoDARPP-32 digest (Fig. 1).Cerenkov counting of the fractions from the phospho-DARPP-32digest indicated that these were the only radioactivelylabeledpeaks.Based on amino acid. DARPP-32 PhoSsiptehorylation Sequence analysis, at least 11peaks (labeled T-1through T-10‘) con-. Amino acid compositions of phosphoryhted peptidefsrom DARPP-32 peptide T-7 was unusual in that itcontained at least 54% Out of a total of about 28) glutamic acid/glutamine residues

Cerenkov counting of all the fractions obtained from the

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DISCUSSION

The remarkable homology of the phosphorylation sites of