Abstract
DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000) is a potent inhibitor of protein phosphatase-1 when it is phosphorylated on Thr-34 by cAMP-dependent protein kinase. DARPP-32 is highly enriched in some specific cell populations such as striatonigral neurons and choroid plexus epithelial cells. Here we show that recombinant rat DARPP-32 is phosphorylated by casein kinase I on seryl residues to a stoichiometry of approximately 2 mol of phosphate/mol of protein. DARPP-32 is one of the best known substrates for casein kinase I (Km = 3.4 +/- 0.3 microM), whereas the homologous phosphatase-1 inhibitor, inhibitor-1, is not. Phosphorylation of DARPP-32 by casein kinase I does not alter its ability to inhibit protein phosphatase-1. Residues phosphorylated by casein kinase I were identified as Ser-137 and Ser-189 by site-directed mutagenesis and by protein sequencing. Ser-137 and the preceding stretch of 16-18 acidic residues are conserved in DARPP-32 among all species examined, whereas Ser-189 is not. Phosphorylation of Ser-137 induces an unusual increase in DARPP-32 electrophoretic mobility in polyacrylamide gels in the presence of SDS. In striatonigral neurons, DARPP-32 is phosphorylated on Ser-137 and the stoichiometry of phosphorylation on this residue in vivo appears to be higher in the substantia nigra (axon terminals) than in the striatum (soma and dendrites). These results indicate that casein kinase I is highly active in striatonigral neurons in which it may play important roles, including in protein phosphatase-1 modulation via phosphorylation of DARPP-32.
Highlights
Since the rat DARPP-32 sequence contains a stretch of 18 acidic amino acids followed by a serine at position 137, which provides a casein kinase I consensus site, we have suggested previously that DARPP-32 might be a substrate for casein kinase I [10]
When DARPP-32 was phosphorylated by casein kinase I to a low stoichiometry, it migrated as a doublet (Fig. 3A), and the thermolytic maps of the lower band contained a major acidic peptide absent from the maps obtained from the upper band (Fig. 4, A and B)
We have identified casein kinase I as a novel DARPP-32 kinase in vitro and, most probably, in vivo
Summary
Materials-Recombinant rat DARPP-32 was produced in Escherichia coli and purified as previously described [18]. DARPP-32 was incubated with 10 J,Lg of casein kinase I and 150 J.LM [-/2P]ATP, at 30 oc for 4 h. Phosphopeptide Sequencing- Three hundred J.Lg of DARPP-32 or S137A-DARPP-32 were phosphorylated with 150 J.LM [-/2P]ATP (specific activity, 950 cpm/pmol) and 4 J,Lg of casein kinase I in a final volume of 1 ml, for 90 min, at 30 °C. For estimation of the stoichiometry of phosphorylation by casein kinase I in vivo, striata and substantia nigra of four rats were rapidly dissected and directly frozen in liquid nitrogen. DARPP-32 in striatum and substantia nigra was analyzed by immunoblotting with a mixture of two monoclonal antibodies (C24-5a and C24-6a), as previously described [26], except that a horseradish peroxidase-coupled donkey-anti-mouse IgG secondary antibody was used and that immunoreactivity was detected with an enhanced chemiluminescence method (Amersham). PP-1c inhibition was studied as described, using phosphorylase a as substrate [10]
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