DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated neuronal phosphoprotein. II. Comparison of the kinetics of phosphorylation of DARPP-32 and phosphatase inhibitor 1.
DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000) is a cytosolic neuronal phosphoprotein enriched in dopamine-innervated brain regions which, in its phosphorylated form, acts as an inhibitor of protein phosphatase 1. We have compared the phosphorylation of purified DARPP-32 with that of purified phosphatase inhibitor 1, a widespread inhibitor of protein phosphatase 1. Purified cyclic AMP-dependent protein kinase and cyclic GMP-dependent protein kinase each catalyzed the maximal incorporation of 0.9-1.1 mol of [32P]phosphate/mol of DARPP-32 or phosphatase inhibitor 1, with phosphorylation occurring on threonine residues. Evidence for the existence of a single phosphorylation site in each substrate protein was obtained by two-dimensional thin-layer phosphopeptide mapping of thermolytic digests. Initial rate studies of the phosphorylation of DARPP-32 yielded an apparent Km of 2.4 microM and a kcat of 2.7 S-1 for the catalytic subunit of cyclic AMP-dependent protein kinase, and an apparent Km of 5.4 microM and a kcat of 2.3 S-1 for cyclic GMP-dependent protein kinase. These in vitro results are compatible with a physiological role for the phosphorylation of DARPP-32 by either protein kinase in vivo. Similar studies with phosphatase inhibitor 1 yielded an apparent Km of 5.0 microM and a kcat of 1.4 S-1 for the catalytic subunit of cyclic AMP-dependent protein kinase, and an apparent Km of 25.0 microM and a kcat of 1.2 S-1 for cyclic GMP-dependent protein kinase. A synthetic nonapeptide, corresponding to the phosphorylation site of DARPP-32, was phosphorylated with apparent Km values of 1.12 mM and 1.86 mM and kcat values of 0.22 S-1 and 3.4 S-1 for cyclic AMP-dependent and cyclic GMP-dependent protein kinase, respectively.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002352:Carrier Proteins; D002417:Cattle; D002421:Caudate Nucleus / Q000378:metabolism*; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D002352:Carrier Proteins; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011506:Proteins; C021042:protein phosphatase inhibitor-1