Mechanism of Inhibition of Protein Phosphatase 1 by DARPP-32: Studies with Recombinant DARPP-32 and Synthetic Peptides

• Published in: Biochemical and biophysical research communications Vol. 206; no. 2; pp. 652 - 658
• Main Authors: Desdouits, F., Cheetham, J.J., Huang, H.B., Kwon, Y.G., Silva, E.F.D.E., Denefle, P., Ehrlich, M.E., Nairn, A.C., Greengard, P., Girault, J.A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.01.1995
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Cattle; Cell Line; Cyclic AMP-Dependent Protein Kinases - metabolism; Dopamine and cAMP-Regulated Phosphoprotein 32; Escherichia coli; Kinetics; Molecular Sequence Data; Muscle, Skeletal - enzymology; Mutagenesis, Site-Directed; Myocardium - enzymology; Nerve Tissue Proteins - pharmacology; Oligodeoxyribonucleotides; Peptide Fragments - chemical synthesis; Peptide Fragments - pharmacology; Phosphopeptides - chemical synthesis; Phosphopeptides - pharmacology; Phosphoprotein Phosphatases - antagonists & inhibitors; Phosphoproteins - pharmacology; Phosphorylation; Protein Binding; Protein Phosphatase 1; Rabbits; Recombinant Proteins - pharmacology; Spodoptera
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.1995.1092

The mechanism of inhibition of protein phosphatase-1 catalytic subunit (PP-1c) by recombinant DARPP-32 and synthetic peptides was studied. DARPP-32 was expressed in Escherichia coli as a non-fusion protein using a pEt-3a plasmid, purified to homogeneity and shown to have physicochemical properties similar to those of the protein purified from bovine brain. Recombinant DARPP-32 phosphorylated on threonine-34 by cAMP-dependent protein kinase inhibited PP-1c with an IC50 ≍ 0.5 nM, comparable to that obtained with bovine DARPP-32. Non-phosphorylated DARPP-32, and mutated forms in which threonine-34 was replaced by an alanine or a glutamic acid, inhibited PP-1c with an IC50 ≍ 1 μM. Surface plasmon resonance analysis showed binding of PP-1c to nonphospho- and phospho-DARPP-32-(8-38) synthetic peptides with apparent Kd values of 1.2 and 0.3 μM,respectively, supporting the existence of an interaction between non-phosphorylated DARPP-32 and PP-1c that is increased by phosphorylation of DARPP-32 at threonine-34. These results suggest a model in which DARPP-32 interacts with PP-1c by at least two low affinity sites, the combination of which is responsible for the high affinity (nM) inhibition.