title: Mechanism of inhibition of protein phosphatase 1 by DARPP-32: studies with recombinant DARPP-32 and synthetic peptides.
abstract: 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 approximately 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 approximately 1 microM. 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 microM, 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.
authors: INSERM U114, Collège de France, Paris.
authors: F
authors: F
authors: Desdouits
authors: J J
authors: JJ
authors: Cheetham
authors: H B
authors: HB
authors: Huang
authors: Y G
authors: YG
authors: Kwon
authors: E F
authors: EF
authors: da Cruz e Silva
authors: P
authors: P
authors: Denefle
authors: M E
authors: ME
authors: Ehrlich
authors: A C
authors: AC
authors: Nairn
authors: P
authors: P
authors: Greengard
authors: J A
authors: JA
authors: Girault
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000201:enzymology; D009419:Nerve Tissue Proteins / Q000494:pharmacology*; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology*; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010750:Phosphoproteins / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D054645:Protein Phosphatase 1; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D018411:Spodoptera
chemical_list: D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1