Mechanism of inhibition of protein phosphatase 1 by DARPP-32: studies with recombinant DARPP-32 and synthetic peptides.
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.
Desdouits
F
F
INSERM U114, Collège de France, Paris.
Cheetham
J J
JJ
Huang
H B
HB
Kwon
Y G
YG
da Cruz e Silva
E F
EF
Denefle
P
P
Ehrlich
M E
ME
Nairn
A C
AC
Greengard
P
P
Girault
J A
JA
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
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