Inhibitor-1 interaction domain that mediates the inhibition of protein phosphatase-1.
Inhibitor-1 (I-1), a cyclic AMP-regulated phosphoprotein, inhibits protein phosphatase-1 (PP1) activity in response to hormones. The molecular mechanism for PP1 inhibition by I-1 remains unknown. Mutation of nine acidic residues lining a proposed I-1-binding channel in rabbit PP1alpha yielded one mutant (E256A) slightly impaired in its inhibition by I-1, with the IC50 increased by 3-fold, and one mutant (E275R) located in the beta12-beta13 loop that showed 4-fold enhanced inhibition by I-1. Substituting Tyr-272, a proposed binding site for the toxins okadaic acid and microcystin-LR, in the beta12-beta13 loop with Trp, Phe, Asp, Arg, or Ala impaired PP1alpha inhibition by I-1 by 8-10-fold. Chemical mutagenesis of the Saccharomyces cerevisiae PP1 gene (GLC7) yielded 20 point mutations in the PP1 coding region. Two-hybrid analyses and biochemical assays of these yeast enzymes identified four additional residues in the beta12-beta13 loop that were required for PP1 binding and inhibition by I-1. Ten-fold higher concentrations of I-1 were required to inhibit these mutants. Finally, deletion of the beta12-beta13 loop from PP1alpha maintained full enzyme activity, but attenuated inhibition by I-1 by >100-fold. These data identified the beta12-beta13 loop in the PP1 catalytic subunit as a domain that mediates binding and enzyme inhibition by I-1.
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J H
JH
Connor
H N
HN
Quan
N T
NT
Ramaswamy
L
L
Zhang
S
S
Barik
J
J
Zheng
J F
JF
Cannon
E Y
EY
Lee
S
S
Shenolikar
D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D004791:Enzyme Inhibitors / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008387:Marine Toxins; D052998:Microcystins; D008958:Models, Molecular; D016296:Mutagenesis; D019319:Okadaic Acid / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D054645:Protein Phosphatase 1; D011506:Proteins / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic; D011506:Proteins; C073882:phosphoprotein phosphatase inhibitor 1; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C057862:cyanoginosin LR