Jh. Connor et al., INHIBITOR-1 INTERACTION DOMAIN THAT MEDIATES THE INHIBITION OF PROTEIN PHOSPHATASE-1, The Journal of biological chemistry, 273(42), 1998, pp. 27716-27724
Inhibitor-1 (I-l), a cyclic AMP-regulated phosphoprotein, inhibits pro
tein phosphatase-l (PP1) activity in response to hormones. The molecul
ar mechanism for PP1 inhibition by I-1 remains unknown. Mutation of ni
ne acidic residues lining a proposed I-1-binding channel in rabbit PP1
alpha yielded one mutant (E256A) slightly impaired in its inhibition
by I-1, with the IC50 increased by 3-fold, and one mutant (E275R) loca
ted in the beta 12-beta 13 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 beta 12-beta 13 loop with Trp,
Phe, Asp, Arg, or Ala impaired PP1 alpha inhibition by I-1 by 8-10-fo
ld, Chemical mutagenesis of the Saccharomyces cerevisiae PP1 gene (GLC
7) yielded 20 point mutations in the PP1 coding region. Two-hybrid ana
lyses and biochemical assays of these yeast enzymes identified four ad
ditional residues in the beta 12-beta 13 loop that were required for P
P1 binding and inhibition by I-1. Ten-fold higher concentrations of I-
1 were required to inhibit these mutants. Finally, deletion of the bet
a 12-beta 13 loop from PP1 alpha maintained full enzyme activity, but
attenuated inhibition by I-1 by >100-fold. These data identified the b
eta 12-beta 13 loop in the PP1 catalytic subunit as a domain that medi
ates binding and enzyme inhibition by I-1.