INHIBITOR-1 INTERACTION DOMAIN THAT MEDIATES THE INHIBITION OF PROTEIN PHOSPHATASE-1

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.