DOMAINS OF PHOSPHATASE INHIBITOR-2 INVOLVED IN THE CONTROL OF THE ATP-MG-DEPENDENT PROTEIN PHOSPHATASE

Inhibitor-2 (I-2) inhibits the free catalytic subunit of type 1 phosph atase (CS1) and controls the cyclic inactivation/activation of CS1 in the ATP-Mg-dependent protein phosphatase complex. We report here the e ffect of mutations on these two properties of I-2. Substitution of Thr -72 with Ala, Asp, or Glu generated complexes with CS1 that could not be activated. Mutation of Ser-86 did not affect activation by glycogen synthase kinase-3 (GSK-3) alone but impaired synergistic activation b y casein kinase II and GSK-3. Mutations in the region between Thr-72 a nd Ser-86 did not alter the inhibitory potency of I-2 but prevented co mplete inactivation of CS1. A mutant without the 35 NH2-terminal resid ues exhibited an IC50 for CS1 200-fold higher than that of wild-type I -2. However, it formed an inactive phosphatase complex with CS1, which was activated by GSK-3. A mutant with the 59 COOH-terminal residues d eleted retained full inhibitory activity and formed an inactive comple x that could not be activated by GSK-3. We conclude that the NH2-termi nal region of I-2 is involved in inhibition, that the sequence between Thr-72 and Ser-86 is necessary for the conversion of CS1 from an acti ve to an inactive conformation, and that the COOH terminus is required for activation by GSK-3. Thus, different functional domains of I-2 ma y interact with distinct regions of CS1.