CONVERSION OF PROTEIN PHOSPHATASE-1 CATALYTIC SUBUNIT TO A MN2-DEPENDENT ENZYME IMPAIRS ITS REGULATION BY INHIBITOR-1()

The phosphorylase phosphatase activity of protein phosphatase 1 (PP1) catalytic subunit from freshly purified rabbit skeletal muscle was inh ibited by MnCl2. Prolonged storage or inhibition by nonspecific phosph atase inhibitors ATP, sodium pyrophosphate, and NaF converted the musc le PP1 to a form that required Mn2+ for enzyme activity. Recombinant P P1 catalytic subunit expressed in Escherichia coli was also a Mn2+-dep endent enzyme. While native PP1 was inhibited by the phosphoprotein in hibitor 1 (I-1), with an IC50 of 1 nM, 40-50-fold higher concentration s of I-1 were required to inhibit the Mn2+-dependent PP1 enzymes. Conv ersion to the Mn2+-dependent state was accompanied by a 20-fold increa se in PP1's ability to dephosphorylate and inactivate I-1. Inhibition by thiophosphorylated I-1 established that dephosphorylation does not play a significant role in I-1's reduced potency as an inhibitor of Mn 2+-dependent PP1. The Mn2+-dependent PP1 enzymes were poorly inhibited by N-terminal phosphopeptides of I-1, indicating their impaired inter action with the I-1 functional domain. Mutation of a residue conserved in I-1 and DARPP-32, a structurally related PP1 inhibitor, preferenti ally attenuated I-1's activity as an inhibitor of Mn2+-dependent PP1. These data showed that, in addition to changes in its catalytic proper ties, Mn2+-dependent PP1 was modified in its interaction with I-1 at a site that was distinct from its catalytic domain. Our studies suggest that conversion to a Mn2+-dependent state alters multiple structural elements in PP1 catalytic subunit that together define its regulation by I-1.