Interaction of Inhibitor-2 with the Catalytic Subunit of Type 1 Protein Phosphatase

Inhibitor-2 (I-2) is the regulatory subunit of a cytosolic type 1 Ser/Thr protein phosphatase (PP1) and potently inhibits the activity of the free catalytic subunit (CS1). Previous work from the laboratory had proposed that the interaction of I-2 with CS1 involved multiple sites (Park, I. K., and DePaoli-Roach, A. A. (1994) J. Biol. Chem. 269, 28919–28928). The present study refines the earlier analysis and arrives at a more detailed model for the interaction between I-2 and CS1. Although the NH2-terminal I-2 regions containing residues 1–35 and 1–64 have no inhibitory activity on their own, they increase the IC50 for I-2 by ∼30-fold, indicating the presence of a CS1-interacting site. Based on several experimental approaches, we have also identified the sequence Lys144-Leu-His-Tyr147 as a second site of interaction that corresponds to the RVXF motif present in many CS1-binding proteins. The peptide I-2(135–151) significantly increases the IC50 for I-2 and attenuates CS1 inhibition. Replacement of Leu and Tyr with Ala abolishes the ability to counteract inhibition by I-2. The I-2(135–151) peptide, but not I-2(1–35), also antagonizes inhibition of CS1 by DARPP-32 in a pattern similar to that of I-2. Furthermore, a peptide derived from the glycogen-binding subunit, RGL/GM(61–80), which contains a consensus CS1-binding motif, completely counteracts CS1 inhibition by I-2 and DARPP-32. The NH2-terminal 35 residues of I-2 bind to CS1 at a site that is specific for I-2, whereas the KLHY sequence interacts with CS1 at a site shared with other interacting proteins. Other results suggest the presence of yet more sites of interaction. A model is presented in which multiple “anchoring interactions” serve to position a segment of I-2 such that it sterically occludes the catalytic pocket but need not make high affinity contacts itself. Inhibitor-2 (I-2) is the regulatory subunit of a cytosolic type 1 Ser/Thr protein phosphatase (PP1) and potently inhibits the activity of the free catalytic subunit (CS1). Previous work from the laboratory had proposed that the interaction of I-2 with CS1 involved multiple sites (Park, I. K., and DePaoli-Roach, A. A. (1994) J. Biol. Chem. 269, 28919–28928). The present study refines the earlier analysis and arrives at a more detailed model for the interaction between I-2 and CS1. Although the NH2-terminal I-2 regions containing residues 1–35 and 1–64 have no inhibitory activity on their own, they increase the IC50 for I-2 by ∼30-fold, indicating the presence of a CS1-interacting site. Based on several experimental approaches, we have also identified the sequence Lys144-Leu-His-Tyr147 as a second site of interaction that corresponds to the RVXF motif present in many CS1-binding proteins. The peptide I-2(135–151) significantly increases the IC50 for I-2 and attenuates CS1 inhibition. Replacement of Leu and Tyr with Ala abolishes the ability to counteract inhibition by I-2. The I-2(135–151) peptide, but not I-2(1–35), also antagonizes inhibition of CS1 by DARPP-32 in a pattern similar to that of I-2. Furthermore, a peptide derived from the glycogen-binding subunit, RGL/GM(61–80), which contains a consensus CS1-binding motif, completely counteracts CS1 inhibition by I-2 and DARPP-32. The NH2-terminal 35 residues of I-2 bind to CS1 at a site that is specific for I-2, whereas the KLHY sequence interacts with CS1 at a site shared with other interacting proteins. Other results suggest the presence of yet more sites of interaction. A model is presented in which multiple “anchoring interactions” serve to position a segment of I-2 such that it sterically occludes the catalytic pocket but need not make high affinity contacts itself. type 1 serine/threonine protein phosphatase catalytic subunit of PP1 phosphatase inhibitor-1 phosphatase inhibitor-2 regulatory subunit of glycogen-associated PP1 polyacrylamide gel electrophoresis Type 1 protein phosphatases (PP1)1 constitute a major proportion of the cellular Ser/Thr phosphatases and play important roles in the regulation of many cellular functions including metabolism, hormone receptor activation, muscle contraction, cell growth and division, and gene expression (1Cohen P. Trends Biochem. Sci. 1992; 17: 408-413Abstract Full Text PDF PubMed Scopus (277) Google Scholar, 2Fischer E.H. Biofactors. PubMed Scopus Google Scholar, Biochem. Biol. 1992; PubMed Scopus Google Scholar, A. A. PubMed Scopus Google The that of of catalytic and J. 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