Multiple interactions within the AKAP220 signaling complex contribute to protein phosphatase 1 regulation

The phosphorylation status of cellular proteins is controlled by the opposi ng actions of protein kinases and phosphatases. Compartmentalization of the se enzymes is critical for spatial and temporal control of these phosphoryl ation/dephosphorylation events. We previously reported that a 220-kDa A-kin ase anchoring protein (AKAP220) coordinates the location of the cAMP-depend ent protein kinase (PKA) and the type 1 protein phosphatase catalytic subun it (PP1c) (Schillace, R. V., and Scott, J. D. (1999) Curr. Biol. 9, 321-324 ). We now demonstrate that an AKAP220 fragment is a competitive inhibitor o f PP1c activity (K-i = 2.9 +/- 0.7 muM). Mapping studies and activity measu rements indicate that several protein-protein interactions act synergistica lly to inhibit PP1. A consensus targeting motif, between residues 1195 and 1198 (Lys-Val-Gln-Phe), binds but does not affect enzyme activity, whereas determinants between residues 1711 and 1901 inhibit the phosphatase. Analys is of truncated PP1c and chimeric PP1/2A catalytic subunits suggests that A KAP220 inhibits the phosphatase in a manner distinct from all known PP1 inh ibitors and toxins. Intermolecular interactions within the AKAP220 signalin g complex further contribute to PP1 inhibition as addition of the PKA regul atory subunit (RII) enhances phosphatase inhibition. These experiments indi cate that regulation of PP1 activity by AKAP220 involves a complex network of intra- and intermolecular interactions.