Multiple interactions within the AKAP220 signaling complex contribute to protein phosphatase 1 regulation.
The phosphorylation status of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases. Compartmentalization of these enzymes is critical for spatial and temporal control of these phosphorylation/dephosphorylation events. We previously reported that a 220-kDa A-kinase anchoring protein (AKAP220) coordinates the location of the cAMP-dependent protein kinase (PKA) and the type 1 protein phosphatase catalytic subunit (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 of PP1c activity (K(i) = 2.9 +/- 0.7 micrometer). Mapping studies and activity measurements indicate that several protein-protein interactions act synergistically 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. Analysis of truncated PP1c and chimeric PP1/2A catalytic subunits suggests that AKAP220 inhibits the phosphatase in a manner distinct from all known PP1 inhibitors and toxins. Intermolecular interactions within the AKAP220 signaling complex further contribute to PP1 inhibition as addition of the PKA regulatory subunit (RII) enhances phosphatase inhibition. These experiments indicate that regulation of PP1 activity by AKAP220 involves a complex network of intra- and intermolecular interactions.
Schillace
R V
RV
Howard Hughes Medical Institute, Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201-3098, USA. schillac@ohsu.edu
Voltz
J W
JW
Sim
A T
AT
Shenolikar
S
S
Scott
J D
JD
D054758:A Kinase Anchor Proteins; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D054645:Protein Phosphatase 1; D015398:Signal Transduction
D054758:A Kinase Anchor Proteins; C099270:AKAP11 protein, human; D002352:Carrier Proteins; D017931:DNA Primers; D004791:Enzyme Inhibitors; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1