title: Identification of AKAP79 as a protein phosphatase 1 catalytic binding protein.
abstract: The ubiquitously expressed and highly promiscuous protein phosphatase 1 (PP1) regulates many cellular processes. Targeting PP1 to specific locations within the cell allows for the regulation of PP1 by conferring substrate specificity. In the present study, we identified AKAP79 as a novel PP1 regulatory subunit. Immunoprecipitaiton of the AKAP from rat brain extract found that the PP1 catalytic subunit copurified with the anchoring protein. This is a direct interaction, demonstrated by pulldown experiments using purified proteins. Interestingly, the addition of AKAP79 to purified PP1 catalytic subunit decreased phosphatase activity with an IC(50) of 811 ± 0.56 nM of the anchoring protein. Analysis of AKAP79 identified a PP1 binding site that conformed to a consensus PP1 binding motif (FxxR/KxR/K) in the first 44 amino acids of the anchoring protein. This was confirmed when a peptide mimicking this region of AKAP79 was able to bind PP1 by both pulldown assay and surface plasmon resonance. However, PP1 was still able to bind to AKAP79 upon deletion of this region, suggesting additional sites of contact between the anchoring protein and the phosphatase. Importantly, this consensus PP1 binding motif was found not to be responsible for PP1 inhibition, but rather enhanced phosphatase activity, as deletion of this domain resulted in an increased inhibition of PP1 activity. Instead, a second interaction domain localized to residues 150-250 of AKAP79 was required for the inhibition of PP1. However, the inhibitory actions of AKAP79 on PP1 are substrate dependent, as the anchoring protein did not inhibit PP1 dephosphorylation of phospho-PSD-95, a substrate found in AKAP79 complexes in the brain. These combined observations suggest that AKAP79 acts as a PP1 regulatory subunit that can direct PP1 activity toward specific targets in the AKAP79 complex.
authors.affiliation: University of Connecticut Health Center, Farmington, CT 06030, USA.
authors.forename: Andrew V
authors.initials: AV
authors.lastname: Le
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Tavalin
authors.forename: Kimberly L
authors.initials: KL
authors.lastname: Dodge-Kafka
mesh_terms: D054758:A Kinase Anchor Proteins / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002478:Cells, Cultured; D008969:Molecular Sequence Data; D054645:Protein Phosphatase 1 / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D020349:Surface Plasmon Resonance
chemical_list: D054758:A Kinase Anchor Proteins; C487775:AKAP5 protein, human; D054645:Protein Phosphatase 1