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Identification of AKAP79 as a Protein Phosphatase 1 Catalytic Binding Protein

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    Identification of AKAP79 as a Protein Phosphatase 1 Catalytic Binding Protein
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    Pat and Jim Calhoun Center for Cardiology, University of Connecticut Health Center, Farmington, Connecticut 06030, United States
    Department of Pharmacology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, United States
    Pat and Jim Calhoun Center for Cardiology, University of Connecticut Health Center, Farmington, Connecticut 06030, United States
    *Tel: 860-679-2452. Fax: 860-679-1426. E-mail: [email protected]
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    Biochemistry

    Cite this: Biochemistry 2011, 50, 23, 5279–5291
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    https://doi.org/10.1021/bi200089z
    Published May 11, 2011
    Copyright © 2011 American Chemical Society
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    Abstract

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    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 IC50 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.

    Copyright © 2011 American Chemical Society

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    Supporting Information

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    To demonstrate that AKAP79 can link PP1 to PP2B, purified PP1 catalytic subunit was subjected to pulldown assay using GST-tagged PP2B A subunit in the presence and absence of AKAP79; as shown in Figure 1, PP1 was only seen in the PP2B complex in the presence of AKAP79, suggesting the anchoring protein organizes a trimeric complex consisting of AKAP79, PP1 and PP2B. This material is available free of charge via the Internet at http://pubs.acs.org.

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    Cited By

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    This article is cited by 17 publications.

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    4. Shannon N. Leslie, Angus C. Nairn. cAMP regulation of protein phosphatases PP1 and PP2A in brain. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2019, 1866 (1) , 64-73. https://doi.org/10.1016/j.bbamcr.2018.09.006
    5. David L. Brautigan, Shirish Shenolikar. Protein Serine/Threonine Phosphatases: Keys to Unlocking Regulators and Substrates. Annual Review of Biochemistry 2018, 87 (1) , 921-964. https://doi.org/10.1146/annurev-biochem-062917-012332
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    11. Seungwoo Kang, Mingxuan Xu, Edward C. Cooper, Naoto Hoshi. Channel-anchored Protein Kinase CK2 and Protein Phosphatase 1 Reciprocally Regulate KCNQ2-containing M-channels via Phosphorylation of Calmodulin. Journal of Biological Chemistry 2014, 289 (16) , 11536-11544. https://doi.org/10.1074/jbc.M113.528497
    12. Matthew D. Fuller, Ying Fu, Todd Scheuer, William A. Catterall. Differential regulation of CaV1.2 channels by cAMP-dependent protein kinase bound to A-kinase anchoring proteins 15 and 79/150. Journal of General Physiology 2014, 143 (3) , 315-324. https://doi.org/10.1085/jgp.201311075
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    14. Debbie Willoughby, Michelle L. Halls, Katy L. Everett, Antonio Ciruela, Philipp Skroblin, Enno Klussmann, Dermot M. F. Cooper. A key phosphorylation site in AC8 mediates regulation of Ca2+-dependent cAMP dynamics by an AC8–AKAP79–PKA signalling complex. Journal of Cell Science 2012, 125 (23) , 5850-5859. https://doi.org/10.1242/jcs.111427
    15. Christian Seeger, Xenia Gorny, Pasham Parameshwar Reddy, Constanze Seidenbecher, U. Helena Danielson. Kinetic and mechanistic differences in the interactions between caldendrin and calmodulin with AKAP79 suggest different roles in synaptic function. Journal of Molecular Recognition 2012, 25 (10) , 495-503. https://doi.org/10.1002/jmr.2215
    16. Xenia Gorny, Marina Mikhaylova, Christian Seeger, Pasham Parameshwar Reddy, Carsten Reissner, Björn H. Schott, U. Helena Danielson, Michael R. Kreutz, Constanze Seidenbecher. AKAP79/150 interacts with the neuronal calcium‐binding protein caldendrin. Journal of Neurochemistry 2012, 122 (4) , 714-726. https://doi.org/10.1111/j.1471-4159.2012.07828.x
    17. Lei Chen, Robert S. Kass. A-Kinase Anchoring Protein 9 and IKs Channel Regulation. Journal of Cardiovascular Pharmacology 2011, 58 (5) , 459-13. https://doi.org/10.1097/FJC.0b013e318232c80c
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    Biochemistry

    Cite this: Biochemistry 2011, 50, 23, 5279–5291
    Click to copy citationCitation copied!
    https://doi.org/10.1021/bi200089z
    Published May 11, 2011
    Copyright © 2011 American Chemical Society
    Request reuse permissions

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