Skip to main content
SpringerLink
Log in

Biochemical Analysis of AKAP-Anchored PKA Signaling Complexes

  • Protocol
  • First Online:
cAMP Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2483))

Abstract

Generation of the prototypic second messenger cAMP instigates numerous signaling events. A major intracellular target of cAMP is Protein kinase A (PKA), a Ser/Thr protein kinase. Where and when this enzyme is activated inside the cell has profound implications on the functional impact of PKA. It is now well established that PKA signaling is focused locally into subcellular signaling “islands” or “signalosomes.” The A-Kinase Anchoring Proteins (AKAPs) play a critical role in this process by dictating spatial and temporal aspects of PKA action. Genetically encoded biosensors, small molecule and peptide-based disruptors of PKA signaling are valuable tools for rigorous investigation of local PKA action at the biochemical level. This chapter focuses on approaches to evaluate PKA signaling islands, including a simple assay for monitoring the interaction of an AKAP with a tunable PKA holoenzyme. The latter approach evaluates the composition of PKA holoenzymes, in which regulatory subunits and catalytic subunits can be visualized in the presence of test compounds and small-molecule inhibitors.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Wilson LJ et al (2018) New perspectives, opportunities, and challenges in exploring the human protein kinome. Cancer Res 78(1):15–29

    Article  CAS  PubMed  Google Scholar 

  2. Newton AC, Bootman MD, Scott JD (2016) Second messengers. Cold Spring Harb Perspect Biol 8(8):a005926

    Article  PubMed  PubMed Central  Google Scholar 

  3. Beuschlein F et al (2014) Constitutive activation of PKA catalytic subunit in adrenal Cushing’s syndrome. N Engl J Med 370(11):1019–1028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Omar MH, Scott JD (2020) AKAP Signaling islands: venues for precision pharmacology. Trends Pharmacol Sci 41(12):933–946

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Bucko PJ, Scott JD (2021) Drugs that regulate local cell signaling: AKAP targeting as a therapeutic option. Annu Rev Pharmacol Toxicol 61:361–379

    Article  CAS  PubMed  Google Scholar 

  6. Carnegie GK, Means CK, Scott JD (2009) A-kinase anchoring proteins: from protein complexes to physiology and disease. IUBMB Life 61(4):394–406

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Scott JD, Pawson T (2009) Cell signaling in space and time: where proteins come together and when they’re apart. Science 326(5957):1220–1224

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Carr DW et al (1991) Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif. J Biol Chem 266(22):14188–14192

    Article  CAS  PubMed  Google Scholar 

  9. Gold MG et al (2006) Molecular basis of AKAP specificity for PKA regulatory subunits. Mol Cell 24(3):383–395

    Article  CAS  PubMed  Google Scholar 

  10. Newlon MG et al (2001) A novel mechanism of PKA anchoring revealed by solution structures of anchoring complexes. EMBO J 20(7):1651–1662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Smith FD et al (2017) Local protein kinase A action proceeds through intact holoenzymes. Science 356(6344):1288–1293

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Welch EJ, Jones BW, Scott JD (2010) Networking with AKAPs: context-dependent regulation of anchored enzymes. Mol Interv 10(2):86–97

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Skroblin P et al (2010) Mechanisms of protein kinase A anchoring. Int Rev Cell Mol Biol 283:235–330

    Article  CAS  PubMed  Google Scholar 

  14. Sanderson JL, Dell’Acqua ML (2011) AKAP signaling complexes in regulation of excitatory synaptic plasticity. Neuroscientist 17(3):321–336

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Dessauer CW (2009) Adenylyl cyclase--A-kinase anchoring protein complexes: the next dimension in cAMP signaling. Mol Pharmacol 76(5):935–941

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Diviani D et al (2011) A-kinase anchoring proteins: scaffolding proteins in the heart. Am J Physiol Heart Circ Physiol 301(5):H1742–H1753

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Klauck TM et al (1996) Coordination of three signaling enzymes by AKAP79, a mammalian scaffold protein. Science 271(5255):1589–1592

    Article  CAS  PubMed  Google Scholar 

  18. Coghlan VM, Hausken ZE, Scott JD (1995) Subcellular targeting of kinases and phosphatases by association with bifunctional anchoring proteins. Biochem Soc Trans 23(3):592–596

    Article  CAS  PubMed  Google Scholar 

  19. Dodge KL et al (2001) mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module. EMBO J 20(8):1921–1930

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tasken KA et al (2001) Phosphodiesterase 4D and protein kinase a type II constitute a signaling unit in the centrosomal area. J Biol Chem 276(25):21999–22002

    Article  CAS  PubMed  Google Scholar 

  21. Bauman AL et al (2006) Dynamic regulation of cAMP synthesis through anchored PKA-adenylyl cyclase V/VI complexes. Mol Cell 23(6):925–931

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Taylor SS et al (2004) PKA: a portrait of protein kinase dynamics. Biochim Biophys Acta 1697(1–2):259–269

    Article  CAS  PubMed  Google Scholar 

  23. Gold MG (2019) Swimming regulations for protein kinase A catalytic subunit. Biochem Soc Trans 47(5):1355–1366

    Article  CAS  PubMed  Google Scholar 

  24. Herberg FW et al (2000) Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding. J Mol Biol 298(2):329–339

    Article  CAS  PubMed  Google Scholar 

  25. Aye TT et al (2009) Selectivity in enrichment of cAMP-dependent protein kinase regulatory subunits type I and type II and their interactors using modified cAMP affinity resins. Mol Cell Proteomics 8(5):1016–1028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Walker-Gray R, Stengel F, Gold MG (2017) Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches. Proc Natl Acad Sci U S A 114(39):10414–10419

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lester LB et al (1996) Cloning and characterization of a novel A-kinase anchoring protein. AKAP 220, association with testicular peroxisomes. J Biol Chem 271(16):9460–9465

    Article  CAS  PubMed  Google Scholar 

  28. Huang LJ et al (1997) Identification of a novel protein kinase A anchoring protein that binds both type I and type II regulatory subunits. J Biol Chem 272(12):8057–8064

    Article  CAS  PubMed  Google Scholar 

  29. Trotter KW et al (1999) Alternative splicing regulates the subcellular localization of A-kinase anchoring protein 18 isoforms. J Cell Biol 147(7):1481–1492

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Scott JD, Dessauer CW, Tasken K (2013) Creating order from chaos: cellular regulation by kinase anchoring. Annu Rev Pharmacol Toxicol 53:187–210

    Article  CAS  PubMed  Google Scholar 

  31. Wang Y et al (2014) Isoform-selective disruption of AKAP-localized PKA using hydrocarbon stapled peptides. ACS Chem Biol 9(3):635–642

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Carr DW et al (1992) Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain. J Biol Chem 267(19):13376–13382

    Article  CAS  PubMed  Google Scholar 

  33. Vijayaraghavan S et al (1997) Protein kinase A-anchoring inhibitor peptides arrest mammalian sperm motility. J Biol Chem 272(8):4747–4752

    Article  CAS  PubMed  Google Scholar 

  34. Alto NM et al (2003) Bioinformatic design of A-kinase anchoring protein-in silico: a potent and selective peptide antagonist of type II protein kinase A anchoring. Proc Natl Acad Sci U S A 100(8):4445–4450

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Faruque OM et al (2009) Cell-permeable peptide-based disruption of endogenous PKA-AKAP complexes: a tool for studying the molecular roles of AKAP-mediated PKA subcellular anchoring. Am J Physiol Cell Physiol 296(2):C306–C316

    Article  CAS  PubMed  Google Scholar 

  36. Hundsrucker C et al (2006) High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides. Biochem J 396(2):297–306

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Christian F et al (2011) Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes. J Biol Chem 286(11):9079–9096

    Article  CAS  PubMed  Google Scholar 

  38. Schafer G et al (2013) Highly functionalized terpyridines as competitive inhibitors of AKAP-PKA interactions. Angew Chem Int Ed Eng 52(46):12187–12191

    Article  Google Scholar 

  39. Verdine GL, Hilinski GJ (2012) Stapled peptides for intracellular drug targets. Methods Enzymol 503:3–33

    Article  CAS  PubMed  Google Scholar 

  40. Gold MG et al (2013) Engineering A-kinase anchoring protein (AKAP)-selective regulatory subunits of protein kinase A (PKA) through structure-based phage selection. J Biol Chem 288(24):17111–17121

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Burns-Hamuro LL et al (2003) Designing isoform-specific peptide disruptors of protein kinase A localization. Proc Natl Acad Sci U S A 100(7):4072–4077

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Carlson CR et al (2006) Delineation of type I protein kinase A-selective signaling events using an RI anchoring disruptor. J Biol Chem 281(30):21535–21545

    Article  CAS  PubMed  Google Scholar 

  43. Torheim EA et al (2009) Design of proteolytically stable RI-anchoring disruptor peptidomimetics for in vivo studies of anchored type I protein kinase A-mediated signalling. Biochem J 424(1):69–78

    Article  CAS  PubMed  Google Scholar 

  44. Sarma GN et al (2010) Structure of D-AKAP2:PKA RI complex: insights into AKAP specificity and selectivity. Structure 18(2):155–166

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Jarnaess E et al (2008) Dual specificity A-kinase anchoring proteins (AKAPs) contain an additional binding region that enhances targeting of protein kinase A type I. J Biol Chem 283(48):33708–33718

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Seamon KB, Padgett W, Daly JW (1981) Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc Natl Acad Sci U S A 78(6):3363–3367

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Patel TB et al (2001) Molecular biological approaches to unravel adenylyl cyclase signaling and function. Gene 269(1–2):13–25

    Article  CAS  PubMed  Google Scholar 

  48. Premont RT et al (1996) Identification and characterization of a widely expressed form of adenylyl cyclase. J Biol Chem 271(23):13900–13907

    Article  CAS  PubMed  Google Scholar 

  49. Dessauer CW, Scully TT, Gilman AG (1997) Interactions of forskolin and ATP with the cytosolic domains of mammalian adenylyl cyclase. J Biol Chem 272(35):22272–22277

    Article  CAS  PubMed  Google Scholar 

  50. Beavo JA et al (1970) Effects of xanthine derivatives on lipolysis and on adenosine 3′,5′-monophosphate phosphodiesterase activity. Mol Pharmacol 6(6):597–603

    CAS  PubMed  Google Scholar 

  51. Omori K, Kotera J (2007) Overview of PDEs and their regulation. Circ Res 100(3):309–327

    Article  CAS  PubMed  Google Scholar 

  52. Robison GA, Butcher RW, Sutherland EW (1968) Cyclic AMP. Annu Rev Biochem 37:149–174

    Article  CAS  PubMed  Google Scholar 

  53. Shear M et al (1976) Agonist-specific refractoriness induced by isoproterenol. Studies with mutant cells. J Biol Chem 251(23):7572–7576

    Article  CAS  PubMed  Google Scholar 

  54. Schwede F et al (2000) Cyclic nucleotide analogs as biochemical tools and prospective drugs. Pharmacol Ther 87(2–3):199–226

    Article  CAS  PubMed  Google Scholar 

  55. Beavo JA, Brunton LL (2002) Cyclic nucleotide research -- still expanding after half a century. Nat Rev Mol Cell Biol 3(9):710–718

    Article  CAS  PubMed  Google Scholar 

  56. Walsh DA et al (1971) Krebs EG. Purification and characterization of a protein inhibitor of adenosine 3′,5′-monophosphate-dependent protein kinases. J Biol Chem 246(7):1977–1985

    Article  CAS  PubMed  Google Scholar 

  57. Dalton GD, Dewey WL (2006) Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function. Neuropeptides 40(1):23–34

    Article  CAS  PubMed  Google Scholar 

  58. Demaille JG, Peters KA, Fischer EH (1977) Isolation and properties of the rabbit skeletal muscle protein inhibitor of adenosine 3′,5′-monophosphate dependent protein kinases. Biochemistry 16(14):3080–3086

    Article  CAS  PubMed  Google Scholar 

  59. Byrne DP et al (2016) cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility-mass spectrometry. Biochem J 473(19):3159–3175

    Article  CAS  PubMed  Google Scholar 

  60. Eyers PA et al (2005) Regulation of the G(2)/M transition in Xenopus oocytes by the cAMP-dependent protein kinase. J Biol Chem 280(26):24339–24346

    Article  CAS  PubMed  Google Scholar 

  61. Scott JD et al (1985) Identification of an inhibitory region of the heat-stable protein inhibitor of the cAMP-dependent protein kinase. Proc Natl Acad Sci U S A 82(13):4379–4383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Scott JD et al (1986) Primary-structure requirements for inhibition by the heat-stable inhibitor of the cAMP-dependent protein kinase. Proc Natl Acad Sci U S A 83(6):1613–1616

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Cheng HC et al (1985) An active twenty-amino-acid-residue peptide derived from the inhibitor protein of the cyclic AMP-dependent protein kinase. Biochem J 231(3):655–661

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Glass DB et al (1986) Differential and common recognition of the catalytic sites of the cGMP-dependent and cAMP-dependent protein kinases by inhibitory peptides derived from the heat-stable inhibitor protein. J Biol Chem 261(26):12166–12171

    Article  CAS  PubMed  Google Scholar 

  65. Cheng HC et al (1986) A potent synthetic peptide inhibitor of the cAMP-dependent protein kinase. J Biol Chem 261(3):989–992

    Article  CAS  PubMed  Google Scholar 

  66. Reed J et al (1989) Conformational analysis of PKI(5-22)amide, the active inhibitory fragment of the inhibitor protein of the cyclic AMP-dependent protein kinase. Biochem J 264(2):371–380

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Knighton DR et al (1991) Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253(5018):414–420

    Article  CAS  PubMed  Google Scholar 

  68. Manschwetus JT et al (2019) A Stapled peptide mimic of the pseudosubstrate inhibitor PKI inhibits protein kinase A. Molecules 24(8):1567

    Article  CAS  PubMed Central  Google Scholar 

  69. Bain J et al (2007) The selectivity of protein kinase inhibitors: a further update. Biochem J 408(3):297–315

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Hidaka H et al (1984) Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23(21):5036–5041

    Article  CAS  PubMed  Google Scholar 

  71. Chijiwa T et al (1990) Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265(9):5267–5272

    Article  CAS  PubMed  Google Scholar 

  72. Byrne DP et al (2020) Aurora A regulation by reversible cysteine oxidation reveals evolutionarily conserved redox control of Ser/Thr protein kinase activity. Sci Signal 13(639):eaax2713

    Article  CAS  PubMed  Google Scholar 

  73. Davies SP et al (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351(Pt 1):95–105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Murray AJ (2008) Pharmacological PKA inhibition: all may not be what it seems. Sci Signal 1(22):re4

    Article  PubMed  Google Scholar 

  75. Yap TA et al (2012) AT13148 is a novel, oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity. Clin Cancer Res 18(14):3912–3923

    Article  CAS  PubMed  Google Scholar 

  76. Rothermel JD et al (1983) Inhibition of glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3′,5′-phosphorothioate. J Biol Chem 258(20):12125–12128

    Article  CAS  PubMed  Google Scholar 

  77. Rothermel JD, Jastorff B, Botelho LH (1984) Inhibition of glucagon-induced glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3′,5′-phosphorothioate. J Biol Chem 259(13):8151–8155

    Article  CAS  PubMed  Google Scholar 

  78. Gjertsen BT et al (1995) Novel (Rp)-cAMPS analogs as tools for inhibition of cAMP-kinase in cell culture. Basal cAMP-kinase activity modulates interleukin-1 beta action. J Biol Chem 270(35):20599–20607

    Article  CAS  PubMed  Google Scholar 

  79. Maller JL, Butcher FR, Krebs EG (1979) Early effect of progesterone on levels of cyclic adenosine 3′:5′-monophosphate in Xenopus oocytes. J Biol Chem 254(3):579–582

    Article  CAS  PubMed  Google Scholar 

  80. Maller JL, Krebs EG (1977) Progesterone-stimulated meiotic cell division in Xenopus oocytes. Induction by regulatory subunit and inhibition by catalytic subunit of adenosine 3′:5′-monophosphate-dependent protein kinase. J Biol Chem 252(5):1712–1718

    Article  CAS  PubMed  Google Scholar 

  81. Mant A et al (2011) Protein kinase A is central for forward transport of two-pore domain potassium channels K2P3.1 and K2P9.1. J Biol Chem 286(16):14110–14119

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Iyer GH, Moore MJ, Taylor SS (2005) Consequences of lysine 72 mutation on the phosphorylation and activation state of cAMP-dependent kinase. J Biol Chem 280(10):8800–8807

    Article  CAS  PubMed  Google Scholar 

  83. Byrne DP et al (2018) New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors. Biochem J 475(15):2435–2455

    Article  CAS  PubMed  Google Scholar 

  84. Buechler YJ, Herberg FW, Taylor SS (1993) Regulation-defective mutants of type I cAMP-dependent protein kinase. Consequences of replacing arginine 94 and arginine 95. J Biol Chem 268(22):16495–16503

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported, in whole or in part, by and BBSRC grants BB/S018514/1, BB/N021703/1, and BB/R000182/1 to D.B. and P.A.E., and National Institutes of Health Grants 1K22CA154600 to EJK, F32DK121415 to MO and NIH DK119186 and DK119192 to J.D.S

Author information

Authors and Affiliations

  1. Department of Biochemistry and Systems Biology, ISMIB, University of Liverpool, Liverpool, UK

    Dominic P. Byrne & Patrick A. Eyers

  2. Department of Pharmacology, University of Washington, Seattle, WA, USA

    Mitchell H. Omar & John D. Scott

  3. Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA, USA

    Eileen J. Kennedy

Authors
  1. Dominic P. Byrne
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mitchell H. Omar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eileen J. Kennedy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Patrick A. Eyers
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. John D. Scott
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Patrick A. Eyers or John D. Scott .

Editor information

Editors and Affiliations

  1. Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, UK

    Manuela Zaccolo

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Byrne, D.P., Omar, M.H., Kennedy, E.J., Eyers, P.A., Scott, J.D. (2022). Biochemical Analysis of AKAP-Anchored PKA Signaling Complexes. In: Zaccolo, M. (eds) cAMP Signaling. Methods in Molecular Biology, vol 2483. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2245-2_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2245-2_19

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2244-5

  • Online ISBN: 978-1-0716-2245-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation