Article | Published:

Phosphorylation by protein kinase A disassembles the caspase-9 core

Cell Death & Differentiationvolume 25pages10251039 (2018) | Download Citation

Abstract

Caspases, the cysteine proteases which facilitate the faithful execution of apoptosis, are tightly regulated by a number of mechanisms including phosphorylation. In response to cAMP, PKA phosphorylates caspase-9 at three sites preventing caspase-9 activation, and suppressing apoptosis progression. Phosphorylation of caspase-9 by PKA at the functionally relevant site Ser-183 acts as an upstream block of the apoptotic cascade, directly inactivating caspase-9 by a two-stage mechanism. First, Ser-183 phosphorylation prevents caspase-9 self-processing and directly blocks substrate binding. In addition, Ser-183 phosphorylation breaks the fundamental interactions within the caspase-9 core, promoting disassembly of the large and small subunits. This occurs despite Ser-183 being a surface residue distal from the interface between the large and small subunits. This phosphorylation-induced disassembly promotes the formation of ordered aggregates around 20 nm in diameter. Similar aggregates of caspase-9 have not been previously reported. This two-stage regulatory mechanism for caspase-9 has likewise not been reported previously but may be conserved across the caspases.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Additional information

Edited by P. Salomoni

References

  1. 1.

    Srinivasula SM, Hegde R, Saleh A, Datta P, Shiozaki E, Chai J, et al. A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis. Nature. 2001;410:112–6.

  2. 2.

    Chęcińska A, Giaccone G, Rodriguez JA, Kruyt FAE, Jimenez CR. Comparative proteomics analysis of caspase-9-protein complexes in untreated and cytochrome c/dATP stimulated lysates of NSCLC cells. J Proteom. 2009;72:575–85.

  3. 3.

    Seaman JE, Julien O, Lee PS, Rettenmaier TJ, Thomsen ND, Wells JA. Cacidases: caspases can cleave after aspartate, glutamate and phosphoserine residues. Cell Death Differ. 2016. https://doi.org/10.1038/cdd.2016.62.

  4. 4.

    Pop C, Salvesen GS. Human caspases: activation, specificity, and regulation. J Biol Chem. 2009;284:21777–81.

  5. 5.

    Renatus M, Stennicke HR, Scott FL, Liddington RC, Salvesen GS. Dimer formation drives the activation of the cell death protease caspase-9. Proc Natl Acad Sci USA. 2001;98:14250–5.

  6. 6.

    Shiozaki EN, Chai J, Rigotti DJ, Riedl SJ, Li P, Srinivasula SM, et al. Mechanism of XIAP-mediated inhibition of caspase-9. Mol Cell. 2003;11:519–27.

  7. 7.

    McIlwain DR, Berger T, Mak TW. Caspase functions in cell death and disease. Cold Spring Harb Perspect Biol. 2013;5:1–27.

  8. 8.

    Friedlander RM. Apoptosis and caspases in neurodegenerative diseases. N Engl J Med. 2003;348:1365–75.

  9. 9.

    Howley B, Fearnhead HO. Caspases as therapeutic targets. J Cell Mol Med. 2008;12:1502–16.

  10. 10.

    Kurokawa M, Kornbluth S. Caspases and kinases in a death grip. Cell. 2009;138:838–54.

  11. 11.

    Franklin RA, McCubrey JA. Kinases: positive and negative regulators of apoptosis. Leukemia. 2000;14:2019–34.

  12. 12.

    López-Otín C, Hunter T. The regulatory crosstalk between kinases and proteases in cancer. Nat Rev Cancer. 2010;10:278–92.

  13. 13.

    Parrish AB, Freel CD, Kornbluth S. Cellular mechanisms controlling caspase activation and function. Cold Spring Harb Perspect Biol. 2013;5:1–24.

  14. 14.

    Dix MM, Simon GM, Wang C, Okerberg E, Patricelli MP, Cravatt BF. Functional interplay between caspase cleavage and phosphorylation sculpts the apoptotic proteome. Cell. 2012;150:426–40.

  15. 15.

    Allan LA, Clarke PR. Apoptosis and autophagy: regulation of caspase-9 by phosphorylation. FEBS J. 2009;276:6063–73.

  16. 16.

    Rossi AG, Cousin JM, Dransfield I, Lawson MF, Chilvers ER, Haslett C. Agents that elevate camp inhibit human neutrophil apoptosis. Biochem Biophys Res Commun. 1995;217:892–9.

  17. 17.

    Martin MC, Dransfield I, Haslett C, Rossi AG. Cyclic AMP regulation of neutrophil apoptosis occurs via a novel protein kinase A-independent signaling pathway. J Biol Chem. 2001;276:45041–50.

  18. 18.

    Orlov SN, Thorin-Trescases N, Dulin NO, Dam T-V, Fortuno MA, Tremblay J, et al. Activation of cAMP signaling transiently inhibits apoptosis in vascular smooth muscle cells in a site upstream of caspase-3. Cell Death Differ. 1999;6:661–72.

  19. 19.

    Insel PA, Zhang L, Murray F, Yokouchi H, Zambon AC. Cyclic AMP is both a pro-apoptotic and anti-apoptotic second messenger. Acta Physiol. 2012;204:277–87.

  20. 20.

    Martin MC, Allan LA, Lickrish M, Sampson C, Morrice N, Clarke PR. Protein kinase A regulates caspase-9 activation by Apaf-1 downstream of cytochrome c. J Biol Chem. 2005;280:15449–55.

  21. 21.

    Lienhard GE. Non-functional phosphorylations? Trends Biochem Sci. 2008;33:351–2.

  22. 22.

    Landry CR, Levy ED, Michnick SW. Weak functional constraints on phosphoproteomes. Trends Genet. 2009;25:193–7.

  23. 23.

    Velázquez-Delgado EM, Hardy JA. Phosphorylation regulates assembly of the caspase-6 substrate-binding groove. Structure. 2012;20:742–51.

  24. 24.

    Eron SJ, Raghupathi K, Hardy JA. Dual site phosphorylation of caspase-7 by PAK2 blocks apoptotic activity by two distinct mechanisms. Structure. 2017;25:27–39.

  25. 25.

    Serrano BP, Szydlo HS, Alfandari DR, Hardy JA. Active-site adjacent phosphorylation at Tyr-397 by c-Abl kinase inactivates caspase-9. J Biol Chem. 2017;292:21352–21365: jbc.M117.811976.

  26. 26.

    Cao Q, Wang X-J, Liu C-W, Liu D-F, Li L-F, Gao Y-Q, et al. Inhibitory mechanism of caspase-6 phosphorylation revealed by crystal structures, molecular dynamics simulations, and biochemical assays. J Biol Chem. 2012;287:15371–9.

  27. 27.

    Zamaraev AV, Kopeina GS, Prokhorova EA, Zhivotovsky B, Lavrik IN. Post-translational modification of caspases: the other side of apoptosis regulation. Trends Cell Biol. 2017;27:322–39.

  28. 28.

    Boatright KM, Renatus M, Scott FL, Sperandio S, Shin H, Pedersen IM, et al. A unified model for apical caspase activation. Mol Cell. 2003;11:529–41.

  29. 29.

    Stennicke HR, Deveraux QL, Humke EW, Reed JC, Dixit VM, Salvesen GS. Caspase-9 can be activated without proteolytic processing. J Biol Chem. 1999;9:8359–62.

  30. 30.

    Pirman NL, Barber KW, Aerni HR, Ma NJ, Haimovich AD, Rogulina S, et al. A flexible codon in genomically recoded Escherichia coli permits programmable protein phosphorylation. Nat Commun. 2015;6:8130.

  31. 31.

    McDonnell MA, Wang D, Khan SM, Vander Heiden MG, Kelekar A. Caspase-9 is activated in a cytochrome c-independent manner early during TNFα-induced apoptosis in murine cells. Cell Death Differ. 2003;10:1005–15.

  32. 32.

    Gyrd-Hansen M, Farkas T, Fehrenbacher N, Bastholm L, Hoyer-Hansen M, Elling F, et al. Apoptosome-independent activation of the lysosomal cell death pathway by caspase-9. Mol Cell Biol. 2006;26:7880–91.

  33. 33.

    Fujita E, Egashira J, Urase K, Kuida K, Momoi T. Caspase-9 processing by caspase-3 via a feedback amplification loop in vivo. Cell Death Differ. 2001;8:335–44.

  34. 34.

    Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, et al. Ordering the cytochrome c-initiated caspase cascade: Hierarchical activation of caspases-2,-3,-6,-7,-8, and -10 in a caspase-9-dependent manner. J Cell Biol. 1999;144:281–92.

  35. 35.

    Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Alnemri ES. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell. 1998;1:949–57.

  36. 36.

    Fuentes-Prior P, Salvesen GS. The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem J. 2004;384:201–32.

  37. 37.

    Chiti F, Dobson CM. Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem. 2006;75:333–66.

  38. 38.

    Biancalana M, Koide S. Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochim Biophys Acta. 2010;1804:1405–12.

  39. 39.

    Groenning M. Binding mode of Thioflavin T and other molecular probes in the context of amyloid fibrils-current status. J Chem Biol. 2010;3:1–18.

  40. 40.

    McStay GP, Salvesen GS, Green DR. Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways. Cell Death Differ. 2008;15:322–31.

  41. 41.

    Caretta A, Mucignat-Caretta C. Protein kinase a in cancer. Cancers. 2011;3:913–26.

  42. 42.

    Sapio L, Di Maiolo F, Illiano M, Esposito A, Chiosi E, Spina A, et al. Targeting protein kinase A in cancer therapy: an update. EXCLI J. 2014;13:843–55.

  43. 43.

    Nesterova MV, Cho-Chung YS. Significance of protein kinase A in cancer. In: Apoptosis, cell signaling, and human diseases. Rakesh Srivastava, Totowa, NJ: Humana Press; 2006. pp 3–30.

  44. 44.

    Mitrea DM, Kriwacki RW. Regulated unfolding of proteins in signaling. FEBS Lett. 2013;587:1081–8.

  45. 45.

    Schultz JE, Natarajan J. Regulated unfolding: a basic principle of intraprotein signaling in modular proteins. Trends Biochem Sci. 2013;38:538–45.

  46. 46.

    Ross CA, Poirier MA. Protein aggregation and neurodegenerative disease. Nat Med. 2004;10:S10–7.

  47. 47.

    Aguzzi A, O’Connor T. Protein aggregation diseases: pathogenicity and therapeutic perspectives. Nat Rev Drug Discov. 2010;9:237–48.

  48. 48.

    Tenreiro S, Eckermann K, Outeiro TF. Protein phosphorylation in neurodegeneration: friend or foe? Front Mol Neurosci. 2014;7:42.

  49. 49.

    Kumar S, Walter J. Phosphorylation of amyloid beta peptides-A trigger for formation of toxic aggregates in Alzheimer’s disease. Aging. 2011;3:803–12.

  50. 50.

    Sato H, Kato T, Arawaka S. The role of Ser129 phosphorylation of α-synuclein in neurodegeneration of Parkinson’s disease: a review of in vivo models. Rev Neurosci. 2013;24:115–23.

  51. 51.

    Samuel F, Flavin WP, Iqbal S, Pacelli C, Sri Renganathan SD, Trudeau L-E, et al. Effects of serine 129 phosphorylation on α-synuclein aggregation, membrane association, and internalization. J Biol Chem. 2016;291:4374–85.

  52. 52.

    Fowler DM, Koulov AV, Balch WE, Kelly JW. Functional amyloid–from bacteria to humans. Trends Biochem Sci. 2007;35:217–224. https://doi.org/10.1016/j.tibs.2007.03.003.

  53. 53.

    Lin S-C, Lo Y-C, Wu H. Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling. Nature. 2010;465:885–90.

  54. 54.

    Qiao Q, Yang C, Zheng C, Fontá L, David L, Yu X, et al. Structural architecture of the CARMA1/Bcl10/MALT1 signalosome: nucleation-induced filamentous assembly. Mol Cell. 2013;51:766–79.

  55. 55.

    Siegel RM, Martin DA, Zheng L, Ng SY, Bertin J, Cohen J, et al. Death-effector filaments: novel cytoplasmic structures that recruit caspases and trigger apoptosis. J Cell Biol. 1998;141:1243–53.

  56. 56.

    Li J, McQuade T, Siemer AB, Napetschnig J, Moriwaki K, Hsiao Y-S, et al. The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell. 2012;150:339–50.

  57. 57.

    Hou F, Sun L, Zheng H, Skaug B, Jiang Q-X, Chen ZJ. MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response. Cell. 2011;146:448–61.

  58. 58.

    Berson JF, Harper DC, Tenza D, Raposo G, Marks MS. Pmel17 initiates premelanosome morphogenesis within multivesicular bodies. Mol Biol Cell. 2001;12:3451–64.

  59. 59.

    Fowler DM, Koulov AV, Alory-Jost C, Marks MS, Balch WE, Kelly JW. Functional amyloid formation within mammalian tissue. PLoS Biol. 2006;4:e6. https://doi.org/10.1371/journal.pbio.0040006.

  60. 60.

    Lu A, Li Y, Schmidt FI, Yin Q, Chen S, Fu T-M, et al. Molecular basis of caspase-1 polymerization and its inhibition by a new capping mechanism. Nat Struct Mol Biol. 2016;23:1–12.

  61. 61.

    Masumoto J, Taniguchi S, Ayukawa K, Sarvotham H, Kishino T, Niikawa N, et al. ASC, a Novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 Cells. J Biol Chem. 1999;274:33835–8.

  62. 62.

    Westphal D, Kluck RM, Dewson G. Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis. Cell Death Differ. 2014;21:196–205.

  63. 63.

    Zhou Q, Snipas S, Orth K, Muzio M, Dixit VM, Salvesen GS. Target protease specificity of the viral serpin CrmA: analysis of five caspases. J Biol Chem. 1997;272:7797–800.

  64. 64.

    Narayana N, Cox S, Shaltiel S, Taylor SS, Xuong N. Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of cAMP-dependent protein kinase complexed with the peptide inhibitor PKI(5-24) and adenosine. Biochemistry. 1997;36:4438–48.

  65. 65.

    Slice LW, Taylor SS. Expression of the catalytic subunit of CAMP-dependent protein kinase. J Biol Chem. 1989;264:20940–6.

  66. 66.

    Yonemoto W, McGlone ML, Grant B, Taylor SS. Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli. Protein Eng. 1997;10:915–25.

Download references

Acknowledgements

This work was supported by the National Institutes of Health (GM 080532) to JH. BS was supported in part by the UMass Chemistry-Biology Interface Training Program (National Research Service Award T32 GM 08515 from the National Institutes of Health). We thank Jesse Rinehart (Yale University) for generously providing the E. coli strain C321.ΔA, the pBAD-GST-AmpR and SepOTSλ plasmids and for advice and helpful discussions about phosphoprotein synthesis. We thank Alex Ribbe, Director of the UMass Electron Microscopy facility for the collection of EM images. We thank Tyler Marcinko for his assistance with staining of protein samples and ThT assay. We also thank Scott Eron for providing casp-8 proteins as samples for caspase cleavage assays.

Author information

Affiliations

  1. Department of Chemistry, University of Massachusetts, 104 LGRT, 710 N. Pleasant Street, Amherst, MA, 01003, USA

    • Banyuhay P. Serrano
    •  & Jeanne A. Hardy

Authors

  1. Search for Banyuhay P. Serrano in:

  2. Search for Jeanne A. Hardy in:

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Jeanne A. Hardy.

Electronic supplementary material

About this article

Publication history

Received

Revised

Accepted

Published

DOI

https://doi.org/10.1038/s41418-017-0052-9