Advertisement

Comprehensive Characterization of the Recombinant Catalytic Subunit of cAMP-Dependent Protein Kinase by Top-Down Mass Spectrometry

  • Zhijie Wu
  • Yutong Jin
  • Bifan Chen
  • Morgan K. Gugger
  • Chance L. Wilkinson-Johnson
  • Timothy N. Tiambeng
  • Song Jin
  • Ying GeEmail author
Focus: 34th Asilomar Conference, Quantitative Analysis of PTMs: Research Article

Abstract

Reversible phosphorylation plays critical roles in cell growth, division, and signal transduction. Kinases which catalyze the transfer of γ-phosphate groups of nucleotide triphosphates to their substrates are central to the regulation of protein phosphorylation and are therefore important therapeutic targets. Top-down mass spectrometry (MS) presents unique opportunities to study protein kinases owing to its capabilities in comprehensive characterization of proteoforms that arise from alternative splicing, sequence variations, and post-translational modifications. Here, for the first time, we developed a top-down MS method to characterize the catalytic subunit (C-subunit) of an important kinase, cAMP-dependent protein kinase (PKA). The recombinant PKA C-subunit was expressed in Escherichia coli and successfully purified via his-tag affinity purification. By intact mass analysis with high resolution and high accuracy, four different proteoforms of the affinity-purified PKA C-subunit were detected, and the most abundant proteoform was found containing seven phosphorylations with the removal of N-terminal methionine. Subsequently, the seven phosphorylation sites of the most abundant PKA C-subunit proteoform were characterized simultaneously using tandem MS methods. Four sites were unambiguously identified as Ser10, Ser11, Ser18, and Ser30, and the remaining phosphorylation sites were localized to Ser2/Ser3, Ser358/Thr368, and Thr[215-224]Tyr in the PKA C-subunit sequence with a 20mer 6xHis-tag added at the N-terminus. Interestingly, four of these seven phosphorylation sites were located at the 6xHis-tag. Furthermore, we have performed dephosphorylation reaction by Lambda protein phosphatase and showed that all phosphorylations of the recombinant PKA C-subunit phosphoproteoforms were removed by this phosphatase.

Keywords

Post-translational modifications Top-down mass spectrometry Protein kinases 

Notes

Acknowledgements

The authors would like to thank Dr. Wenxuan Cai for preparing the construct for PKA C-subunit for bacterial expression. Financial support was provided by NIH R01 GM117058 (to S. J. and Y. G.) and R01 GM125085 (to Y. G.). Y. G. also would like to acknowledge the NIH grants, R01 HL096971, R01 HL109810, and S10 OD018475.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

13361_2019_2341_MOESM1_ESM.pdf (756 kb)
ESM 1 (PDF 755 kb)

References

  1. 1.
    Hunter, T.: Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell. 80, 225–236 (1995)PubMedCrossRefGoogle Scholar
  2. 2.
    Olsen, J.V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P., Mann, M.: Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell. 127, 635–648 (2006)PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Grimes, M., Hall, B., Foltz, L., Levy, T., Rikova, K., Gaiser, J., Cook, W., Smirnova, E., Wheeler, T., Clark, N.R., Lachmann, A., Zhang, B., Hornbeck, P., Ma'ayan, A., Comb, M.: Integration of protein phosphorylation, acetylation, and methylation data sets to outline lung cancer signaling networks. Sci. Signal. 11, 531 (2018)CrossRefGoogle Scholar
  4. 4.
    Hanger, D.P., Anderton, B.H., Noble, W.: Tau phosphorylation: the therapeutic challenge for neurodegenerative disease. Trends Mol. Med. 15, 112–119 (2009)PubMedCrossRefGoogle Scholar
  5. 5.
    Peng, Y., Gregorich, Z.R., Valeja, S.G., Zhang, H., Cai, W., Chen, Y.C., Guner, H., Chen, A.J., Schwahn, D.J., Hacker, T.A., Liu, X., Ge, Y.: Top-down proteomics reveals concerted reductions in myofilament and Z-disc protein phosphorylation after acute myocardial infarction. Mol. Cell. Proteomics. 13, 2752–2764 (2014)PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Dong, X.T., Sumandea, C.A., Chen, Y.C., Garcia-Cazarin, M.L., Zhang, J., Balke, C.W., Sumandea, M.P., Ge, Y.: Augmented phosphorylation of cardiac troponin I in hypertensive heart failure. J. Biol. Chem. 287, 848–857 (2012)PubMedCrossRefGoogle Scholar
  7. 7.
    Zhang, J., Guy, M.J., Norman, H.S., Chen, Y.C., Xu, Q.G., Dong, X.T., Guner, H., Wang, S.J., Kohmoto, T., Young, K.H., Moss, R.L., Ge, Y.: Top-down quantitative proteomics identified phosphorylation of cardiac troponin I as a candidate biomarker for chronic heart failure. J. Proteome Res. 10, 4054–4065 (2011)PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Chen, I.H., Xue, L., Hsu, C.C., Paez, J.S.P., Pan, L., Andaluz, H., Wendt, M.K., Iliuk, A.B., Zhu, J.K., Tao, W.A.: Phosphoproteins in extracellular vesicles as candidate markers for breast cancer. Proc. Natl. Acad. Sci. U. S. A. 114, 3175–3180 (2017)PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Manning, G., Whyte, D.B., Martinez, R., Hunter, T., Sudarsanam, S.: The protein kinase complement of the human genome. Science. 298, 1912–1934 (2002)PubMedCrossRefGoogle Scholar
  10. 10.
    Hanks, S.K., Quinn, A.M., Hunter, T.: The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 241, 42–52 (1988)PubMedCrossRefGoogle Scholar
  11. 11.
    Sacco, F., Silvestri, A., Posca, D., Pirro, S., Gherardini, P.F., Castagnoli, L., Mann, M., Cesareni, G.: Deep proteomics of breast cancer cells reveals that metformin rewires signaling networks away from a pro-growth state. Cell Syst. 2, 159–171 (2016)PubMedCrossRefGoogle Scholar
  12. 12.
    Dhillon, A.S., Hagan, S., Rath, O., Kolch, W.: MAP kinase signalling pathways in cancer. Oncogene. 26, 3279–3290 (2007)PubMedCrossRefGoogle Scholar
  13. 13.
    Chatterjee, K.: Neurohormonal activation in congestive heart failure and the role of vasopressin. Am. J. Cardiol. 95, 8b–13b (2005)PubMedCrossRefGoogle Scholar
  14. 14.
    Vlahos, C.J., McDowell, S.A., Clerk, A.: Kinases as therapeutic targets for heart failure. Nat. Rev. Drug Discov. 2, 99–113 (2003)PubMedCrossRefGoogle Scholar
  15. 15.
    Zhang, J.M., Yang, P.L., Gray, N.S.: Targeting cancer with small molecule kinase inhibitors. Nat. Rev. Cancer. 9, 28–39 (2009)PubMedCrossRefGoogle Scholar
  16. 16.
    Ferguson, F.M., Gray, N.S.: Kinase inhibitors: the road ahead. Nat. Rev. Drug Discov. 17, 353–376 (2018)PubMedCrossRefGoogle Scholar
  17. 17.
    Roskoski Jr., R.: ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol. Res. 66, 105–143 (2012)PubMedCrossRefGoogle Scholar
  18. 18.
    Steichen, J.M., Iyer, G.H., Li, S., Saldanha, S.A., Deal, M.S., Woods Jr., V.L., Taylor, S.S.: Global consequences of activation loop phosphorylation on protein kinase A. J. Biol. Chem. 285, 3825–3832 (2010)PubMedCrossRefGoogle Scholar
  19. 19.
    Yonemoto, W., McGlone, M.L., Grant, B., Taylor, S.S.: Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli. Protein Eng. 10, 915–925 (1997)PubMedCrossRefGoogle Scholar
  20. 20.
    Wheeler-Jones, C.P.: Cell signalling in the cardiovascular system: an overview. Heart. 91, 1366–1374 (2005)PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Ruehr, M.L., Russell, M.A., Ferguson, D.G., Bhat, M., Ma, J., Damron, D.S., Scott, J.D., Bond, M.: Targeting of protein kinase A by muscle A kinase-anchoring protein (mAKAP) regulates phosphorylation and function of the skeletal muscle ryanodine receptor. J. Biol. Chem. 278, 24831–24836 (2003)PubMedCrossRefGoogle Scholar
  22. 22.
    Bauman, A.L., Scott, J.D.: Kinase- and phosphatase-anchoring proteins: harnessing the dynamic duo. Nat. Cell Biol. 4, E203–E206 (2002)PubMedCrossRefGoogle Scholar
  23. 23.
    Taylor, S.S., Knighton, D.R., Zheng, J.H., Teneyck, L.F., Sowadski, J.M.: Structural framework for the protein-kinase family. Annu. Rev. Cell Biol. 8, 429–462 (1992)PubMedCrossRefGoogle Scholar
  24. 24.
    Yonemoto, W., Garrod, S.M., Bell, S.M., Taylor, S.S.: Identification of phosphorylation sites in the recombinant catalytic subunit of cAMP-dependent protein kinase. J. Biol. Chem. 268, 18626–18632 (1993)PubMedGoogle Scholar
  25. 25.
    Byrne, D.P., Vonderach, M., Ferries, S., Brownridge, P.J., Eyers, C.E., Eyers, P.A.: cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility-mass spectrometry. Biochem. J. 473, 3159–3175 (2016)PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Roy, J., Cyert, M.S.: Cracking the phosphatase code: docking interactions determine substrate specificity. Sci. Signal. 2, re9 (2009)PubMedCrossRefGoogle Scholar
  27. 27.
    Toby, T.K., Fornelli, L., Kelleher, N.L.: Progress in top-down proteomics and the analysis of proteoforms. Annu Rev Anal Chem (Palo Alto, Calif). 9, 499–519 (2016)CrossRefGoogle Scholar
  28. 28.
    Cai, W.X., Tucholski, T.M., Gregorich, Z.R., Ge, Y.: Top-down proteomics: technology advancements and applications to heart diseases. Expert Rev. Proteomics. 13, 717–730 (2016)PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Chen, B., Brown, K.A., Lin, Z., Ge, Y.: Top-down proteomics: ready for prime time? Anal. Chem. 90, 110–127 (2018)PubMedCrossRefGoogle Scholar
  30. 30.
    Tran, J.C., Zamdborg, L., Ahlf, D.R., Lee, J.E., Catherman, A.D., Durbin, K.R., Tipton, J.D., Vellaichamy, A., Kellie, J.F., Li, M.X., Wu, C., Sweet, S.M.M., Early, B.P., Siuti, N., LeDuc, R.D., Compton, P.D., Thomas, P.M., Kelleher, N.L.: Mapping intact protein isoforms in discovery mode using top-down proteomics. Nature. 480, 254–U141 (2011)PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Brown, K.A., Chen, B., Guardado-Alvarez, T.M., Lin, Z., Hwang, L., Ayaz-Guner, S., Jin, S., Ge, Y.: A photocleavable surfactant for top-down proteomics. Nat. Methods. 16, 417–420 (2019)PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Chen, B., Hwang, L., Ochowicz, W., Lin, Z., Guardado-Alvarez, T.M., Cai, W., Xiu, L., Dani, K., Colah, C., Jin, S., Ge, Y.: Coupling functionalized cobalt ferrite nanoparticle enrichment with online LC/MS/MS for top-down phosphoproteomics. Chem. Sci. 8, 4306–4311 (2017)PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Roberts, D.S., Chen, B.F., Tiambeng, T.N., Wu, Z.J., Ge, Y., Jin, S.: Reproducible large-scale synthesis of surface silanized nanoparticles as an enabling nanoproteomics platform: enrichment of the human heart phosphoproteome. Nano Res. 12, 1473–1481 (2019)PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Ge, Y., Rybakova, I.N., Xu, Q.G., Moss, R.L.: Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state. Proc. Natl. Acad. Sci. U. S. A. 106, 12658–12663 (2009)PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Major, L.L., Denton, H., Smith, T.K.: Coupled enzyme activity and thermal shift screening of the maybridge rule of 3 fragment library against Trypanosoma brucei choline kinase; a genetically validated drug target. In: El-Shemy, H.A. (ed.) Rijeka (HR) (2013)Google Scholar
  36. 36.
    Smith, L.M., Kelleher, N.L.: Proteoforms as the next proteomics currency. Science. 359, 1106–1107 (2018)PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Aebersold, R., Agar, J.N., Amster, I.J., Baker, M.S., Bertozzi, C.R., Boja, E.S., Costello, C.E., Cravatt, B.F., Fenselau, C., Garcia, B.A., Ge, Y., Gunawardena, J., Hendrickson, R.C., Hergenrother, P.J., Huber, C.G., Ivanov, A.R., Jensen, O.N., Jewett, M.C., Kelleher, N.L., Kiessling, L.L., Krogan, N.J., Larsen, M.R., Loo, J.A., Loo, R.R.O., Lundberg, E., MacCoss, M.J., Mallick, P., Mootha, V.K., Mrksich, M., Muir, T.W., Patrie, S.M., Pesavento, J.J., Pitteri, S.J., Rodriguez, H., Saghatelian, A., Sandoval, W., Schluter, H., Sechi, S., Slavoff, S.A., Smith, L.M., Snyder, M.P., Thomas, P.M., Uhlen, M., Van Eyk, J.E., Vidal, M., Walt, D.R., White, F.M., Williams, E.R., Wohlschlager, T., Wysocki, V.H., Yates, N.A., Young, N.L., Zhang, B.: How many human proteoforms are there? Nat. Chem. Biol. 14, 206–214 (2018)PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Narayana, N., Cox, S., Shaltiel, S., Taylor, S.S., 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. 36, 4438–4448 (1997)PubMedCrossRefPubMedCentralGoogle Scholar
  39. 39.
    Wu, Z., Tiambeng, T.N., Cai, W., Chen, B., Lin, Z., Gregorich, Z.R., Ge, Y.: Impact of phosphorylation on the mass spectrometry quantification of intact phosphoproteins. Anal. Chem. 90, 4935–4939 (2018)PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Chen, Y.C., Ayaz-Guner, S., Peng, Y., Lane, N.M., Locher, M., Kohmoto, T., Larsson, L., Moss, R.L., Ge, Y.: Effective top-down LC/MS+ method for assessing actin isoforms as a potential cardiac disease marker. Anal. Chem. 87, 8399–8406 (2015)PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Cai, W., Guner, H., Gregorich, Z.R., Chen, A.J., Ayaz-Guner, S., Peng, Y., Valeja, S.G., Liu, X., Ge, Y.: MASH suite pro: a comprehensive software tool for top-down proteomics. Mol. Cell. Proteomics. 15, 703–714 (2016)PubMedCrossRefGoogle Scholar
  42. 42.
    Hirel, P.H., Schmitter, M.J., Dessen, P., Fayat, G., Blanquet, S.: Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc. Natl. Acad. Sci. U. S. A. 86, 8247–8251 (1989)PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Herberg, F.W., Bell, S.M., Taylor, S.S.: Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli: multiple isozymes reflect different phosphorylation states. Protein Eng. 6, 771–777 (1993)PubMedCrossRefGoogle Scholar
  44. 44.
    Siuti, N., Kelleher, N.L.: Decoding protein modifications using top-down mass spectrometry. Nat. Methods. 4, 817–821 (2007)PubMedPubMedCentralCrossRefGoogle Scholar
  45. 45.
    Haydon, C.E., Eyers, P.A., Aveline-Wolf, L.D., Resing, K.A., Maller, J.L., Ahn, N.G.: Identification of novel phosphorylation sites on Xenopus laevis Aurora A and analysis of phosphopeptide enrichment by immobilized metal-affinity chromatography. Mol. Cell. Proteomics. 2, 1055–1067 (2003)PubMedCrossRefGoogle Scholar
  46. 46.
    Iakoucheva, L.M., Radivojac, P., Brown, C.J., O'Connor, T.R., Sikes, J.G., Obradovic, Z., Dunker, A.K.: The importance of intrinsic disorder for protein phosphorylation. Nucleic Acids Res. 32, 1037–1049 (2004)PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Barraud, P., Banerjee, S., Mohamed, W.I., Jantsch, M.F., Allain, F.H.: A bimodular nuclear localization signal assembled via an extended double-stranded RNA-binding domain acts as an RNA-sensing signal for transportin 1. Proc. Natl. Acad. Sci. U. S. A. 111, E1852–E1861 (2014)PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Comstock, M.J., Whitley, K.D., Jia, H., Sokoloski, J., Lohman, T.M., Ha, T., Chemla, Y.R.: Protein structure. Direct observation of structure-function relationship in a nucleic acid-processing enzyme. Science. 348, 352–354 (2015)PubMedPubMedCentralCrossRefGoogle Scholar
  49. 49.
    Waugh, D.S.: An overview of enzymatic reagents for the removal of affinity tags. Protein Expr. Purif. 80, 283–293 (2011)PubMedPubMedCentralCrossRefGoogle Scholar
  50. 50.
    Tholey, A., Pipkorn, R., Bossemeyer, D., Kinzel, V., Reed, J.: Influence of myristoylation, phosphorylation, and deamidation on the structural behavior of the N-terminus of the catalytic subunit of cAMP-dependent protein kinase. Biochemistry. 40, 225–231 (2001)PubMedCrossRefGoogle Scholar
  51. 51.
    Toner-Webb, J., van Patten, S.M., Walsh, D.A., Taylor, S.S.: Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase. J. Biol. Chem. 267, 25174–25180 (1992)PubMedGoogle Scholar
  52. 52.
    Meng, F.Y., Cargile, B.J., Miller, L.M., Forbes, A.J., Johnson, J.R., Kelleher, N.L.: Informatics and multiplexing of intact protein identification in bacteria and the archaea. Nat. Biotechnol. 19, 952–957 (2001)PubMedCrossRefGoogle Scholar
  53. 53.
    Zubarev, R.A., Horn, D.M., Fridriksson, E.K., Kelleher, N.L., Kruger, N.A., Lewis, M.A., Carpenter, B.K., McLafferty, F.W.: Electron capture dissociation for structural characterization of multiply charged protein cations. Anal. Chem. 72, 563–573 (2000)PubMedCrossRefGoogle Scholar
  54. 54.
    Holden, D.D., Sanders, J.D., Weisbrod, C.R., Mullen, C., Schwartz, J.C., Brodbelt, J.S.: Implementation of fragment ion protection (FIP) during ultraviolet photodissociation (UVPD) mass spectrometry. Anal. Chem. 90, 8583–8591 (2018)PubMedCrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  • Zhijie Wu
    • 1
  • Yutong Jin
    • 1
  • Bifan Chen
    • 1
  • Morgan K. Gugger
    • 1
  • Chance L. Wilkinson-Johnson
    • 1
  • Timothy N. Tiambeng
    • 1
  • Song Jin
    • 1
  • Ying Ge
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of ChemistryUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Department of Cell and Regenerative BiologyUniversity of Wisconsin-MadisonMadisonUSA
  3. 3.Human Proteomics ProgramUniversity of Wisconsin-MadisonMadisonUSA

Personalised recommendations