Skip to main content
SpringerLink
Log in

Pharmacologic inhibition of the CK2-mediated phosphorylation of B23/NPM in cancer cells selectively modulates genes related to protein synthesis, energetic metabolism, and ribosomal biogenesis

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

B23/NPM is a multifunctional nucleolar protein frequently overexpressed, mutated, or rearranged in neoplastic tissues. B23/NPM is involved in diverse biological processes and is mainly regulated by heteroligomer association and posttranslational modification, phosphorylation being a major posttranslational event. While the role of B23/NPM in supporting and/or driving malignant transformation is widely recognized, the particular relevance of its CK2-mediated phosphorylation remains unsolved. Interestingly, the pharmacologic inhibition of such phosphorylation event by CIGB-300, a clinical-grade peptide drug, was previously associated to apoptosis induction in tumor cell lines. In this work, we sought to identify the biological processes modulated by CIGB-300 in a lung cancer cell line using subtractive suppression hybridization and subsequent functional annotation clustering. Our results indicate that CIGB-300 modulates a subset of genes involved in protein synthesis (ES = 8.4, p < 0.001), mitochondrial ATP metabolism (ES = 2.5, p < 0.001), and ribosomal biogenesis (ES = 1.5, p < 0.05). The impairment of these cellular processes by CIGB-300 was corroborated at the molecular and cellular levels by Western blot (P-S6/P-4EBP1, translation), confocal microscopy (JC-1, mitochondrial potential), qPCR (45SrRNA/p21, ribosome biogenesis), and electron microscopy (nucleolar structure, ribosome biogenesis). Altogether, our findings provide new insights on the potential relevance of the CK2-mediated phosphorylation of B23/NPM in cancer cells, revealing at the same time the potentialities of its pharmacological manipulation for cancer therapy. Finally, this work also suggests several candidate gene biomarkers to be evaluated during the clinical development of the anti-CK2 peptide CIGB-300.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Grisendi S, Mecucci C, Falini B, Pandolfi PP (2006) Nucleophosmin and cancer. Nat Rev Cancer 6:493–505

    Article  CAS  PubMed  Google Scholar 

  2. Borer RA, Lehner CF, Eppenberger HM, Nigg EA (1989) Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell 56:379–390

    Article  CAS  PubMed  Google Scholar 

  3. Yun JP, Chew EC, Liew CT, Chan JY, Jin ML, Ding MX, Fai YH, Li HK, Liang XM, Wu QL (2003) Nucleophosmin/B23 is a proliferate shuttle protein associated with nuclear matrix. J Cell Biochem 90:1140–1148

    Article  CAS  PubMed  Google Scholar 

  4. Hingorani K, Szebeni A, Olson MOJ (2000) Mapping the functional domains of nucleolar protein B23. J Biol Chem 275:24451–24457

    Article  CAS  PubMed  Google Scholar 

  5. Murano TK, Okuwaki M, Hisaoka M, Nagata K (2008) Transcription regulation of the rRNA gene by a multifunctional nucleolar protein, B23/nucleophosmin, through its histone chaperone activity. Mol Cell Biol 28:3114–3126

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Wu MH, Yung BYM (2002) UV Stimulation of nucleophosmin/B23 expression is an immediate-early gene response induced by damaged DNA. J Biol Chem 277:48234–48240

    Article  CAS  PubMed  Google Scholar 

  7. Li J, Zhang X, Sejas DP, Bagby GC, Pang Q (2004) Hypoxia-induced nucleophosmin protects cell death through inhibition of p53. J Biol Chem 279:41275–41279

    Article  CAS  PubMed  Google Scholar 

  8. Zhang H, Shi X, Paddon H, Hampong M, Dai W, Pelech S (2004) B23/nucleophosmin serine 4 phosphorylation mediates mitotic functions of polo-like kinase 1. J Biol Chem 279:35726–35734

    Article  CAS  PubMed  Google Scholar 

  9. Takemura M, Sato K, Nishio M, Akiyama T, Umekawa H, Yoshida S (1999) Nucleolar protein B23.1 binds to retinoblastoma protein and synergistically stimulates DNA polymerase alpha activity. J Biochem 125:904–909

    Article  CAS  PubMed  Google Scholar 

  10. Dhar SK, Lynn BC, Daosukho C, St. Clair DK (2004) Identification of nucleophosmin as an NF-κB co-activator for the induction of the human SOD2 gene. J Biol Chem 279:28209–28219

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Okuwaki M (2008) The structure and functions of NPM1/nucleophsmin/B23, a multifunctional nucleolar acidic protein. J Biochem 222:1–7

    Google Scholar 

  12. Okuda M, Horn HF, Tarapore P, Tokuyama Y, Smulian AG, Chan PK, Knudsen ES, Hofmann IA, Snyder JD, Bove KE, Fukasawa K (2000) Nucleophosmin/B23 is a target of CDK2/cyclin E in centrosome duplication. Cell 103:127–140

    Article  CAS  PubMed  Google Scholar 

  13. Okuwaki M, Tsujimoto M, Nagata K (2002) The RNA binding activity of a ribosome biogenesis factor, nucleophosmin/B23, is modulated by phosphorylation with a cell cycle-dependent kinase and by association with its subtype. Mol Biol Cell 13:2016–2030

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Chan PK, Liu QR, Durban E (1990) The major phosphorylation site of nucleophosmin (B23) is phosphorylated by a nuclear kinase II. Biochem J 270:549–552

    PubMed Central  CAS  PubMed  Google Scholar 

  15. Jiang PS, Chang JH, Yung YB (2000) Different kinases phosphorylate nucleophosmin/B23 at different sites during G(2) and M phases of the cell cycle. Cancer Lett 153:151–160

    Article  CAS  PubMed  Google Scholar 

  16. Negi S, Olson MO (2006) Effects of interphase and mitotic phosphorylation on the mobility and location of nucleolar protein B23. J Cell Sci 401:616–620

    Google Scholar 

  17. Louvet E, Junera HR, Berthuy I, Hernandez-Verdun D (2006) Compartmentation of the nucleolar processing proteins in the granular component is a CK2-driven process. Mol Biol Cell 17:2537–2546

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Cozza G, Pinna LA, Moro S (2013) Kinase CK2 inhibition: an update. Curr Med Chem 20:671–693

    Article  CAS  PubMed  Google Scholar 

  19. Perea SE, Reyes O, Puchades Y, Mendoza O, Vispo NS, Torrens I, Santos A, Silva R, Acevedo B, López E, Falcón V, Alonso DF (2004) Antitumor effect of a novel proapoptotic peptide that impairs the fosforilación by the protein kinase 2 (casein kinase 2). Cancer Res 64:7127–7129

    Article  CAS  PubMed  Google Scholar 

  20. Perera Y, Farina HG, Gil J, Rodriguez A, Benavent F, Castellanos L, Gómez RE, Acevedo BE, Alonso DF, Perea SE (2009) Anticancer peptide CIGB-300 binds to nucleophosmin/B23, impairs its CK2-mediated phosphorylation, and leads to apoptosis through its nucleolar disassembly activity. Mol Cancer Ther 8:1189–1196

    Article  CAS  PubMed  Google Scholar 

  21. Perera Y, Costales HC, Diaz Y, Reyes O, Farina HG, Mendez L, Gómez RE, Acevedo BE, Gomez DE, Alonso DF, Perea SE (2012) Sensitivity of tumor cells towards CIGB-300 anticancer peptide relies on its nucleolar localization. J Pept Sci 18:215–223

    Article  CAS  PubMed  Google Scholar 

  22. Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci 93:6025–6030

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57

    Article  CAS  Google Scholar 

  24. Rodriguez-Ulloa A, Ramos Y, Gil J, Perera Y, Castellanos-Serra L, Garcia Y, Betancourt L, Besada V, Gonzalez LJ, Fernandez-de-Cossio J, Sanchez A, Serrano JM, Farina HG, Alonso DF, Acevedo BE, Padron G, Musacchio A, Perea SE (2010) Proteomic profile regulated by the anticancer peptide CIGB-300 in non-small cell lung cancer (NSCLC) cells. J Proteome Res 9:5473–5483

    Article  CAS  PubMed  Google Scholar 

  25. Boyd MR (1997) The NCI in vitro anticancer drug discovery screen: concept, implementation, and operation, 1985–1995, in anticancer drug development guide: preclinical screening, clinical trials, and approval. Humana Press, Totowa, pp 23–42

    Google Scholar 

  26. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386

    CAS  PubMed  Google Scholar 

  27. Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1:1559–1582

    Article  CAS  PubMed  Google Scholar 

  28. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:1–12

    Article  Google Scholar 

  29. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36

    Article  PubMed Central  PubMed  Google Scholar 

  30. Iskar M, Campillos M, Kuhn M, Jensen LJ, van Noort V, Bork P (2010) Drug-induced regulation of target expression. PLoS Comput Biol 6:e1000925

    Article  PubMed Central  PubMed  Google Scholar 

  31. Vassiliou GS, Cooper JL, Rad R, Li J, Rice S, Uren A, Rad L, Ellis P, Andrews R, Banerjee R, Grove C, Wang W, Liu P, Wright P, Arends M, Bradley A (2011) Mutant nucleophosmin and cooperating pathways drive leukemia initiation and progression in mice. Nat Genet 43:470–475

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Mupo A, Celani L, Dovey O, Cooper JL, Grove C, Rad R, Sportoletti P, Falini B, Bradley A, Vassiliou GS (2013) A powerful molecular synergy between mutant nucleophosmin and Flt3-ITD drives acute myeloid leukemia in mice. Leukemia 27:1917–1920

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Szebeni A, Hingorani K, Negi S, Olson MOJ (2003) Role of protein kinase CK2 phosphorylation in the molecular chaperone activity of nucleolar protein B23. J Biol Chem 278:9107–9115

    Article  CAS  PubMed  Google Scholar 

  34. Savkur RS, Olson MOJ (1998) Preferential cleavage in pre-ribosomal RNA by protein B23 endoribonuclease. Nucleic Acids Res 26:4508–4515

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Maggi LB, Kuchenruether M, Dadey DYA, Schwope RM, Grisendi S, Townsend RR, Pandolfi PP, Weber JD (2008) Nucleophosmin serves as a rate-limiting nuclear export chaperone for the Mammalian ribosome. Mol Cell Biol 28:7050–7065

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Li X, Rao V, Jin J, Guan B, Anderes KL, Bieberich CJ (2012) Identification and validation of inhibitor-responsive kinase substrates using a new paradigm to measure kinase-specific protein phosphorylation index. J Proteome Res 11:3637–3649

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Hsieh AC, Costa M, Zollo O, Davis C, Feldman ME, Testa JR, Meyuhas O, Shokat KM, Ruggero D (2010) Genetic dissection of the oncogenic mTOR pathway reveals druggable addiction to translational control via 4EBP-eIF4E. Cancer Cell 17:249–261

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Dennis PB, Fumagalli S, Thomas G (1999) Target of rapamycin (TOR): balancing the opposing forces of protein synthesis and degradation. Curr Opin Genet Dev 9:49–54

    Article  CAS  PubMed  Google Scholar 

  39. Meyuhas O (2000) Synthesis of the translational apparatus is regulated at the translational level. Eur J Biochem 267:6321–6330

    Article  CAS  PubMed  Google Scholar 

  40. Chedin S, Laferte A, Hoang T, Lafontaine DL, Riva M, Carles C (2007) Is ribosome synthesis controlled by pol I transcription? Cell Cycle 6:11–15

    Article  CAS  PubMed  Google Scholar 

  41. Opferman JT, Zambetti GP (2006) Translational research? ribosome integrity and a new p53 tumor suppressor checkpoint. Cell Death Differ 13:898–901

    Article  CAS  PubMed  Google Scholar 

  42. http://p53.iarc.fr/TP53GeneVariations.aspx?mutant=R249S. Accessed 9 Oct 2014

  43. Thomas G (2000) An encore for ribosome biogenesis in the control of cell proliferation. Nat Cell Biol 2:E71–E72

    Article  CAS  PubMed  Google Scholar 

  44. Schwanhausser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, Selbach M (2011) Global quantification of mammalian gene expression control. Nature 473:337–342

    Article  PubMed  Google Scholar 

  45. Meggio F, Pinna LA (2003) One-thousand-and-one substrates of protein kinase CK2. FASEB J 17:349–368

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by C.I.G.B. and Biorec: Grant CIGB-300.

Conflict of interest

The authors declare no conflict of interests.

Author information

Authors and Affiliations

  1. Laboratory of Molecular Oncology, Division of Pharmaceuticals, Center for Genetic Engineering and Biotechnology (CIGB), PO BOX 6162, CP10600, Havana, Cuba

    Yasser Perera & Silvio E. Perea

  2. Department of Genomics, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba

    Seidy Pedroso, Dania M. Vázquez & Adelaida Villareal

  3. Plant Division, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba

    Orlando Borras-Hidalgo

  4. Bioinformatics Department, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba

    Jamilet Miranda

  5. Chemical-Physical Division, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba

    Viviana Falcón

  6. Center for Advanced Studies of Cuba (CEAC), Carretera San Antonio, km 1-1/2, Puentes Grandes, Havana, Cuba

    Luis D. Cruz

  7. Laboratory of Molecular Oncology, Quilmes National University, R. Sáenz Peña 352, Bernal B1876BXD, Buenos Aires, Argentina

    Hernán G. Farinas

Authors
  1. Yasser Perera
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Seidy Pedroso
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Orlando Borras-Hidalgo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dania M. Vázquez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jamilet Miranda
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Adelaida Villareal
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Viviana Falcón
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Luis D. Cruz
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hernán G. Farinas
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Silvio E. Perea
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yasser Perera.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (XLSX 11 kb)

Supplementary material 2 (XLSX 10 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Perera, Y., Pedroso, S., Borras-Hidalgo, O. et al. Pharmacologic inhibition of the CK2-mediated phosphorylation of B23/NPM in cancer cells selectively modulates genes related to protein synthesis, energetic metabolism, and ribosomal biogenesis. Mol Cell Biochem 404, 103–112 (2015). https://doi.org/10.1007/s11010-015-2370-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-015-2370-x

Keywords

Search

Navigation