Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Acute Leukemias

Multiple forms of PKR present in the nuclei of acute leukemia cells represent an active kinase that is responsive to stress

Leukemia volume 25, pages 236–245 (2011)Cite this article

Subjects

Abstract

A number of cancers possess constitutive activity of the dsRNA-dependent kinase, PKR. Inhibition of PKR in these cancers leads to tumor cell death. We recently reported the increased presence of PKR phosphorylated on Thr451 (p-T451 PKR) in clinical samples from myelodysplastic syndrome (MDS) patients and acute leukemia cell lines. Whereas p-T451 PKR in low-risk patient samples or PTEN-positive acute leukemia cell lines was mostly cytoplasmic, in high-risk patient samples and acute leukemia cell lines deficient in PTEN, p-T451 PKR was mainly nuclear. As nuclear activity of PKR has not been previously characterized, we examined the status of nuclear PKR in acute leukemia cell lines. Using antibodies to N-terminus, C-terminus and the kinase domain in conjunction with a proteomics approach, we found that PKR exists in diverse molecular weight forms in the nucleus. Analysis of PKR transcripts by reverse transcriptase-PCR, and PKR-derived peptides by MS/MS revealed that these forms were the result of post-translational modifications (PTMs). Biochemical analysis demonstrated that nuclear PKR is an active kinase that can respond to stress. Given the association of PKR with PTEN and the Fanconi complex, these results indicate that PKR likely has other previously unrecognized roles in nuclear signaling that may contribute to leukemic development.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Roberts WK, Clemens MJ, Kerr IM . Interferon-induced inhibition of protein synthesis in L-cell extracts: an ATP-dependent step in the activation of an inhibitor by double-stranded RNA. Proc Natl Acad Sci USA 1976; 73: 3136–3140.

    Article  CAS  Google Scholar 

  2. Williams BR . PKR; a sentinel kinase for cellular stress. Oncogene 1999; 18: 6112–6120.

    Article  CAS  Google Scholar 

  3. Sadler AJ, Latchoumanin O, Hawkes D, Mak J, Williams BR . An antiviral response directed by PKR phosphorylation of the RNA helicase A. PLoS Pathog 2009; 5: e1000311.

    Article  Google Scholar 

  4. Blalock WL, Bavelloni A, Piazzi M, Faenza I, Cocco L . A role for PKR in hematologic malignancies. J Cell Physiol 2010; 223: 572–591.

    CAS  Google Scholar 

  5. Yoon CH, Miah MA, Kim KP, Bae YS . New Cdc2 Tyr 4 phosphorylation by dsRNA-activated protein kinase triggers Cdc2 polyubiquitination and G2 arrest under genotoxic stresses. EMBO Rep 2010; 11: 393–399.

    Article  CAS  Google Scholar 

  6. Nakamura T, Furuhashi M, Li P, Cao H, Tuncman G, Sonenberg N et al. Double-stranded RNA-dependent protein kinase links pathogen sensing with stress and metabolic homeostasis. Cell 2010; 140: 338–348.

    Article  CAS  Google Scholar 

  7. Mounir Z, Krishnamoorthy JL, Robertson GP, Scheuner D, Kaufman RJ, Georgescu MM et al. Tumor suppression by PTEN requires the activation of the PKR-eIF2alpha phosphorylation pathway. Sci Signal 2009; 2: ra85.

    Article  Google Scholar 

  8. Blalock WL, Grimaldi C, Fala F, Follo M, Horn S, Basecke J et al. PKR activity is required for acute leukemic cell maintenance and growth: a role for PKR-mediated phosphatase activity to regulate GSK-3 phosphorylation. J Cell Physiol 2009; 221: 232–241.

    Article  CAS  Google Scholar 

  9. Follo MY, Finelli C, Mongiorgi S, Clissa C, Bosi C, Martinelli G et al. PKR is activated in MDS patients and its subcellular localization depends on disease severity. Leukemia 2008; 22: 2267–2269.

    Article  CAS  Google Scholar 

  10. Kim SH, Gunnery S, Choe JK, Mathews MB . Neoplastic progression in melanoma and colon cancer is associated with increased expression and activity of the interferon-inducible protein kinase, PKR. Oncogene 2002; 21: 8741–8748.

    Article  CAS  Google Scholar 

  11. Kim SH, Forman AP, Mathews MB, Gunnery S . Human breast cancer cells contain elevated levels and activity of the protein kinase, PKR. Oncogene 2000; 19: 3086–3094.

    Article  CAS  Google Scholar 

  12. Basu S, Panayiotidis P, Hart SM, He LZ, Man A, Hoffbrand AV et al. Role of double-stranded RNA-activated protein kinase in human hematological malignancies. Cancer Res 1997; 57: 943–947.

    CAS  Google Scholar 

  13. Pataer A, Swisher SG, Roth JA, Logothetis CJ, Corn P . Inhibition of RNA-dependent protein kinase (PKR) leads to cancer cell death and increases chemosensitivity. Cancer Biol Ther 2009; 8: 245–252.

    Article  CAS  Google Scholar 

  14. Eley HL, McDonald PS, Russell ST, Tisdale MJ . Inhibition of activation of dsRNA-dependent protein kinase and tumour growth inhibition. Cancer Chemother Pharmacol 2009; 63: 651–659.

    Article  CAS  Google Scholar 

  15. Jeffrey IW, Kadereit S, Meurs EF, Metzger T, Bachmann M, Schwemmle M et al. Nuclear localization of the interferon-inducible protein kinase PKR in human cells and transfected mouse cells. Exp Cell Res 1995; 218: 17–27.

    Article  CAS  Google Scholar 

  16. Paquet C, Bose A, Polivka M, Peoc′h K, Brouland JP, Keohane C et al. Neuronal Phosphorylated RNA-Dependent Protein Kinase in Creutzfeldt-Jakob Disease. J Neuropathol Exp Neurol 2009; 68: 190–198.

    Article  CAS  Google Scholar 

  17. Follo MY, Mongiorgi S, Bosi C, Cappellini A, Finelli C, Chiarini F et al. The Akt/mammalian target of rapamycin signal transduction pathway is activated in high-risk myelodysplastic syndromes and influences cell survival and proliferation. Cancer Res 2007; 67: 4287–4294.

    Article  CAS  Google Scholar 

  18. Nyakern M, Tazzari PL, Finelli C, Bosi C, Follo MY, Grafone T et al. Frequent elevation of Akt kinase phosphorylation in blood marrow and peripheral blood mononuclear cells from high-risk myelodysplastic syndrome patients. Leukemia 2006; 20: 230–238.

    Article  CAS  Google Scholar 

  19. Zhang F, Romano PR, Nagamura-Inoue T, Tian B, Dever TE, Mathews MB et al. Binding of double-stranded RNA to protein kinase PKR is required for dimerization and promotes critical autophosphorylation events in the activation loop. J Biol Chem 2001; 276: 24946–24958.

    Article  CAS  Google Scholar 

  20. Romano PR, Garcia-Barrio MT, Zhang X, Wang Q, Taylor DR, Zhang F et al. Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2alpha kinases PKR and GCN2. Mol Cell Biol 1998; 18: 2282–2297.

    Article  CAS  Google Scholar 

  21. Su Q, Wang S, Baltzis D, Qu LK, Wong AH, Koromilas AE . Tyrosine phosphorylation acts as a molecular switch to full-scale activation of the eIF2alpha RNA-dependent protein kinase. Proc Natl Acad Sci USA 2006; 103: 63–68.

    Article  CAS  Google Scholar 

  22. Dey M, Cao C, Dar AC, Tamura T, Ozato K, Sicheri F et al. Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition. Cell 2005; 122: 901–913.

    Article  CAS  Google Scholar 

  23. Dephoure N, Zhou C, Villen J, Beausoleil SA, Bakalarski CE, Elledge SJ et al. A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci USA 2008; 105: 10762–10767.

    Article  CAS  Google Scholar 

  24. Taylor DR, Tian B, Romano PR, Hinnebusch AG, Lai MM, Mathews MB . Hepatitis C virus envelope protein E2 does not inhibit PKR by simple competition with autophosphorylation sites in the RNA-binding domain. J Virol 2001; 75: 1265–1273.

    Article  CAS  Google Scholar 

  25. Bennett RL, Blalock WL, Abtahi DM, Pan Y, Moyer SA, May WS . RAX, the PKR activator, sensitizes cells to inflammatory cytokines, serum withdrawal, chemotherapy, and viral infection. Blood 2006; 108: 821–829.

    Article  CAS  Google Scholar 

  26. McCubrey JA, Steelman LS, Hoyle PE, Blalock WL, Weinstein-Oppenheimer C, Franklin RA et al. Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells. Leukemia 1998; 12: 1903–1929.

    Article  CAS  Google Scholar 

  27. Liu TC, Lin PM, Chang JG, Lee JP, Chen TP, Lin SF . Mutation analysis of PTEN/MMAC1 in acute myeloid leukemia. Am J Hematol 2000; 63: 170–175.

    Article  CAS  Google Scholar 

  28. Dahia PL, Aguiar RC, Alberta J, Kum JB, Caron S, Sill H et al. PTEN is inversely correlated with the cell survival factor Akt/PKB and is inactivated via multiple mechanismsin haematological malignancies. Hum Mol Genet 1999; 8: 185–193.

    Article  CAS  Google Scholar 

  29. Sakai A, Thieblemont C, Wellmann A, Jaffe ES, Raffeld M . PTEN gene alterations in lymphoid neoplasms. Blood 1998; 92: 3410–3415.

    CAS  Google Scholar 

  30. Li S, Koromilas AE . Dominant negative function by an alternatively spliced form of the interferon-inducible protein kinase PKR. J Biol Chem 2001; 276: 13881–13890.

    Article  CAS  Google Scholar 

  31. Jammi NV, Whitby LR, Beal PA . Small molecule inhibitors of the RNA-dependent protein kinase. Biochem Biophys Res Commun 2003; 308: 50–57.

    Article  CAS  Google Scholar 

  32. Onuki R, Bando Y, Suyama E, Katayama T, Kawasaki H, Baba T et al. An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer′s disease. Embo J 2004; 23: 959–968.

    Article  CAS  Google Scholar 

  33. Zhang X, Li J, Sejas DP, Rathbun KR, Bagby GC, Pang Q . The Fanconi anemia proteins functionally interact with the protein kinase regulated by RNA (PKR). J Biol Chem 2004; 279: 43910–43919.

    Article  CAS  Google Scholar 

  34. von Holzen U, Pataer A, Raju U, Bocangel D, Vorburger SA, Liu Y et al. The double-stranded RNA-activated protein kinase mediates radiation resistance in mouse embryo fibroblasts through nuclear factor kappaB and Akt activation. Clin Cancer Res 2007; 13: 6032–6039.

    Article  CAS  Google Scholar 

  35. Planchon SM, Waite KA, Eng C . The nuclear affairs of PTEN. J Cell Sci 2008; 121: 249–253.

    Article  CAS  Google Scholar 

  36. Fan C, He L, Kapoor A, Rybak AP, De Melo J, Cutz JC et al. PTEN inhibits BMI1 function independently of its phosphatase activity. Mol Cancer 2009; 8: 98.

    Article  Google Scholar 

  37. Garcia MA, Collado M, Munoz-Fontela C, Matheu A, Marcos-Villar L, Arroyo J et al. Antiviral action of the tumor suppressor ARF. Embo J 2006; 25: 4284–4292.

    Article  CAS  Google Scholar 

  38. Zhao C, Denison C, Huibregtse JM, Gygi S, Krug RM . Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways. Proc Natl Acad Sci USA 2005; 102: 10200–10205.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the Cassa di Risparmio di Modena (CARISMO) for purchasing the Chip Cube-QTOF Instrumentation (Agilent). This work was supported by the Italian MIUR-FIRB Human ProteomeNet, Italian MIUR-PRIN, and CARISBO Foundation (to L.C.).

Author information

Authors and Affiliations

  1. Department of Human Anatomical Sciences, Cell Signalling Laboratory, University of Bologna, Bologna, Italy

    W L Blalock, M Piazzi, F Tagliavini, I Faenza, A M Martelli, M Y Follo & L Cocco

  2. Cell Biology Laboratory, Rizzoli Orthopedic Institute, Bologna, Italy

    A Bavelloni

  3. Department of Histology and Embryology, University of Modena, Modena, Italy

    M Piazzi

  4. IGM-CNR, Bologna Unit c/o Rizzoli Orthopedic Institute, Bologna, Italy

    A M Martelli

Authors
  1. W L Blalock
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A Bavelloni
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M Piazzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. F Tagliavini
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. I Faenza
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A M Martelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M Y Follo
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. L Cocco
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to L Cocco.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Leukemia website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Blalock, W., Bavelloni, A., Piazzi, M. et al. Multiple forms of PKR present in the nuclei of acute leukemia cells represent an active kinase that is responsive to stress. Leukemia 25, 236–245 (2011). https://doi.org/10.1038/leu.2010.264

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/leu.2010.264

Keywords

This article is cited by

Search

Advanced search

Quick links