Skip to main content

Advertisement

SpringerLink
Log in

Proteomic identification of C/EBP-DBD multiprotein complex: JNK1 activates stem cell regulator C/EBPα by inhibiting its ubiquitination

  • Original Article
  • Published:
Oncogene Submit manuscript

Abstract

Functional inactivation of transcription factors in hematopoietic stem cell development is involved in the pathogenesis of acute myeloid leukemia (AML). Stem cell regulator C/enhancer binding protein (EBP)α is among such transcription factors known to be inactive in AML. This is either due to mutations or inhibition by protein–protein interactions. Here, we applied a mass spectrometry-based proteomic approach to systematically identify putative co-activator proteins interacting with the DNA-binding domain (DBD) of C/EBP transcription factors. In our proteomic screen, we identified c-Jun N-terminal kinase (JNK) 1 among others such as PAK6, MADP-1, calmodulin-like skin proteins and ZNF45 as proteins interacting with DBD of C/EBPs from nuclear extract of myelomonocytic U937 cells. We show that kinase JNK1 physically interacts with DBD of C/EBPα in vitro and in vivo. Furthermore, we show that active JNK1 inhibits ubiquitination of C/EBPα possibly by phosphorylating in its DBD. Consequently, JNK1 prolongs C/EBPα protein half-life leading to its enhanced transactivation and DNA-binding capacity. In certain AML patients, however, the JNK1 mRNA expression and its kinase activity is decreased which suggests a possible reason for C/EBPα inactivation in AML. Thus, we report the first proteomic screen of C/EBP-interacting proteins, which identifies JNK1 as positive regulator of C/EBPα.

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

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

Similar content being viewed by others

References

  • Balkhi MY, Trivedi AK, Geletu M, Christopeit M, Bohlander SK, Behre HM et al. (2006). Proteomics of acute myeloid leukaemia: cytogenetic risk groups differ specifically in their proteome, interactome and post-translational protein modifications. Oncogene May 29: [Epub ahead of print].

  • Behre G, Reddy VA, Tenen DG, Hiddemann W, Zada AA, Singh SM . (2002a). Proteomic analysis of transcription factor interactions in myeloid stem cell development and leukaemia. Expert Opin Ther Targets 6: 491–495.

    Article  CAS  PubMed  Google Scholar 

  • Behre G, Singh SM, Liu H, Bortolin LT, Christopeit M, Radomska HS et al. (2002b). Ras signaling enhances the activity of C/EBP alpha to induce granulocytic differentiation by phosphorylation of serine 248. J Biol Chem 277: 26293–26299.

    Article  CAS  PubMed  Google Scholar 

  • Bogoyevitch MA, Ketterman AJ, Sugden PH . (1995). Cellular stresses differentially activate c-Jun N-terminal protein kinases and extracellular signal-regulated protein kinases in cultured ventricular myocytes. J Biol Chem 270: 29710–29717.

    Article  CAS  PubMed  Google Scholar 

  • Cleaves R, Wang QF, Friedman AD . (2004). C/EBPalphap30, a myeloid leukemia oncoprotein, limits G-CSF receptor expression but not terminal granulopoiesis via site-selective inhibition of C/EBP DNA binding. Oncogene 23: 716–725.

    Article  CAS  PubMed  Google Scholar 

  • Cristea IM, Gaskell SJ, Whetton AD . (2004). Proteomics techniques and their application to hematology. Blood 103: 3624–3634.

    Article  CAS  PubMed  Google Scholar 

  • D’Alo F, Johansen LM, Nelson EA, Radomska HS, Evans EK, Zhang P et al. (2003). The amino terminal and E2F interaction domains are critical for C/EBP alpha-mediated induction of granulopoietic development of hematopoietic cells. Blood 102: 3163–3171.

    Article  PubMed  Google Scholar 

  • Friedman AD . (1996). GADD153/CHOP, a DNA damage-inducible protein, reduced CAAT/enhancer binding protein activities and increased apoptosis in 32D c13 myeloid cells. Cancer Res 56: 3250–3256.

    CAS  PubMed  Google Scholar 

  • Frohling S, Schlenk RF, Stolze I, Bihlmayr J, Benner A, Kreitmeier S et al. (2004). CEBPA mutations in younger adults with acute myeloid leukemia and normal cytogenetics: prognostic relevance and analysis of cooperating mutations. J Clin Oncol 22: 624–633.

    Article  PubMed  Google Scholar 

  • Fuchs SY, Dolan L, Davis RJ, Ronai Z . (1996). Phosphorylation-dependent targeting of c-Jun ubiquitination by Jun N-kinase. Oncogene 13: 1531–1535.

    CAS  PubMed  Google Scholar 

  • Fuchs SY, Fried VA, Ronai Z . (1998). Stress-activated kinases regulate protein stability. Oncogene 17: 1483–1490.

    Article  CAS  PubMed  Google Scholar 

  • Fuchs SY, Xie B, Adler V, Fried VA, Davis RJ, Ronai Z . (1997). c-Jun NH2-terminal kinases target the ubiquitination of their associated transcription factors. J Biol Chem 272: 32163–32168.

    Article  CAS  PubMed  Google Scholar 

  • Gombart AF, Hofmann WK, Kawano S, Takeuchi S, Krug U, Kwok SH et al. (2002). Mutations in the gene encoding the transcription factor CCAAT/enhancer binding protein alpha in myelodysplastic syndromes and acute myeloid leukemias. Blood 99: 1332–1340.

    Article  CAS  PubMed  Google Scholar 

  • Han Z, Boyle DL, Chang L, Bennett B, Karin M, Yang L et al. (2001). c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Invest 108: 73–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hattori T, Ohoka N, Inoue Y, Hayashi H, Onozaki K . (2003). C/EBP family transcription factors are degraded by the proteasome but stabilized by forming dimer. Oncogene 22: 1273–1280.

    Article  CAS  PubMed  Google Scholar 

  • Iakova P, Awad SS, Timchenko NA . (2003). Aging reduces proliferative capacities of liver by switching pathways of C/EBPalpha growth arrest. Cell 113: 495–506.

    Article  CAS  PubMed  Google Scholar 

  • Kyriakis JM, Avruch J . (1990). pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-L-lysine. J Biol Chem 265: 17355–17363.

    CAS  PubMed  Google Scholar 

  • Landschulz WH, Johnson PF, McKnight SL . (1989). The DNA binding domain of the rat liver nuclear protein C/EBP is bipartite. Science 243: 1681–1688.

    Article  CAS  PubMed  Google Scholar 

  • Mahoney CW, Shuman J, McKnight SL, Chen HC, Huang KP . (1992). Phosphorylation of CCAAT-enhancer binding protein by protein kinase C attenuates site-selective DNA binding. J Biol Chem 267: 19396–19403.

    CAS  PubMed  Google Scholar 

  • Mann M, Hendrickson RC, Pandey A . (2001). Analysis of proteins and proteomes by mass spectrometry. Annu Rev Biochem 70: 437–473.

    Article  CAS  PubMed  Google Scholar 

  • Moriguchi T, Toyoshima F, Masuyama N, Hanafusa H, Gotoh Y, Nishida E . (1997). A novel SAPK/JNK kinase, MKK7, stimulated by TNFalpha and cellular stresses. EMBO J 16: 7045–7053.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Morton S, Davis RJ, McLaren A, Cohen P . (2003). A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun. EMBO J 22: 3876–3886.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Muller C, Calkhoven CF, Sha X, Leutz A . (2004). The CCAAT enhancer-binding protein alpha (C/EBPalpha) requires a SWI/SNF complex for proliferation arrest. J Biol Chem 279: 7353–7358.

    Article  PubMed  Google Scholar 

  • Muller C, Kowenz-Leutz E, Grieser-Ade S, Graf T, Leutz A . (1995). NF-M (chicken C/EBP beta) induces eosinophilic differentiation and apoptosis in a hematopoietic progenitor cell line. EMBO J 14: 6127–6135.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Musti AM, Treier M, Bohmann D . (1997). Reduced ubiquitin-dependent degradation of c-Jun after phosphorylation by MAP kinases. Science 275: 400–402.

    Article  CAS  PubMed  Google Scholar 

  • Nordeen SK . (1988). Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques 6: 454–458.

    CAS  PubMed  Google Scholar 

  • Pabst T, Mueller BU, Harakawa N, Schoch C, Haferlach T, Behre G et al. (2001a). AML1-ETO downregulates the granulocytic differentiation factor C/EBPalpha in t(8;21) myeloid leukemia. Nat Med 7: 444–451.

    Article  CAS  PubMed  Google Scholar 

  • Pabst T, Mueller BU, Zhang P, Radomska HS, Narravula S, Schnittger S et al. (2001b). Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia. Nat Genet 27: 263–270.

    Article  CAS  PubMed  Google Scholar 

  • Perrotti D, Cesi V, Trotta R, Guerzoni C, Santilli G, Campbell K et al. (2002). BCR-ABL suppresses C/EBPalpha expression through inhibitory action of hnRNP E2. Nat Genet 30: 48–58.

    Article  CAS  PubMed  Google Scholar 

  • Perrotti D, Marcucci G, Caligiuri MA . (2004). Loss of C/EBP alpha and favorable prognosis of acute myeloid leukemias: a biological paradox. J Clin Oncol 22: 582–584.

    Article  CAS  PubMed  Google Scholar 

  • Porse BT, Pedersen TA, Xu X, Lindberg B, Wewer UM, Friis-Hansen L et al. (2001). E2F repression by C/EBPalpha is required for adipogenesis and granulopoiesis in vivo. Cell 107: 247–258.

    CAS  PubMed  Google Scholar 

  • Raitano AB, Halpern JR, Hambuch TM, Sawyers CL . (1995). The Bcr-Abl leukemia oncogene activates Jun kinase and requires Jun for transformation. Proc Natl Acad Sci USA 92: 11746–11750.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rangatia J, Vangala RK, Treiber N, Zhang P, Radomska H, Tenen DG et al. (2002). Downregulation of c-Jun expression by transcription factor C/EBPalpha is critical for granulocytic lineage commitment. Mol Cell Biol 22: 8681–8694.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reddy VA, Iwama A, Iotzova G, Schulz M, Elsasser A, Vangala RK et al. (2002). Granulocyte inducer C/EBPalpha inactivates the myeloid master regulator PU.1: possible role in lineage commitment decisions. Blood 100: 483–490.

    Article  CAS  PubMed  Google Scholar 

  • Ronai Z . (2004). JNKing Revealed. Mol Cell 15: 843–844.

    Article  CAS  PubMed  Google Scholar 

  • Ross SE, Erickson RL, Hemati N, MacDougald OA . (1999). Glycogen synthase kinase 3 is an insulin-regulated C/EBPalpha kinase. Mol Cell Biol 19: 8433–8441.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ross SE, Radomska HS, Wu B, Zhang P, Winnay JN, Bajnok L et al. (2004). Phosphorylation of C/EBPalpha inhibits granulopoiesis. Mol Cell Biol 24: 675–686.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schoch C, Kohlmann A, Schnittger S, Brors B, Dugas M, Mergenthaler S et al. (2002). Acute myeloid leukemias with reciprocal rearrangements can be distinguished by specific gene expression profiles. Proc Natl Acad Sci USA 99: 10008–10013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Scott LM, Civin CI, Rorth P, Friedman AD . (1992). A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells. Blood 80: 1725–1735.

    CAS  PubMed  Google Scholar 

  • Shim M, Smart RC . (2003). Lithium stabilizes the CCAAT/enhancer-binding protein alpha (C/EBPalpha) through a glycogen synthase kinase 3 (GSK3)-independent pathway involving direct inhibition of proteasomal activity. J Biol Chem 278: 19674–19681.

    Article  CAS  PubMed  Google Scholar 

  • Sieweke MH, Graf T . (1998). A transcription factor party during blood cell differentiation. Curr Opin Genet Dev 8: 545–551.

    Article  CAS  PubMed  Google Scholar 

  • Smith ML, Cavenagh JD, Lister TA, Fitzgibbon J . (2004). Mutation of CEBPA in familial acute myeloid leukemia. N Engl J Med 351: 2403–2407.

    Article  CAS  PubMed  Google Scholar 

  • Subramanian L, Benson MD, Iniguez-Lluhi JA . (2003). A synergy control motif within the attenuator domain of CCAAT/enhancer-binding protein alpha inhibits transcriptional synergy through its PIASy-enhanced modification by SUMO-1 or SUMO-3. J Biol Chem 278: 9134–9141.

    Article  CAS  PubMed  Google Scholar 

  • Tenen DG . (2003). Disruption of differentiation in human cancer: AML shows the way. Nat Rev Cancer 3: 89–101.

    Article  CAS  PubMed  Google Scholar 

  • Terrance A Stadheim GLK . (2002). c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) is required for mitoxantrone- and anisomycin-induced apoptosis in HL-60 cells. Leukemia Res 26: 55–65.

    Article  Google Scholar 

  • Vangala RK, Heiss-Neumann MS, Rangatia JS, Singh SM, Schoch C, Tenen DG et al. (2003). The myeloid master regulator transcription factor PU.1 is inactivated by AML1-ETO in t(8;21) myeloid leukemia. Blood 101: 270–277.

    Article  CAS  PubMed  Google Scholar 

  • Wang H, Iakova P, Wilde M, Welm A, Goode T, Roesler WJ et al. (2001). C/EBPalpha arrests cell proliferation through direct inhibition of Cdk2 and Cdk4. Mol Cell 8: 817–828.

    Article  CAS  PubMed  Google Scholar 

  • Westendorf JJ, Yamamoto CM, Lenny N, Downing JR, Selsted ME, Hiebert SW . (1998). The t(8;21) fusion product, AML-1-ETO, associates with C/EBP-alpha, inhibits C/EBP-alpha-dependent transcription, and blocks granulocytic differentiation. Mol Cell Biol 18: 322–333.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu S, Cobb MH . (1997). MEKK1 binds directly to the c-Jun N-terminal kinases/stress-activated protein kinases. J Biol Chem 272: 32056–32060.

    Article  CAS  PubMed  Google Scholar 

  • Yoon K, Smart RC . (2004). C/EBPalpha is a DNA damage-inducible p53-regulated mediator of the G1 checkpoint in keratinocytes. Mol Cell Biol 24: 10650–10660.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zada AA, Singh SM, Reddy VA, Elsasser A, Meisel A, Haferlach T et al. (2003). Downregulation of c-Jun expression and cell cycle regulatory molecules in acute myeloid leukemia cells upon CD44 ligation. Oncogene 22: 2296–2308.

    Article  CAS  PubMed  Google Scholar 

  • Zhang DE, Zhang P, Wang ND, Hetherington CJ, Darlington GJ, Tenen DG . (1997). Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice. Proc Natl Acad Sci USA 94: 569–574.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zheng R, Friedman AD, Levis M, Li L, Weir EG, Small D . (2004). Internal tandem duplication mutation of FLT3 blocks myeloid differentiation through suppression of C/EBPalpha expression. Blood 103: 1883–1890.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr Roger Davis for providing pCDNA3-MKK7, pCDNA3-MEKK1-flag; Dr Daniel G Tenen and Dr Claus Nerlov for GST, GST-DBD and GST-C/EBPα expressin plasmids. This work was supported by Deutsche José Carreras Leukamie-Stiftung e.V. grant, DJCLS F03/03 to Dr Trivedi AK.

Author information

Authors and Affiliations

  1. Department of Internal Medicine IV – Hematology and Oncology, Bone Marrow Transplantation Section, State Center for Cell and Gene Therapy, Martin-Luther-University Halle-Wittenberg, Halle, SA, Germany

    A K Trivedi, D Bararia, M Christopeit, A A PeerZada, S M Singh & G Behre

  2. Department of Gene Vectors, GSF-National Research Center for Environment and Health, Munich, Germany

    A Kieser

  3. Department of Internal Medicine III Grosshadern, University of Munich, Munich, Germany

    W Hiddemann

  4. Andrology Section, Martin-Luther-University Halle-Wittenberg, Halle, Germany

    H M Behre

Authors
  1. A K Trivedi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D Bararia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M Christopeit
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A A PeerZada
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S M Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A Kieser
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. W Hiddemann
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. H M Behre
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. G Behre
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G Behre.

Additional information

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Trivedi, A., Bararia, D., Christopeit, M. et al. Proteomic identification of C/EBP-DBD multiprotein complex: JNK1 activates stem cell regulator C/EBPα by inhibiting its ubiquitination. Oncogene 26, 1789–1801 (2007). https://doi.org/10.1038/sj.onc.1209964

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.onc.1209964

  • Springer Nature Limited

Keywords

This article is cited by

Search

Navigation