Chimeric oncogenic interferon regulatory factor-2 (IRF-2): Degradation products are biologically active

Krishna Prakash, Pardeep Kumar, Pramod C. Rath

Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Radiation and Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Recombinant DNA Technology Laboratory, Centre for Biological Science, Central University of Bihar, BIT Patna Campus, P.O. B.V. College, Patna, India.

DOI: 10.4236/ajmb.2012.24038                

Abstract

Interferon Regulatory Factor-2 (IRF-2) belongs to IRF family, was identified as a mammalian transcription factor involved in Interferon beta (IFN_β) gene regulation. Besides that IRF-2 is involved in immunomodulation, hematopoietic differentiation, cell cycle regulation and oncogenesis. We have done molecular sub-cloning and expression of recombinant murine IRF-2 as GST (Glutathione-S-Transferase)- IRF-2 fusion protein in E. coli/XL-1blue cells. Recombinant IRF-2 with GST moiety at N-terminus expressed as GST-IRF-2 (~66 kd) in E. coli along with different low molecular mass degradation products revealed approximately 30, 42, 60 and 62 kd by SDS-PAGE and Western blot, respectively. We further confirm that degradation takes place at C-terminus of the fusion protein not at N-terminus as anti-GST antibody was detecting all bands in the immunoblot. The recombinant IRF-2 was biologically active along with their degradation products in terms of their DNA binding activity as assessed by Electrophoretically Mobility Shift Assay (EMSA). We observed three different molecular mass DNA/protein complexes (1 - 3) with Virus Response Element (VRE) derived from human Interferon IFN_β gene and five different molecular mass complexes (1 - 5) with IRF-E motif (GAAAGT)₄ in EMSA gel. GST only expressed from empty vector did not bind to these DNA elements. To confirm that the binding is specific, all complexes were competed out completely when challenged with 100-X fold molar excess of IRF-E oligo under cold competition. It means degradation products along with full-length protein are able to interact with VRE_β as well as IRF-E motif. This means degradation products may regulate the target gene (s) activation/repression via interacting with VRE/IRF-E.

Keywords

Recombinant Interferon Regulatory Factor-2 (IRF-2); DNA Binding Domain (DBD); C-Terminus of IRF-2; EMSA

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Mamane, Y., Heylbroeck, C., Genin, P., Algarte, M., Servant, M. J., LePage, C., DeLuca, C., Kwon, H., Lin, R. and Hiscott, J. (1999). Interferon regulatory factors: the next generation. Gene 237, 1–14. doi:10.1016/S0378-1119(99)00262-0
[2]	Taniguchi, T., Ogasawara, K., Takaoka, A. and Tanaka, N. (2001). IRF family of transcription factors as regulators of host defense. Annual Review of Immunology 19, 623-655. doi:10.1146/annurev.immunol.19.1.623
[3]	Harada, H., Fujita, T., Miyamoto, M., Kimura, Y., Maruyama, M., Furia, A., Miyata, T. and Taniguchi, T. (1989). Structurally similar but functionally distinct factors, IRF-1 and IRF-2, bind to the same regulatory elements of IFN and IFN-inducible genes. Cell 4, 729-39. doi:10.1016/0092-8674(89)90107-4
[4]	Aziz, F., vanWijnen, A. J., Stein, J. L. and Stein, G. S. (1998). HiNF-D (CDP-cut/CDC2/cyclin A/pRB-complex) influences the timing of IRF-2-dependent cell cycle activation of human histone H4 gene transcription at the G1/S phase transition. J Cell Physiology 3, 453-64. DOI: doi:10.1002/(SICI)1097-4652(199812)177:3<453::AID-JCP8>3.0.CO;2-F
[5]	Yamamoto, H., Lauphier, M. S., Taniguchi, T. and Harada, H. (1994). The oncogenic transcription factor IRF-2 possess a transcriptional repression and a activation domain. Oncogene 9, 1423-1428. http://www.ncbi.nlm.nih.gov/pubmed/8152803
[6]	Cohen, L. and Hiscott, J. (1992). Characterization of TH3, an induction-specific protein interacting with the interferon beta promoter. Virology 2, 589-99. http://www.ncbi.nlm.nih.gov/pubmed/1448916
[7]	Cui, L., Deng, Y., Rong, Y., Lou, W., Mao, Z., Feng, Y., Xie, D. and Jin, D. (2012). IRF-2 is over-expressed in pancreatic cancer and promotes the growth of pancreatic cancer cells. Tumour Biology 1, 247-55. doi:10.1007/s13277-011-0273-3
[8]	Xi, H., and Blanck, G. (2000). Interferon regulatory fac-tor-2 point mutations in human pancreatic tumors. International J. Cancer 87, 6: 803-8. doi:10.1002/1097-0215(20000915)87:6<803::AID-IJC7>3.0.CO;2-E
[9]	Doherty, G. M., Boucher, L., Sorenson and K., Lowney, J. (2001). Interferon regulatory factor expression in human breast cancer. Annals Surgery 5, 623-9. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1421301/
[10]	Wang, Y., Liu, D., Chen, P., Koeffler, H. P., Tong, X. and Xie, D. (2008). Negative feedback regulation of IFN-gamma pathway by IFN regulatory factor 2 in esophageal cancers. Cancer Res. 4, 1136-43.
[11]	Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
[12]	Childs, K.S., and Goodbourn, S. (2003). Identification of novel corepressor molecules for Interferon Regulatory Factor-2. Nucleic Acids Res. 12, 3016-26. doi:10.1093/nar/gkg431
[13]	Taniguchi, T., Tanaka, N., Ogasawara, K., Taki, S., Sato, M. and Takaoka, A. (2000). Transcription factor IRF-1 and its family members in the regulation of host defense. Cold Spring Harb. Symp. Quant. Biol. 64, 465-72. doi:10.1146/annurev.immunol.19.1.623
[14]	Masumi, A., Wang, I.M., Lefebvre, B., Yang, X.J., Nakatani, Y. and Ozato, K. (1999). The histone acetylase PCAF is a phorbolester-inducible coactivator of the IRF family that confers enhanced interferon responsiveness. Mol. Cell Biol. 3, 1810-20. http://mcb.asm.org/content/19/3/1810.long
[15]	Upreti, M. and Rath, P.C. (2005). Expression and DNA binding activity of the recombinant interferon regulatory factor-1 (IRF-1) of mouse. Mol. Biol. Rep. 2, 103-16. doi:10.1007/s11033-004-6940-3
[16]	Lin, R., Mustafa, A., Nguyen, H., Gewert, D. and Hiscott, J. (1994). Mutational analysis of interferon (IFN) regulatory factors 1 and 2 effects on the induction of IFN-beta gene expression. J. Biol. Chem. 26,17542-9. http://www.jbc.org/content/269/26/17542.