Key Event Title
|Level of Biological Organization|
Key Event Components
|negative regulation of DNA binding||protein-DNA complex||decreased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Immune dysfunction induced by JAK3 inhibition||KeyEvent|
|Homo sapiens||Homo sapiens||High||NCBI|
|Mus musculoides||Mus musculoides||High||NCBI|
|All life stages||High|
Key Event Description
Interleukin-2 (IL-2) and other cytokines rapidly activate the Janus-Associated-Kinase (JAK)1 and JAK3 (21-Beadling-1994) in peripheral blood lymphocytes (PBLs). The activation of JAK kinases and signal transducer and activator of transcription (STAT) proteins by IL-2 and interferon (IFN)-α does not include the T cell antigen receptor in human T lymphocytes. (EMBO J. 13:5606–5615). After activation of JAKs, latent STAT transcription factors make dimeric STAT proteins (44-Gaffen-1995). The STAT proteins then translocate to the nucleus, where they bind to and regulate the transcriptional activation of the promoters of target genes. Dimeric STAT proteins can bind to the palindromic gamma interferon-activated (GAS) sequence TTCNmGAA, where m is 3 for all the STATs except Stat6, which can additionally bind to GAS motifs where m is 4 (45-Darnell-1997,46-Decker-1997,51-Ihle-1996,52-Leonard-1998).
How It Is Measured or Detected
Electrophoretic mobility shift assays (EMSAs) using nuclear extracts and specific oligo including transcription factor binding sites such as cytokine-inducible SH2-containing proteins (CIS) gene promoters is useful for evaluation of DNA binding activity (30-Johnston-1995). Yoshida et al. demonstrated that activated Stat5 binds specific DNA-probe in splenocytes (53-Liu-2010). A cell-permeable nonpeptidic nicotinoyl hydrazone compound that selectively targets the SH2 domain of STAT5 (IC50 = 47 µM against STAT5b SH2 domain EPO peptide binding activity), while exhibiting much less effect towards the SH2 domain of STAT1, STAT3, or Lck (IC50 >500 µM). It shows blockage STAT5/STAT5 DNA binding activity in K562 nuclear extract and inhibit IFN-α-stimulated STAT5, but not STAT1 or STAT3, tyrosine phosphorylation in Daudi cells (49-Muller-2008).
Nuclear extracts were prepared from untreated YT cells or cells that had been treated with recombinant IL-2 (2 nM) for 30 min at 37°C. EMSAs were performed by using glycerol-containing 5% polyacrylamide gels (29:1) containing 0.5X Tris-borate-EDTA buffer. For supershifting assays, nuclear extracts were preincubated for 10 min with antibodies to STAT5. Oligonucleotide sequences from PRRIFV were used as probes (54-Maeshima-2012).
Supershifting was performed by preincubating the whole-cell extract with 3 μl of a pan-STAT5 antiserum that recognizes both STAT5a and STAT5b. Electrophoresis was carried out at room temperature using 5% or 6% polyacrylamide gels (39-Heidt-2010).
Domain of Applicability
STAT5 is expressed in hematopoietic cells such as T, B cells from humans, rodents and other mammalian species (55-Gilmour-1995).
Evidence for Perturbation by Stressor
21. Beadling, C., Guschin, D., Witthuhn, B. A., Ziemiecki, A., Ihle, J. N., Kerr, I. M., and Cantrell, D. A. (1994) Activation of JAK kinases and STAT proteins by interleukin-2 and interferon alpha, but not the T cell antigen receptor, in human T lymphocytes. EMBO J 13, 5605-5615
30. Johnston, J. A., Bacon, C. M., Finbloom, D. S., Rees, R. C., Kaplan, D., Shibuya, K., Ortaldo, J. R., Gupta, S., Chen, Y. Q., Giri, J. D., and et al. (1995) Tyrosine phosphorylation and activation of STAT5, STAT3, and Janus kinases by interleukins 2 and 15. Proc Natl Acad Sci U S A 92, 8705-8709
39. Heidt, S., Roelen, D. L., Eijsink, C., Eikmans, M., van Kooten, C., Claas, F. H., and Mulder, A. (2010) Calcineurin inhibitors affect B cell antibody responses indirectly by interfering with T cell help. Clin Exp Immunol 159, 199-207
44. Gaffen, S. L., Lai, S. Y., Xu, W., Gouilleux, F., Groner, B., Goldsmith, M. A., and Greene, W. C. (1995) Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity. Proc Natl Acad Sci U S A 92, 7192-7196
45. Darnell, J. E., Jr. (1997) STATs and gene regulation. Science 277, 1630-1635
46. Decker, T., Kovarik, P., and Meinke, A. (1997) GAS elements: a few nucleotides with a major impact on cytokine-induced gene expression. J Interferon Cytokine Res 17, 121-134
49. Muller, J., Sperl, B., Reindl, W., Kiessling, A., and Berg, T. (2008) Discovery of chromone-based inhibitors of the transcription factor STAT5. Chembiochem : a European journal of chemical biology 9, 723-727
51. Ihle, J. N. (1996) STATs: signal transducers and activators of transcription. Cell 84, 331-334
52. Leonard, W. J., and O'Shea, J. J. (1998) Jaks and STATs: biological implications. Annu Rev Immunol 16, 293-322
53. Liu, J., Yoshida, Y., Kunugita, N., Noguchi, J., Sugiura, T., Ding, N., Arashidani, K., Fujimaki, H., and Yamashita, U. (2010) Thymocytes are activated by toluene inhalation through the transcription factors NF-kappaB, STAT5 and NF-AT. Journal of applied toxicology : JAT 30, 656-660
54. Maeshima, K., Yamaoka, K., Kubo, S., Nakano, K., Iwata, S., Saito, K., Ohishi, M., Miyahara, H., Tanaka, S., Ishii, K., Yoshimatsu, H., and Tanaka, Y. (2012) The JAK inhibitor tofacitinib regulates synovitis through inhibition of interferon-gamma and interleukin-17 production by human CD4+ T cells. Arthritis Rheum 64, 1790-1798
55. Gilmour, K. C., Pine, R., and Reich, N. C. (1995) Interleukin 2 activates STAT5 transcription factor (mammary gland factor) and specific gene expression in T lymphocytes. Proc Natl Acad Sci U S A 92, 10772-10776