Advertisement

Analysis of Janus Tyrosine Kinase Phosphorylation and Activation

  • Jeremy A. Ross
  • Georgialina Rodriguez
  • Robert A. KirkenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 967)

Abstract

Activation of Janus kinases (Jaks) occurs through autophosphorylation of key tyrosine residues located primarily within their catalytic domain. Phosphorylation of these tyrosine residues facilitates access of substrates to the active site and serves as an intrinsic indicator of Jak activation. Here, we describe the methods and strategies used for analyzing Jak phosphorylation and activation. Tyrosine-phosphorylated (active) Jaks are primarily detected from cell extracts using anti-phosphotyrosine-directed Western blot analysis of Jak-specific immunoprecipitates. Additionally, receptor pull-down and in vitro kinase assays can also be utilized to measure cellular Jak catalytic activity. In addition to tyrosine phosphorylation, recent evidence indicates Jaks can be serine phosphorylated upon cytokine stimulation, however the lack of commercially available antibodies to detect these sites has hindered their analysis by Western blot. However, phosphoamino acid analysis (PAA) has been employed to monitor Jak serine and threonine phosphorylation. Over the past decade, remarkable advances have been made in our understanding of Jak function and dysfunction, however much remains to be learned about their complex regulatory mechanisms.

Key words

Janus kinase (Jak) Tyrosine phosphorylation Immunoprecipitation SDS-PAGE Immunoblot Kinase assay Phosphoamino acid analysis (PAA) 

Notes

Acknowledgments

This work was supported by grants from the Lizanell and Colbert Coldwell Foundation, Edward N. and Margaret G. Marsh Foundation, and grant G12MD007592 from the National Center on Minority Health and Health Disparities (NCMHD), a component of the National Institutes of Health (NIH).

References

  1. 1.
    Ihle JN, Witthuhn B, Quelle FW, Yamamoto K, Silvennoinen O (1995) Signaling through the hematopoietic cytokine receptors. Annu Rev Immunol 13:369–398PubMedCrossRefGoogle Scholar
  2. 2.
    Ghoreschi K et al (2009) Janus kinases in immune cell signaling. Immunol Rev 228:273–287PubMedCrossRefGoogle Scholar
  3. 3.
    Ihle JN et al (1997) Jaks and Stats in cytokine signaling. Stem Cells 15(Suppl 1):105–111, discussion 112PubMedCrossRefGoogle Scholar
  4. 4.
    Kawamura M et al (1994) Molecular cloning of L-JAK, a Janus family protein-tyrosine kinase expressed in natural killer cells and activated leukocytes. Proc Natl Acad Sci U S A 91:6374–6378PubMedCrossRefGoogle Scholar
  5. 5.
    Tortolani PJ et al (1995) Regulation of JAK3 expression and activation in human B cells and B cell malignancies. J Immunol 155:5220–5226PubMedGoogle Scholar
  6. 6.
    Musso T et al (1995) Regulation of JAK3 expression in human monocytes: phosphorylation in response to interleukins 2, 4, and 7. J Exp Med 181:1425–1431PubMedCrossRefGoogle Scholar
  7. 7.
    Ross JA et al (2007) Regulation of T cell homeostasis by JAKs and STATs. Arch Immunol Ther Exp (Warsz) 55:231–245CrossRefGoogle Scholar
  8. 8.
    Leonard WJ, O’Shea JJ (1998) Jaks and STATs: biological implications. Annu Rev Immunol 16:293–322PubMedCrossRefGoogle Scholar
  9. 9.
    Tam L et al (2007) Expression levels of the JAK/STAT pathway in the transition from hormone-sensitive to hormone-refractory prostate cancer. Br J Cancer 97:378–383PubMedCrossRefGoogle Scholar
  10. 10.
    Lai SY, Johnson FM (2010) Defining the role of the JAK-STAT pathway in head and neck and thoracic malignancies: implications for future therapeutic approaches. Drug Resist Updat 13:67–78PubMedCrossRefGoogle Scholar
  11. 11.
    Neilson LM et al (2007) Coactivation of janus tyrosine kinase (Jak)1 positively modulates prolactin-Jak2 signaling in breast cancer: recruitment of ERK and signal transducer and activator of transcription (Stat)3 and enhancement of Akt and Stat5a/b pathways. Mol Endocrinol 21:2218–2232PubMedCrossRefGoogle Scholar
  12. 12.
    Gao B et al (2001) Constitutive activation of JAK-STAT3 signaling by BRCA1 in human prostate cancer cells. FEBS Lett 488:179–184PubMedCrossRefGoogle Scholar
  13. 13.
    Pesu M et al (2008) Therapeutic targeting of Janus kinases. Immunol Rev 223:132–142PubMedCrossRefGoogle Scholar
  14. 14.
    Zhou YJ et al (1997) Distinct tyrosine phosphorylation sites in JAK3 kinase domain positively and negatively regulate its enzymatic activity. Proc Natl Acad Sci U S A 94:13850–13855PubMedCrossRefGoogle Scholar
  15. 15.
    Feng J et al (1997) Activation of Jak2 catalytic activity requires phosphorylation of Y1007 in the kinase activation loop. Mol Cell Biol 17:2497–2501PubMedGoogle Scholar
  16. 16.
    Gauzzi MC et al (1996) Interferon-alpha-dependent activation of Tyk2 requires phosphorylation of positive regulatory tyrosines by another kinase. J Biol Chem 271:20494–20500PubMedCrossRefGoogle Scholar
  17. 17.
    Wang R et al (2003) Mechanism of Janus kinase 3-catalyzed phosphorylation of a Janus kinase 1 activation loop peptide. Arch Biochem Biophys 410:7–15PubMedCrossRefGoogle Scholar
  18. 18.
    Cheng H et al (2008) Phosphorylation of human Jak3 at tyrosines 904 and 939 positively regulates its activity. Mol Cell Biol 28:2271–2282PubMedCrossRefGoogle Scholar
  19. 19.
    Argetsinger LS et al (2011) Tyrosines 868, 966, and 972 in the kinase domain of JAK2 are autophosphorylated and required for maximal JAK2 kinase activity. Mol Endocrinol 24:1062–1076CrossRefGoogle Scholar
  20. 20.
    Funakoshi-Tago M et al (2008) Negative regulation of Jak2 by its auto-phosphorylation at tyrosine 913 via the Epo signaling pathway. Cell Signal 20:1995–2001PubMedCrossRefGoogle Scholar
  21. 21.
    Li Z et al (2007) SH2B1 enhances leptin signaling by both Janus kinase 2 Tyr813 phosphorylation-dependent and -independent mechanisms. Mol Endocrinol 21:2270–2281PubMedCrossRefGoogle Scholar
  22. 22.
    Kurzer JH et al (2004) Tyrosine 813 is a site of JAK2 autophosphorylation critical for activation of JAK2 by SH2-B beta. Mol Cell Biol 24:4557–4570PubMedCrossRefGoogle Scholar
  23. 23.
    Argetsinger LS et al (2004) Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity. Mol Cell Biol 24:4955–4967PubMedCrossRefGoogle Scholar
  24. 24.
    Robertson SA et al (2009) Regulation of Jak2 function by phosphorylation of Tyr317 and Tyr637 during cytokine signaling. Mol Cell Biol 29:3367–3378PubMedCrossRefGoogle Scholar
  25. 25.
    Mazurkiewicz-Munoz AM et al (2006) Phosphorylation of JAK2 at serine 523: a negative regulator of JAK2 that is stimulated by growth hormone and epidermal growth factor. Mol Cell Biol 26:4052–4062PubMedCrossRefGoogle Scholar
  26. 26.
    Ross JA et al (2010) Protein phosphatase 2A regulates interleukin-2 receptor complex formation and JAK3/STAT5 activation. J Biol Chem 285:3582–3591PubMedCrossRefGoogle Scholar
  27. 27.
    Cacalano NA et al (1999) Autosomal SCID caused by a point mutation in the N-terminus of Jak3: mapping of the Jak3-receptor interaction domain. EMBO J 18:1549–1558PubMedCrossRefGoogle Scholar
  28. 28.
    Zhou YJ et al (2001) Unexpected effects of FERM domain mutations on catalytic activity of Jak3: structural implication for Janus kinases. Mol Cell 8:959–969PubMedCrossRefGoogle Scholar
  29. 29.
    Funakoshi-Tago M et al (2006) Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2. EMBO J 25:4763–4772PubMedCrossRefGoogle Scholar
  30. 30.
    Sayyah J et al (2011) Phosphorylation of Y372 is critical for Jak2 tyrosine kinase activation. Cell Signal 23:1806–1815PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Jeremy A. Ross
    • 1
    • 2
  • Georgialina Rodriguez
    • 1
    • 2
  • Robert A. Kirken
    • 1
    • 2
    Email author
  1. 1.Department of Biological SciencesThe University of Texas at El PasoEl PasoUSA
  2. 2.Border Biomedical Research CenterThe University of Texas at El PasoEl PasoUSA

Personalised recommendations