The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway plays a critical role in the signaling of a wide array of cytokines and growth factors. These cytokines and growth factors are responsible for various cellular functions, including proliferation, growth, hematopoiesis, and immune response [1-4]. Thermo Scientific™ has a wide range of products to help with JAK-STAT research. The binding of cytokines and growth factors to their corresponding receptors activates JAK, which then phosphorylates the receptor and STAT proteins on specific tyrosine residues. STATs then dimerize, translocate to the nucleus, bind to the consensus DNA sequence of 5’-TT(N4–6)AA-3’ and initiate the transcription of target genes [1-4].

Key JAK-STAT Pathway Targets

Four JAK family kinases and seven STAT family members have been identified:

JAK1STAT4
JAK2STAT5a
JAK3STAT5b
STAT1STAT6 
STAT2TYK2
STAT3 

Common targets in the JAK/STAT Pathway:

EGFIL-6
GM-CSFIL-12
GRB2IL-13
IFN-α/βPIAS
IFN-γShc
IL-2SHP
IL-3SOCS3
IL-4 

JAK1, JAK2, and TYK2 appear to be universally expressed, while JAK3 expression is normally limited to lymphoid cells. The JAKs are structurally unique in having a C-terminal kinase domain (JH1) preceded by a pseudokinase domain (JH2), which lacks catalytic activity but has a critical regulatory function. JAKs also have a Src homology 2 (SH2) domain and an N-terminal band four-point-one, ezrin, radixin, moesin (FERM) domain that is critical for mediating the association with cytokine receptors.

STAT proteins contain a SH2 domain for dimerization and a DNA-binding domain. The amino acid sequence diversity and their tissue-specific distributions account for the diverse roles of STATs in response to extracellular cytokines. [1-4] The JAK-STAT pathways are up-regulated by a vast array of cytokines/growth factors. One mechanism for negative regulation of JAK-STAT pathways is through suppresser of cytokine signaling (SOCS) proteins, which directly bind to and inactivate JAKs [5], and protein inhibitors of activated STATs (PIAS) that bind to phosphorylated STAT dimers, preventing DNA binding [6].

Abnormal constitutive activation of JAK-STAT pathways has been implicated in various cancers and immune disorders. For example, STAT3 and STAT5 is overactive in many tumors, including major carcinomas and some hematologic tumors [7, 8]. Activating mutations in JAK2 have been linked to leukemia. TEL-JAK2 fusion due to chromosomal translocation was identified in a small set of human T cell acute lymphoblast leukemia patients [9]. The V617F mutation in the JH2 pseudo-kinase domain of JAK2 was found in a high percentage of patients with myeloproliferative disorders, including polycythaemia vera [10]. Inhibitors of JAK-STAT pathways are currently being developed in the areas of oncology and immune disorders. 

Figures

Events leading to STAT activation
 
     Events leading to STAT activation. (A) Extracellular IFN (blue triangle) prior to binding to its receptor. (B) Upon binding, JAK kinases constitutively associated with the receptor subunits interact. The interacting JAK proteins activate one another by reciprocal tyrosine phosphorylation, and phosphorylate a tyrosine on two subunits contained in the receptor complex. These phosphorylated tyrosine residues provide paired docking sites for STAT via its SH2 domain (C). STAT, recruited to the receptor complex, is then phosphorylated at a tyrosine residue by the JAKs. This tyrosine phosphorylation promotes STAT homo and heterodimerization mediated by reciprocal phosphotyrosine SH2 domain interaction and the dissociation of the dimers from the receptor complex (D). The STAT dimer is then translocated to the cell nucleus, where they complex with other nuclear proteins and regulate gene expression by binding to promoters or other response elements on DNA.

Data

Thermo Scientific™ offers antibodies, ELISAs, Luminex® multiplex assays and growth factors for key targets in the JAK-STAT signaling pathway. 

Featured below is flow cytometry and ELISA data using Thermo Scientific™ products. 

Flow cytometry analysis of STAT4 on human Jurkat cells

Flow cytometry analysis of STAT4 on human Jurkat cells.  Cells were fixed and permeabilized with FIX & PERM® reagents.  Cells were stained with an STAT4 Abfinity recombinant rabbit monoclonal antibody (Product # 700185, black histogram) or left unstained (blue histogram).  After incubation of the primary antError! Hyperlink reference not valid.ibody for 1 hour on ice, the cells were stained with a Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody. Note that pre-incubation with immunogenic peptide resulted in partial block of the STAT4 signal (red). 

STAT5a

Cell extracts were prepared and analyzed with the STAT5a [pY694] ELISA and STAT5a Total ELISA kits. Phosphorylation of STAT5a is increased in sodium vanadate-treated HEL cells. The total level of STAT5a remains relatively constant in the treated vs. untreated control. Data was generated using a Novex® STAT5a [pY694] ELISA kit (Cat. No. KHO0761) and STAT5a (total) ELISA kit (Cat. No. KHO0751).

STAT5b

Analysis of STAT5b [pY699] phosphorylation in TF-1 cells. TF-1 cells were treated with IFN-α, IL-3, or left untreated . Cell lysates were analyzed using the STAT5b [pY699] ELISA kit. Treatment with IFN-α or IL-3 results in activation of the JAK/STAT pathway, as seen by the upregulation of phosphorylated STAT5b. Data was generated using a Novex® IFN-α Pure Recombinant protein (Cat. No. PHC4014).

Resources

Our scientists have been hard at work trying to better understand the JAK-STAT cell signaling pathway over the years. For further information on this pathway please see our publications, white papers and posters:

References

  1. Aaronson, D.S., et al. (2002) A road map for those who don’t know JAK-STAT. Science 296: 1653-1655.
  2. Rybinski, M. (2012) Model-based selection of the robust JAK-STAT activation mechanism.   J Theoretical Bio 309: 34-46.
  3. O’Shea, J.J., et al. (2004) A new modality for immunosuppression: targeting the JAK/STAT pathway. Nat Rev Drug Discovery 3: 555-564.
  4. Liongue, C., et al. (2012) Evolution of JAK-STAT pathway components: mechanisms and role in immune system development. PLoS ONE 7: e32777.
  5. Kishimoto, T., et al. Knocking the SOCS off a tumor suppressor. (2001) Nature Genetics 28: 4-5.
  6. Shuai, K,. et al. (2000) Modulation of STAT signaling by STAT-interacting proteins. Oncogene 19 : 2638-2645.
  7. Darnell, J.E., et al. (2005) Validating stat3 in cancer therapy. Nature Medicine 11: 595-596.
  8. Yu, H., et al. (2004) The STATs of cancer – new molecular targets come of age. Nat Rev Cancer 4: 97-105.
  9. Lacronique, V., et al. (1997) A TEL-JAK2 fusion protein with constitutive kinase activity in human leukemia. Science 278: 1309-1312.
  10. Ferrajoli, A., et al. (2006) The JAK-STAT pathway: a therapeutic target in hematological malignancies. Current Cancer Drug Targets 6: 671-679.

Featured Products

Catalog NumberProduct Name
AHC4539IFN-gamma
AHC2919GM-CSF
M621BIL-6
AHC0939IL-3
ARC0132IL-13
135300STAT5b
133600STAT5a
M450IL-4
M600BIL-2
M122IL-12
333400Phospho-STAT1 pTyr701
44829MPhospho-SHC pTyr239/pTyr240
137000STAT3
M806EGF
AHO1352JAK2
44422GPhospho-JAK1 pTyr1022/pTyr1023
717900Phospho-STAT4 pTyr693
AHC4815IFN-alpha
MA515561JAK3
700247Phospho-STAT6 pTyr641

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