Janus Kinase (JAK) / Signal Transducer and Activator of Transcription (STAT) Signaling Pathway explained at molecular level.

1. DELHI INSTITUTE OF PHARMACEUTICAL SCIENCES AND RESEARCH
JAK/STAT SIGNALING PATHWAY
PRESENTED TO:
DR. SACHIN KUMAR
ASSISTANT PROFESSOR
DIPSAR
PRESENTED BY:
KUMARI KALA SAH
M.PHARM (PHARMACOLOGY)
58/MPL/DIPSAR/2022

2. 2
Table of Contents
 Introduction
 Discovery of JAK/STAT Pathway
 Components of JAK/STAT Pathway
 Composition of JAKs
 Composition of STATs
 Activation and Regulation of JAK/STAT
Signaling Pathway
 Positive Regulations of JAK/STAT Signaling Pathway
 Negative Regulations of JAK/STAT Signaling Pathway
 Inhibitors and Clinical Application
 Clinical Applications of Cytokine Antibodies
 Clinical Application of STATs Inhibitor
 Clinical Application of JAKs Inhibitor
 Conclusion
 References
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3. INTRODUCTION
 JAK/STAT is an intracellular cell signaling pathway activated by cytokines and
growth Hormones.
 It is involved in wide range of biological process including hematopoiesis, immune
fitness, tissue repair, inflammation, apoptosis and adipogenesis. (1)
 Dysregulation or mutation of JAK/STAT component is linked with many human
diseases including malignancies and autoimmune disorder.
 Targeting JAK or STAT protein with a small molecule inhibitor is a promising
strategy for treating various diseases. (2)
3
(1) O’Shea, J. J. et al. The JAK-STAT pathway: impact on human disease and therapeutic intervention. Annu.
Rev. Med. 66, 311–328 (2015).
(2) Xin, P. et al. The role of JAK/STAT signaling pathway and its inhibitors in diseases. Int.
Immunopharmacol. 80, 106210 (2020).

4. DISCOVERY OF JAK/STAT PATHWAY
 STATs were firstly found in 1988 as protein that binds to interferons- stimulated
response elements of DNA sequences to stimulate the transcription of type I
Interferons. (3)
 JAKs were discovered in1992 in three separate labs. (4)
 In the late 1980s to early 1990s, the components and outliners of JAK/STAT
signaling pathway were completed with the discovery of JAKs and STATs.
 Research has continued to the present days, revealing more protein and function
involved in the pathway.
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(3) Zhong, Z., Wen, Z. & Darnell, J. E. Jr Stat3 and Stat4: members of the family of signal transducers and
activators of transcription. Proc. Natl Acad. Sci. USA 91, 4806–4810 (1994)
(4) Wilks, A. F. Two putative protein-tyrosine kinases identified by application of the polymerase chain
reaction. Proc. Natl Acad. Sci. USA 86, 1603–1607 (1989).

5. COMPONENTS OF JAK/STAT PATHWAY
 Ligand-Receptor complex.
 JAKs (Janus Kinases): 4 members in the family - JAK1, JAK2, JAK3 and
TYK2. (5)
 STATs (Signal transducer and activator of transcription): 7 members in
the family- STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6. (6)
 See Figure (1) and (2)
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(5) Lai, K. S. et al. A kinase-deficient splice variant of the human JAK3 is expressed in hematopoietic and epithelial
cancer cells. J. Biol. Chem. 270, 25028–25036 (1995).
(6) Fu, X. Y. A transcription factor with SH2 and SH3 domains is directly activated by an interferon alpha-induced
cytoplasmic protein tyrosine kinase(s). Cell 70, 323–335 (1992).

6. COMPOSITION OF JAKs
 JAKs has 7 homology Domain- JH1 to JH7
 JH1 is the first JH starting from carboxyl terminal. It is a kinase domain.
 JH2 is a pseudokinase domain
 JH3 with one half of JH4 constitute SH2 domain.
 The combination of one half of JH4, JH5, JH6 and JH7 constitute a
Ferm domain. (7)
6
(7) Wilks, A. F. et al. Two novel protein-tyrosine kinases, each with a second phosphotransferase-related
catalytic domain, define a new class of protein kinase. Mol. Cell. Biol. 11, 2057–2065 (1991).

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Figure (1): JAK family and
structure

8. JAKs MEMBER Phosphorylation site Receptor activating
JAKs
Functions
JAK1 Y1038/Y1039 Cytokine receptor II,
GP130 receptor
LIF, CNFR
Hematopoietic
JAK2 Y1007/Y1008 Single chain receptors
such as erythropoietin,
Prolactin,
Thrombopoietin.
Cell growth and
proliferation
JAK3 Y980/Y981 Interleukin-2, IL-4, IL-7,
IL-9, IL-15, IL-21
Maintenance of normal
phenotype and
function of peripheral T
cells.
TYK2 Y1054/Y1055 Interferons alfa/ beta ,
IL-6, IL-10, IL-12, IL-13,
IL-23
Important role in
human innate and
acquired immunity.
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Table (1): JAK family, phosphorylation site,receptor activating JAKs, Function

9. COMPOSITION OF STATs
 STATs consist of 750-900 amino acids
 From N-Terminal to C-terminal is consist of 6 domains. (8)
 N-Terminal domain, Coiled coin domain, DNA binding domain, linker
domain, SH2 domain, Transcription activator Domain. (9)
 See Figure (2) at page 6
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(8) Fu, X. Y. A transcription factor with SH2 and SH3 domains is directly activated by an interferon alpha-induced
cytoplasmic protein tyrosine kinase(s). Cell 70, 323–335 (1992).
(9) Horvath, C. M. STAT proteins and transcriptional responses to extracellular signals. Trends Biochem. Sci. 25, 496–502
(2000).

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Figure (2): STAT family and
structure

11. STATs member Phosphoryla
tion site
Transduction of signals Function
STAT1 Y701 IL-2, IL-6, PGF, EGF, HGF, TNF Inhibits tumor occurrence,
early development of β cells
STAT2 Y690 Type I INF ( INF α and INF β) Antiviral effects, Immune
regulation, maintenance of
memory cells
STAT3 Y705 IL-6, IL-10, IL-21, IL-27, G-
CSF, leptins
Immunosuppression activity
STAT4 Y693 Type I INF, IL-12, IL-23 Development of β cells,
humoral immune response
STAT5a, STAT5b Y694,Y699 IL-3, Prolactin, IL-2 Growth, differentiation,
apoptosis, immune system,
tumor immunity
STAT6 Y641 IL-4, IL-13 Innate immunity, proliferation
and maturation of β cells,
Activation of mast cells
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Table (2): STAT family, phosphorylation site,receptor activating STATs, Function

12. Activation and Regulation of JAK/STAT signaling
pathway
1. Binding of cytokine to cytokine receptors bring two JAK protein close enough so that
they phosphorylate each other.
2. Phosphorylated JAK proteins then phosphorylate tyrosine kinase on cytokine receptor
creating phosphor tyrosine docking sites for STAT.
3. STAT proteins moving freely in the cytoplasm binds to these docking sites.
4. JAK phosphorylate the STAT protein.
5. Phosphorylated STATs dissociates from the receptors.
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Figure (3): JAK/STAT Signaling

14. 6. The STAT protein binds to another STAT protein forming a dimer.
7. The STAT dimer enter into the nucleus bind to the DNA.
8. As STAT is a transcription factor, it initiates the transcription of a
particular gene
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15. Positive Regulation of JAK/STAT signaling
pathway
 Glucocorticoid Receptor: Acts as Transcriptional coactivator of STAT5 to
Promote STAT5 dependent transcription. (10)
 CBP and P300: Acts as a auxiliary activator of STAT to regulate the
response of JAK/STAT (11)
 SH2-2B can promote the activation of JAK2 induced by GH.
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(10) Stöcklin, E., Wissler, M., Gouilleux, F. & Groner, B. Functional interactions between Stat5 and the glucocorticoid
receptor. Nature 383, 726–728 (1996).
(11) Horvai, A. E. et al. Nuclear integration of JAK/STAT and Ras/AP-1 signaling by CBP and p300. Proc. Natl Acad. Sci.
USA 94, 1074–1079 (1997).

16. Negative Regulation of JAK/STAT signaling
pathway
 Protein Tyrosine Phosphatase:
-PTPB1 dephosphorylate JAK protein and simultaneously STAT
proteins
 PIAS ( Protein Inhibitor of activated STAT):
-Inhibits transactivation of STATs protein,
-Inhibit binding to GAS elements in DNA, thus prohibit STAT
dimer from inducing gene expression.
 SOCs (Suppressor of cytokine signaling):
-Inhibit JAK from becoming activated.
-Activate ubiquitin protease pathway which leads to
ubiquitination of JAK, leads to degradation of JAK 16

17. INHIBITORS AND CLINICAL APPLICATION
Medication that targets Jak/Stat pathway can be classified into 3 types
1. Cytokine antibodies or receptor antibodies
2. JAK Inhibitors
3. STAT Inhibitors
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Figure (4): Medications
that target the JAK/STAT
pathway can be classified
into three types

19. Cytokine Antibodies Clinical Application
Recombinant IL-2 metastatic melanoma and renal cell
carcinoma
Recombinant TNF Cancer
Type I INF Hepatitis C Virus
INF α Treatment of viral infections
Daclizumab Decrease transplant rejection
Siltuximab Treatment of idiopathic multicentric
Castleman’s disease.
CLINICAL APPLICATION OF CYTOKINE
ANTIBODIES
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Table (3): Cytokine Antibodies, and its clinical
application

20. Drugs Selectivity Clinical Application
Curcumin STAT3 Glioblastoma and squamous
cell carcinoma
Resveratrol STAT1, STAT3 Leukemia
Artemesinin STAT3 Various cancer, viral disease
and inflammation
CLINICAL APPLICATION OF STATs INHIBITOR
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Table (4): STATs Inhibitors and its clinical application

21. Drugs Selectivity Clinical Application
Tofacitinib JAK1, JAK3 RA , Ulcerative colitis,
polyarticular juvenile idiopathic
arthritis
Baricitinib JAK1, JAK2 RA
Ruxolitinib JAK1, JAK2 Myelofibrosis , polycythemia
vera, aGVHD
Filgotinib JAK1 RA(EU)
Upadacitini
b
JAK1 RA
Next Generation JAKs Inhibitor which are under phase II / phase III clinical trials are:
 Acrocitinib
 Itacitinib
 Fedracitinib
 Pacritinib
 Gandotinib
 Lestairtinib
First generation JAKs inhibitor approved by FDA/EU and its clinical
Application
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 Decernotinib
 Peficitinib
Table (5): JAKs Inhibitor, and
its clinical application

22. CONCLUSION
 The JAK/STAT Pathway is involved in cellular proliferation and differentiation, organ development
and immune homeostasis.
 Dysregulation of JAK/STAT signaling pathway is a major contributor to various diseases especially
malignant tumors and autoimmune diseases. (1)
 JAK/STAT inhibitors are currently used in autoimmune diseases, malignant tumors, GVHD and
infectious diseases.
 Research into more clinical indication is ongoing, including IL-6 driven diseases and Type I INF
related diseases.
 Future studies should aim to maximize efficacy and minimize adverse effect in patients in different
diseases, and to explore biomarkers that predict efficacy and offer prognoses.
(1) O’Shea, J. J. et al. The JAK-STAT pathway: impact on human disease and therapeutic intervention. Annu. Rev. Med. 66, 311–
328 (2015).
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REFERENCES
(1) O’Shea, J. J. et al. The JAK-STAT pathway: impact on human disease and
therapeutic intervention. Annu. Rev. Med. 66, 311–328 (2015).
(2) Xin, P. et al. The role of JAK/STAT signaling pathway and its inhibitors in
diseases. Int. Immunopharmacol. 80, 106210 (2020).
(3) Zhong, Z., Wen, Z. & Darnell, J. E. Jr Stat3 and Stat4: members of the family of
signal transducers and activators of transcription. Proc. Natl Acad. Sci. USA 91, 4806–
4810 (1994)
(4) Wilks, A. F. Two putative protein-tyrosine kinases identified by application of the
polymerase chain reaction. Proc. Natl Acad. Sci. USA 86, 1603–1607 (1989).
5) Lai, K. S. et al. A kinase-deficient splice variant of the human JAK3 is expressed in
hematopoietic and epithelial cancer cells. J. Biol. Chem. 270, 25028–25036 (1995).
(6) Fu, X. Y. A transcription factor with SH2 and SH3 domains is directly activated by
an interferon alpha-induced cytoplasmic protein tyrosine kinase(s). Cell 70, 323–335
(1992).

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(7) Wilks, A. F. et al. Two novel protein-tyrosine kinases, each with a second
phosphotransferase-related catalytic domain, define a new class of protein
kinase. Mol. Cell. Biol. 11, 2057–2065 (1991).
(8) Fu, X. Y. A transcription factor with SH2 and SH3 domains is directly activated by
an interferon alpha-induced cytoplasmic protein tyrosine kinase(s). Cell 70, 323–335
(1992).
(9) Horvath, C. M. STAT proteins and transcriptional responses to extracellular
signals. Trends Biochem. Sci. 25, 496–502 (2000).
(10) Stöcklin, E., Wissler, M., Gouilleux, F. & Groner, B. Functional interactions between
Stat5 and the glucocorticoid receptor. Nature 383, 726–728 (1996).
(11) Horvai, A. E. et al. Nuclear integration of JAK/STAT and Ras/AP-1 signaling by
CBP and p300. Proc. Natl Acad. Sci. USA 94, 1074–1079 (1997).
(12) The JAK/STAT signaling pathway: from bench to clinic, Xiaoyi Hu, Jing li, Maorong
Fu, Xia Zhao & Wei Wang

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Thank you!