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Molecular Cell Biology

Prof. D. Karunagaran
Department of Biotechnology
Indian Institute of Technology Madras

Module 7
Cell Signaling Mechanisms
Lecture 10
JAK-STAT Signaling








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The J anus kinase/signal transducers and activators of transcription (J AK/STAT)
pathway has important roles in immune functions, neuronal, hematopoietic and
embryonal development
Often acts locally, on cells of the same or similar type as the cytokine-producing
cells
JAK
J AK family of tyrosine kinases has four members, J AK1-3 and tyrosine kinase 2
(TYK2)
All members possess conserved kinase domain and catalytically inactive
pseudokinase domain at the C-terminus, regulating kinase activity of J AKs
Signal Transducers and Acti vators of Transcription (STATs)
The known STATs include STAT 1-4, STAT5A&5B and STAT-6
STATs contain a SRC homology 2 (SH2) domain involved in activation and
dimerization, a DNA-binding domain and a trans-activation domain which is located
at the C-terminus
Amino-terminal region of STATs is involved regulating STAT activity for the
formation of STAT tetramers and tyrosine dephosphorylation

Key regulators of the pathway
SOCS-Suppressor of cytokine signaling
PIAS- Protein Inhibitor of Activated STAT
PTPs-Protein tyrosine phosphatases







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JAK-STAT Signaling
Cytokine activates J AK, J H1 kinase domain contains two tyrosines which get
phosphorylated
J H2 is pseudo domain
J H6 and 7 mediate binding of J AK to the receptors
Results in phosphorylation of STATs
STATs dimerize and translocate to nucleus and initiate transcription




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Regulation of JAKs
SH2 domain of SOCS1 binds directly to phosphorylated J AKs leading to J AKs
inhibition
SOCS3 mediated inhibition requires its binding to activated J AK receptor.
CIS-Cytokine-inducible SH2 domain protein compete for STAT binding to J AK
SOCS can also bind to components of ligase and signal the protein for
degradation
During cytokine signaling J AKs and RTK can phosphorylate SOCS accelerating
its degradation
Kinetics of J AK-STAT signaling is affected by SOCS but not its initial activation
PTPs regulate J AKs
SHP1,2,CD45,PTP1B and T-cell PTP regulate J AKs
Mainly involve in dephosphorylation steps
SOCS and tyrosine phosphorylation on J AKs by inhibiting cytokines lead to
ubiquitylation of J AKs
J AKs are conjugated with interferon-stimulated gene 15 (ISG15)-a ubiquitin like
protein box
UBP43, ubiquitin specific protease removes ISG15 proteins








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Regulation of STATs
Phosphorylation, methylation, acetylation, ubiquitylation, ISGylation and sumoylation
modifies STATs post translationally.
Tyrosine phosphorylation of STATs is required for dimerization and nuclear
translocation
Dephosphorylation is required to translocate STATs from nucleus to cytoplasm
Serine phosphorylation at the specific site is required for the constitutive
activation of STATs1-4, STATs5A and 5B.
Serine kinases include ERK, J NK, PKC involved in phosphorylation of STATs
STAT1 methylation at specific arginine residue by protein arginine methyl-
transferase 1 is required for its increased DNA binding.
STATs interact with the CREB binding protein for gene activation.
After cytokine stimulation PIAS interacts with dimeric STAT and inhibit its DNA
binding activity thereby blocking the pathway
PTPs inactivate STATs in cytoplasm and in nucleus by dephosphorylation
STATs are able to form either homo or hetero dimer with other STATs which has
different DNA-binding activity
JAK-STAT pathway regulation and cross talks
Based on cytokine stimulation, signaling has either antagonistic or synergistic
effect
Example: Interferons (IFNs) inhibit IL4 response in monocytes. This inhibitory
effect is due to SOCS expression by IFNs that inhibits IL4-induced STAT6
During viral and other foreign antigen mediated immune responses NF-B and
STATs cross talk with each other
Example: NF-B can be activated by TNF. STAT1 is required for TNF-induced
apoptosis during immune regulation
SOCS1 mediates J AK-STAT and NF-B cross talk
IFN--activated STAT pathway negatively regulates SMAD signaling by inducing
inhibitory SMAD7
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JAK-STAT deregulation in immune diseases
In SCID patients, various mutations have been detected in J AK3 that is required
for signaling by cytokines like IL-2, 4, 7 etc.
In Asthma patients increase in levels of STAT6, required for IL-4 signaling and
T
H
2 cell responses
During allergic immune disease like dermatitis and asthma, SOCS3 is involved in
onset and maintenance of T
H
2 cells
During infection, virus can evade host immune system by inactivating IFN
activated J AK-STAT pathway
Paramyxovirus family of RNA viruses target STATs for degradation
Epstein-Barr viruses inhibit IFN receptor expression
Human cytomegalovirus inhibits IFN- induced expression of MHC-II by
degrading J AK1
Varicella-Zoster virus inhibits STAT1 and J AK2 expression
Individuals with defects in IFN-J AK-STAT pathway show increased susceptibility
to viruses and intracellular bacteria
STAT3 deficient mice show Crohns disease, a chronic inflammatory bowel
disease like symptoms. STAT3 deregulation was observed in patients with this
disease






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JAK-STAT inhibitors
Cryptotanshinone, herbal constituent of Salvia miltiorrhizainhibit the constitutive
STAT3 Tyr
705
phosphorylation prostate cancer cells blockage of STAT3
dimerization, nuclear translocation, and STAT3-dependent transcription
J AK Inhibitor I ,2-(1,1-Dimethylethyl)-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-
f]isoquinolin-7-one DBI , P6 Pyridone 6- ATP-competitive inhibitor of J anus
protein tyrosine kinases
J AK2 Inhibitor II, 1, 2, 3, 4, 5, 6-Hexabromocyclohexane, interacts with a solvent-
accessible pocket near the activation loop of J AK2 and direct specific inhibitor of
J AK2 autophosphorylation
PpYLKTK-mts, STAT3 Inhibitor IV, analog of the Stat3-SH2 domain-binding
phosphopeptide that contains a C-terminal membrane translocating sequence
and block of Stat3 activation
STAT5 Inhibitor N-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide,
targets the SH2 domain of STAT5
Study Questions
1. What are the key regulators of the J AK-STAT pathway?
2. Write a note on the J AK-STAT signaling
3. J AK-STAT signaling cross talk negatively regulates a) TGF- b) AKT c) p53
d) none of the above pathway
4. Match the following
Cryptotanshinone STAT5
1, 2, 3, 4, 5, 6-Hexabromocyclohexane peptide
PpYLKTK-mts J AK
N-((4-Oxo-4H-chromen-3-
yl)methylene) nicotinohydrazide
STAT3
5. Varicella-Zoster virus inhibits and expression
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