JAK STAT Flashcards
In simple terms, what is the JAK/STAT signalling pathway
Transmembrane receptors have an intracellular domain that is associated with JAK (a kinase). Ligand mediated dimerisation creates a receptor/JAK complex causes activation of the complex, activating tyrosine kinase, phosphorylation of tyrosines in correct context recruits STATs (TFs) which are activated by tyrosine phosphorylation and dimerise. The STAT dimer translocates to the nucleus, bind to DNA and activate transcription
How many JAKs are there?
4: JAK1,2,3 and JAK type 2
How many STAT TFs are there?
7: STAT1,2,3,4,5a,5b,6
What does the pathway do
Key role in immune system development - specifically the differentiation of naive T cells.
Also has a role in haematopoiesis
Which STATs are involved in differentiation of T helper and T regulatory cells?
STAT3,4,5 and 6
What somatic JAK mutation causes SCID
A somatic JAK3 mutation
Which JAK/STAT ligands have an anti inflammatory effect
IL-4, IL-5 and IL-10
Which JAK/STAT ligands have proinflammatory effects
INF-y, IL-2, IL-6 and IL-12
What is the role of the JAK/STAT pathway in haematopoiesis
HPCs undergo self-renewal throughout life, the process of maintaining and differentiating HPCs is controlled by JAK/STAT pathway ligands (Thrombopoietin and erythropoietin)
What is the result of an embryonic JAK2 KO mouse
Embryonic lethal due to the absence of erythropoiesis i.e. no red blood cells.
What is the result of a JAK2 gain of function mutation
Causes blood cancers - Polycythaemia, Primary Myelofibrosis, Essential thrombocythaemia
Which STATs are involved in erythropoiesis
5a and 5b
what is the result of an EPO receptor KO
Embryonic lethal due to an absence of erythropoiesis
What is the result of a thrombopoietin receptor KO (AKA c-MPL receptor)
Knockout is viable but has a reduced number of HPCs and require thrombopoietin for their maintenance.
What is thrombocytopenia
A condition where the individual has a low platelet blood count. Caused by a mutation in the TPOR
Which three receptors dimerise and function as homodimers
TPO-R, EPO-R and G-CSFR
Do JAK/STAT signalling components experience crosstalk and how was this investigated
Antibodies specific to STAT1 and STAT3 as well as their phosphorylated forms gives an indication of receptor activation - IL-6 is thought to activate STAT3 but also leads to some STAT1 activation
Conversely IFN-y is thought to activate STAT1 but leads to a small amount of STAT3 activation.
Oncostatin M (OSM) strongly activates both STAT1 and STAT3 alluding to cross talk
Why is JAK/STAT analysis difficult in vertebrates
Because of their complexity and compensatory mechanisms through cross talk.
Name three homologues of IL-6
IL-11, LIF (leukemia inhibitory factor), OSM (oncostatin M)
What is the role of the cytokine IL-6
Proinflammatory cytokine inducing an inflammatory response.
What is the structure of IL-6 and IL-6 like ligands
Comprise four long alpha helices (A, B, C, and D) which are arranged in a way that gives them an up-up-down-down topology.
Do ligands express crossover between the receptors they bind
Yes, carrying out the same biological processes.
How is specificity for an individual ligand inferred by the receptor
By a different combination of transmembrane receptors (one must be gp130 containing)
What are the two types of gp130 containing receptor complexes
gp130 heterodimerising with a short receptor monomer - an alpha receptor
Or a gp130 heterodimerising with a long receptor - a beta receptor
What is the difference between the alpha receptor and the beta receptor for IL-6 like ligands
The beta receptor monomers are capable of signalling as they posses the JAK binding domain, whereas the alpha monomers are not
Name two alpha monomers
IL-6R alpha
IL-11R alpha
Name two beta monomer signalling receptors
OSMR-beta
LIFR- beta
Why does the affinity for the IL-6 like ligands have to be extremely high to its receptor
Ligands are present in the blood at vanishingly small amounts
What event follows ligand binding
The receptor-ligand complex is rapidly internalised via cytoplasmic dileucine motifs on the beta receptors
Which site on the IL-6 ligand binds to the extracellular domain of either the alpha or beta receptor
Site I
Which site binds with the long gp130
Sites II and III
What do the extracellular domains of the IL-6 binding receptors contain, and what is their importance
Made up of a structurally repeated motif - fibronectin type III (FNIII)
Ligand dependent receptor activation and signal transduction depends on the three, membrane proximal FNIII domains
What structures does JAK bind to
the intracellular Box1, 2, and 3 domains, present in both gp130 and beta receptors.
What is the benefit of using drosophila to study the JAK/STAT pathway
Simplified version of the human pathway with only 1 JAK and 1 STAT, so if you prevent its signalling there are no compensatory mechanisms/cross talk.
What are the three ligands for the drosophila JAK/STAT pathway
Unpaired-1, 2, 3
What are the two receptors for the drosphila JAK/STAT pathway
Domeless and Latran
What is the drosophila JAK kinase called
Hopscotch
What is the STAT TF for drosophila called
STAT92E
How do the phenotypes of STAT unpaired and domeless mutants differ in drosophila
They don’t - all are extremely similar showing gaps with missing segments and a lack of posterior respiracles
How was JAK/STAT signalling location discovered in the drosophila
Fusion of half disabled B-galactosidase proteins to the intracellular domain of the domeless receptor
Upon dimerization of the JAK/STAT receptor full activity of the B-galactosidase is enabled.
These proteins were ubiquitously expressed
Upon addition of X-gal nblue staining is seen in the areas where the two receptors come into contact. i.e where signalling is present.
How is JAK/STAT signalling regulated in the fruit fly
Temporally and spatially
What 3 cell types can fly HPCs differentiate into
Plasmocytes - essentially macrophages that clean up cell debris, bacteria and fungi
Crystal cells - involved in melanysation response (blood clotting) - includes wound healing
Lamellocytes - large cells that engulf the eggs of parasitic wasps
What is the role of the blood in drosophila
To circulate blood cells that fight infection and promote clotting in the event of injury - not for O2 transport - gaseous oxygen diffuses into the trachea
When are the two rounds of haematopoiesis
- Embryonic stage
2. Larval stage - occurs in the lymph gland located either side of the heart
Describe larval haematopoiesis
The lymph gland has a number of lobes: the primary, secondary and tertiary. Within the primary lobe there is an inner medullar and an outer cortical
Differentiating cells move out and end up in the cortical zone - JAK/STAT pathway ligands and receptors expressed in the lymph gland
Following pupation the lymph gland bursts and all the blood cells are released into circulation
Plasmocytes phagocytose all apoptotic cells that were involved in metamorphis into the adult fly
Does the adult fruit fly make new blood
No
When do parastic flieslay their eggs in drosophila larvae
Late L2 or L3 larvae
How does the drosophila larvae overcome the parasitic fly
Lamellocytes encapsulate the wasp larvae
What is the result of the hopscotch tumour-less-lethal gain of function termperature sensitive JAK kinase mutation
Causes melanotic tumours from increased lamellocytes
Larvae wrongly think they are being attacked by the parasitic wasps
How is the hopscotch mutation temperature sensitive
At 17 degrees C - JAK/STAT signalling has somewhat increased, as have the number of lamellocytes but also the number of overall cells
Whereas at 25 degrees C - Signalling has massively increased with a huge increase in overall cell count especially that of lamellocytes
How do HPCs differentiate at 17 degrees C
Stem cells over self renew but the progenitors give rise to normal blood cells
How do HPCs differentiate at 25 degrees C
Stem cells over self renew and the progenitors differentiate to give rise to massive numbers of lamellocytes - creating lethal tumours.
What is the pathway for Tpo signalling
TPO acts on the MPL receptos, activating JAK2 - this activates STAT3/5a/5b and leads to transcription of target genes (platelet formation)
What is the pathway for Epo signalling
Epo acts on EPO-R - activating JAK2 - Activates STAT3/5a/5b leading to target gene expression (erythrocyte formation)
What are the three “classic” myeloproliferative neoplasms
Primary myelofibrosis, Essential thrombocythemia and polycythemia Vera
What is primary myelofibrosis (MF)
Chronic blood cancer in which the bone marrow function is impacted by becoming scar tissue. Leads to a reduction in the amount of red blood cells being produced
Why do MF patients have an enlarged spleen
Secondary haematopoiesis in the spleen trying to make up for the fact patients aren’t producing enough blood
What is essential thrombocythemia
A blood malignancy that is typically characterised by an elevation in platelets in the blood. Common symptoms of blood clotting and bleeding
What is polycythemia Vera
Elevation of red blood cells - patients often exhibit elevated white blood cells and platelet counts as well as a enlarged spleen
What is the result of the V617F mutation (valine to phenylalanine)
A six fold increase in JAK activity in the absence of a ligand - when combine with EPO the activity is even greater
How did STAT5 levels differ in the V617F mutant compared to WT and how was this measured
Measured by western blot - total STAT levels remained roughly the same however phosphorylated STAT5 levels saw an increase in the mutant JAK2
How does the JAK2 V617F mutant differ to WT
The JH1 domain is the kinase domain that phosphorylates STAT - JH2 domain is a pseudo-kinase domain which has an amino acid 617 substitution in the mutant but still retains some kinase activity, acting as a negative regulator of the activity of the JH1 domain.
How can the TPO receptor MPL be made to be constutively active
Exists as a monomer which, upon ligand binding causes JAK2 phosphorylation. Mutants that cause dimerisation activate the receptor even in the absence of a ligand.
What do all somatic mutations of calreticulin in Myeloproliferative neoplasms ultimately result in
A +1 frame shift mutation that replaces the C terminus of wild type calreticulin with a common novel peptide sequence
What is the role of calreticulin and what is the role of the mutant CALR
Chaperone protein found in the endoplasmic reticulum - Mutant acts to dimerise the MPL receptor even in the absence of ligand.
What was the mouse model used to investigate the action of methotrexate
Gain of function human JAK2 knock in mouse model - comparing WT, heterozygous and homozygous mice.
What is ruxolitinib
A JAK2 inhibitor currently used in the treatment of MPNs
What was the effect of MTX treatment on a patient with essential thrombocythemia with a scleroderma after not responding to normal treatments.
Platelet count was normalised within 45 days
Why is MTX an attractive alternative to ruxolitinib
£32 a year compared to £43,000. Also seems to act via the same pathway - inhibition of JAK/STAT signalling.
