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Flashcards in JAK STAT Deck (69)
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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



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

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



What is the STAT TF for drosophila called



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

1. 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



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.