L3: cell communication 3 (tyrosine kinase signalling pathways)

describe the structure of the EC domain of a transmembrane receptor

hydrophilic

interacts with ligand

often contains conserved elements (e.g. IG-like domains, EGF-like domains, fibronectin type III repeats, cystein-rich regions)

describe the structure of the transmembrane segment of a transmembrane receptor

usually 20-25 aa of alpha-helical structure made up of hydrophobic aas stabilised via interactions with fas of the lipid bilayer

describe the structure of the IC domain of a transmembrane receptor

hydrophilic domain that interacts with downstream signalling machinery

what specifies a receptor tyrosine kinase

tyrosine kinase domain in the IC domain of a transmembrane receptor

how are transmembrane receptors inserted into the plasma membrane

genes encoding transmembrane proteins contain a single peptide which directs the newly synthesised protein to the ER

how is information transmitted from an EC ligand binding site to the IC effector side of a receptor

one of two ways:

conformation change to multi-pass transmembrane receptors (often GPCRs)

dimerization/multimerization of a single-pass transmembrane receptor

how many receptor tyrosine kinases are there

58 single-pass transmembrane receptors

name the 2 types of protein tyrosine kinases

receptor tyrosine kinases (RTK)

cytokine receptors (JAK kinase)

in what state are RTKs active

dimerisation

describe how a ligand binding to a RTK activates it

in resting state, TK poorly active due to being blocked by the activation lip

ligand binding causes conformational change, bringing 2 RTKs together + inducing dimerization

RTKs in dimer phosphorylate each other to activate kinase activity

how was the Ras/Raf/MEP/ERK pathway originally discovered

research into drosophila eye development

describe how Ras is activated following activation of a RTK/CR

ligand binding causes dimerisation and phosphorylation of cytosolic tyrosine residues (Y residues)

p-Y residues act as docking sites for SH2 domain of adaptor protein Grb2

SH3 domains of Grb2 bind proline-rich regions of Sos (a GEF/GDP→GTP exchange factor)

Sos GEF activity swaps GDP→GTP in membrane associated Ras proteins

describe the process of the Ras/Raf/MEK/ERK kinase cascade pathway

Ras activated via exchange of GDP → GTP by GEF

active Ras recruits, binds, + activates Raf (STK)

Ras hydrolysed to release active Raf which phosphorylates + activates MEK (STK)

MEK phosphorylates + activates ERK (STK)

ERK translocates to nucleus to interact with TFs and trigger gene expression

name teh 3 levels of negative regulation in the Ras/Raf/MEK/ERK pathway 

1. Raf -vely regulated by its own N-terminal region, the 14-3-3 protein, and its own phosphorylation. all of these need to be removed before the molecule can be activated

2. ERK contributes to multiple levels of -ve regulation including ‘-ve phosphorylation’ of Sos to prevent binding to Grb2 → prevents Ras activation

3. activated PDGFR recruits multiple -ve regulators of the pathway including a RasGAP, phosphates, and triggering endocytic internalisation

how are small GTPases activated

being bound to GTP

what regulates small GTPases

GEFs and GAPs

what are activating mutations in the Ras/Raf/MEK/ERK pathway associated with

melanomas (activated in 80% of cutaneous melanomas)

how have BRAF inhibitors been used clinically

used to treat malignant melanoma:

early inhibitors associated with spectacular initial results followed by distressingly rapid development of resistance, disease relapse, and progression

more recent inhibitors have selectivity for mutant BRAF and reduced off target effects (vemurafenib + dabrafenib)

patients often treated with both BRAF and MEK inhibitors (like trametinib) to reduce resistance development + improve results

immunotherapies also now widely used

name the ligands of cytokine receptors

interleukins, interferons, cytokines, chemokines, GFs

name the 3 non RTK single-pass transmembrane receptors with receptor associated tyrosine kinases

cytokine receptors - signal via JAK/STAT

T cell receptors - binds to MHC/antigens presented to them + signal to nucleus

integrins - responsible for IC adhesion + focal adhesions

how is JAK regulates

inhibited by SOCS + PTP 

ubiquitinated in proteosome

how is STAT regulated

inhibited by PIAS in the cytoplasm and PIAS + PTP in nucleus

name an example of a cycle mediated by JAK/STAT signalling

inflammation/immunity

haematopoiesis

what disease is caused by mutated JAK/STAT pathway activation

myeloproliferative neoplasms

what do all myeloproliferative neoplasms feature

JAK/STAT pathway activation

what is the most common JAK/STAT mutation

JAK2 V617F

describe myeloproliferative neoplasm treatment

no curative treatment other than BM transplant

name the 4 domains in JAKs

FERM domain

SH2-related domain

kinase domain

pseudokinase domain

how do type I inhibitors work

bind to active conformation of the kinase with the aspartate residue (with backbone) of the DFG motif pointing into the ATP-binding pocket

how do type II inhibitors work

bind and stabilise the inactive conformation of the kinase with the flipped aspartate residue facing outward of the binding pocket

how do type III inhibitors work

occupy an allosteric pocket adjacent to the ATP-binding pocket but doesnt overlap with it

how do type IV inhibitors work

bind to an allosteric pocket remote from the ATP binding pocket

name a JAK inhibitor and how it works

ruxolitinib (inhibits JAK1/2 - binds to ATP binding pocket to prevent activity/downregulate)