Cell signalling Flashcards
how is cell signalling altered in cancer?
3
- signalling turned on or unresponsive to inhibition
- volume of signalling increased (more receptors or more ligand)
- signalling at wrong time or place
alteration of cell signalling enable the cell to evade normal regulation
what is gefitinib?
a targeted therapy for EGFR mutations in non small cell lung cancer. It works by binding to the intracellular enzyme (tyrosine kinase) of the EGFR to directly block signals turned on by triggers outside or inside the cell.
what is cell signalling?
the mechanisms that cells use to perceive and adapt to their state and surroundings
- an interconnected series of molecular events that enables perceptions and transmission of information
what is signal transduction?
the biochemical process that facilitates information processing by the cell
how is information transmitted through pathways?
4| changes in protein state
- ligand and receptor (binding and dissociation)
- protein phosphorylation (post translational modification)
- G protein bound to GTP/GDP (conformational change)
- nucelocytoplasmic shuttling (localisation)
altered protein state is mediated by what two types of proteins
writers (eg kinases)
erasers (eg phosphatases)
cellular regulation is controlled by cell signalling and gene expression, compare these two parts
cell signalling
- enables transmission from outside cell to nuc;eus
- fast ON and OFF (secs to mins)
- transient changes (mins to hrs)
- spatial/directional repsonses and organisation
- energenticallt cheap (no protein synthesis)
gene expression
- slow ON and OFF (mins to hrs)
- stable changes (hrs to yrs)
- limited spatial responses
- energentically costly (transcription and translation)
whats the difference between positive and negative feedback?
Positive feedback occurs to increase the change or output: the result of a reaction is amplified to make it occur more quickly.
Negative feedback occurs to reduce the change or output: the result of a reaction is reduced to bring the system back to a stable state
what are the reasons to think signalling occurs in a network rather than a pathway?
- cross talk (interaction of signalling pathways)
- convergence (multiple signals activate one, integration of multiple signals)
- divergence (one signal elicits multple outcomes, many proteins function in multiple pathways)
what are the reasons to think signalling occurs in a network rather than a pathway?
-cross talk occurs between signalling pathways
-integration of multiple signals
-many proteins function in multiple pathways
what three ways can information cross the membrane?
- membrane permeable signals (eg lpid soluble hormones (steroids))
- gated channels (eg ions)
- transmembrane receptors (eg growth factors)
what are the three domains in transmembrane receptors?
- Discriminator domain binds specific ligand
- Transmembrane domain anchors in membrane
- Effector domain directly or indirectly linked to intracellular enzymatic activities
describe the indirect and direct function of the effector domain in transmembrane receptors?
Indirect: receptor associates with separate kinase protein
Direct: kinase domain in same protein structure as transmembrane receptor
after ligand binding to receptor tyrosine kinases, what do conformational changes in the cytoplasmic domains lead to?
- auto-phosphorylation and recruitment of signalling proteins
- phosphorylation of scaffold proteins that organises signalling complexes
oncogenic mutations in receptor tyrosine kinases increase signalling through what
over expression of receptor and mutations affecting structure lead to ligand independent firing
mutations in receptor tyrosine kinases lead to
ligand independent firing
what two ways can ligand independent firing occur?
- overexpression of receptor
- mutations affecting structure
Egfr mutations are found in what 4 exons of the egfr gene
18 19 20 21.
Mutations in exons 19 and 21 (L858R) account for 90%
what are the three most commonly modified amino acids in eukaryotes
phosphorylated
Ser > Thr > Tyr
give five possible consequences of phosphorylation
o Addition of phosphate group induces conformational changes
o Activation/inactivation
o Form/hinder protein interactions
o Sub-cellular relocalization
o Degradation via proteasome
what percentage of cellular protein may be ubiquitylated at any one time
20%
whats the function of E1, E2 and E3 in ubiquitylation?
E1 - recruits ubiqitin protein (2 in humans)
E2 - activated ubiquitin transfers E2 conjugating enzyme (50 in humans)
E3 - ligase that transfers ubiquitin to lysin group on target protein, primary determinants of substrate specificity (500 in humans)
give 4 different protein modifications that occur in signalling
Phosphorylation
Acetylation
Methylation
Ubiquitylation
give 4 different protein modifications that occur in signalling
Phosphorylation
Acetylation
Methylation
Ubiquitylation
give a method of identifying ubiquitylation sites
using proteomics
mass spectrometry proteomics;
o Digest proteins into peptides leaves a di-Glycine tag at ubiquitylated sites
o This tag can be identified on peptides using mass spectrometry
o Measure abundance and exact sites of ubiquitylation of 1000s proteins
give two trancriptional targets of beta catenin in the wnt pathway
- Cyclin D1 (CCND1)
Drives G1 to S phase transitions in cell cycle - Myc
Proto-oncogene encoding multifunctional transcription factor involved in cell cycle, apoptosis
Wnt pathway activation induces expression of genes that lead to proliferation
wnt pathway activation induces expression of what type of genes
genes that lead to proliferation
give four functions of wnt during embryogenesis
Determination of body axis (which way is up)
- Injecting Wnts into frog embryo causes axis duplication (dual headed embryos)
Cell fate decisions
- Stem cell renewal and differentiation
Cell proliferation
- Wnt signalling activated proteins that promote cell cycle
Cell migration
- Promotes epithelial mesenchymal transition (EMT)
what diseases involve aberrant wnt signalling?
- Cancer(s)
Wnt regulates stem cell homeostasis in the gut; inappropriate activation leads to colorectal cancer - Neural tube defects
Neural tube does not close (e.g. spina bifida) - Bone density defects
Mutations that promote Wnt signaling lead to high bone density and vice-versa - Tooth agenesis
Dental abnormalities in which teeth fail to develop - Diabetes
Mutations in Wnt transcription factor link to elevated risk for type II diabetes
what diseases involve aberrant wnt signalling?
- Cancer(s)
Wnt regulates stem cell homeostasis in the gut; inappropriate activation leads to colorectal cancer - Neural tube defects
Neural tube does not close (e.g. spina bifida) - Bone density defects
Mutations that promote Wnt signaling lead to high bone density and vice-versa - Tooth agenesis
Dental abnormalities in which teeth fail to develop - Diabetes
Mutations in Wnt transcription factor link to elevated risk for type II diabetes
why is Wnt involved in so many different diseases?
because they regulate such fundamental biological processes as cell differentiation proliferation which are intrinsic to all biological processes
what are the two types of Wnt signalling pathways?
what activities are each involved in & what disease are they related to
“Canonical”
* β-catenin dependent Wnt signaling (key effector protein)
* Embryogenesis; differentiation and proliferation
* Important role in many cancers (e.g. colorectal)
“Non-canonical”
* Planar Cell Polarity
* β-catenin independent Wnt signaling
* Tissue patterning, cell polarization and migration
* Neural tube defects (e.g. spina bifida)
* Metastasis?
what is the destruction complex and what are its constituents?
the Wnt pathway
desstruction complex responsible for breaking down beta catenin and direct towards proteosome ubiquitin system
core components include the scaffold proteins axin and adenomatous polyposis coli (APC), as well as the Ser/Thr kinases casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3)
what happens when the Wnt ligand binds to its receptor (LRP5/6)
- When ligand binds, a key component of the destruction complex, Axin is sequestered to the membrane.
- Destruction complex disbanded and beta catenin is no longer broken down so that it can get to work in the nucleus to regulate transcription
give three features of wnt ligands
- Small, secreted glycoproteins with specific lipid modifications to target to membrane
- May function as short- or longer-range morphogens through paracrine and/or autocrine mechanisms
- 19 Wnt genes in human genome encoding Wnt proteins (highly conserved gene family)
what receptos do wnt ligands bind?
Wnt ligands forms trimeric complex with Frizzles receptor and co-receptor LRP5/6
(may also bind to certain RTK type receptors to activate other pathways)
what are Dickkopf proteins?
3
Dickkopf (Dkk) proteins are Wnt antagonists (through binding LRP receptor)
Dkk are transcriptional targets (turned on) by canonical Wnt signaling
Dkks provide negative feedback to regulate Wnt activity
what is Romosozumab ?
an anti-sclerostin antibody that promotes bone formation used as a treatment for osteoporosis
(recessive mutations of sclerostin assosciated with increase bone mass)
what is Romosozumab ?
an anti-sclerostin antibody that promotes bone formation used as a treatment for osteoporosis
(recessive mutations of sclerostin assosciated with increase bone mass)
how are constituents of the wnt pathway involved in bone formation/ increase in bone mass
Wnt signalling promotes bone formation
Dkk1 and sclerostin bind Wnt co-receptors and inhibit signalling
Recessive mutations of sclerostin associated with increased bone mass
whats the role of dishevelled in the wnt pathway
Disshevelled protein recruited to Frizzled receptor on Wnt activation, preventing action of destruction complex
Disshevelled proteins fulfil roles in canonical and non-canonical signalling (cross-talk between pathways?)
what are the three critical components of transduction of the wnt signal?
- Phosphorylation of Beta-catenin by GSK3B kinase leads to degradation via the proteasome
- The destruction complex facilitates the phosphorylation of Beta-catenin by GSK3B
- Binding of Wnt ligand induces recruitment of two critical components, dishevelled and Axin to the membrane receptor components, which alters destruction complex, allowing Beta-catenin to evade destruction.
oncogenic and tumor suppressor mutations occur to which proteins in the wnt pathway
oncogenic mutations in beta catenin
tumor suppressor mutations in APC or axin
give one potetntial therapeutic stretegy for maintaining beta catenin destruction
A molecule that Inhibits Tankyrase
Tankyrase is an enzyme that degrades Axin through parsylation (addition of ADP-ribose units) - when inhibited it preserves the destruction complex and prevents beta-catenin accumulation
what are the two main functions of beta-catenin
signalling and structural
Signalling function - central effector of Wnt signalling pathway
Structural function - component of cell-cell junction (adherins - E-cadherin)
breifly describe beta catenin function in the nucleus
after beta catenin is imported into the nucleus where it binds to TCF transcription factors and releases inhibitory factors
what is a classical means of measuring Wnt signalling?
using a transcriptional reporter consisting of TCF binding regions linked to luciferase
what could be an early gatekeeper event in development of colon cancer?
Activation of Wnt signalling (through APC mutations)
How is the Wnt pathway linked to colon cancer?
Wnt signalling plays a key role in the development and homeostasis of the villus epithelium - activation of which is a gatekeeper in development of colon cancer
what happens in the villus crypt when APC is mutated?
Progenitor like phenotype accumulation at the site of future polyp formation
failure of mutant cell to continue outward migration
Describe the location of stabilising mutations in beta cateinin
stabilising mutations of beta catenin delete codon encoding phosphorylation site (various serin residues)- allow beta catenin to escape destruction via the proteosome
how frequent are truncation mutations in colorectal cancer?
60%
(truncation mutations remove important binding domains allowing beta-catenin accumulation (not complete knockout of the protein)
how frequent are truncation mutations in colorectal cancer?
60%
(truncation mutations remove important binding domains allowing beta-catenin accumulation (not complete knockout of the protein)
What are the three experiental systems used in cancer signalling?
Clinical samples: human tumour or biofluids
Model organisms
Cultured human cells: immortalised cells or primary cells from tumours
give four ways to experimentally perturb signalling
- addition of a ligand
- genetic deletion to remove key proteins
- pharmacological (small molecular inhibitor)
- mutant kinase + ATP analog
What ways can Wnt signalling be activated/inhibited
- ligand (wnt conditioned media, or recombinant purified wnt ligand)
- pharmacological inhibition (inhibition of GSK3B using lithium)
- genetic (kockout cells, RNAi)
What is the role of GSK3B in the wnt pathway?
A kinase that phosphorylates beta catenin, targeting it for destruction
describe how to measure wnt signalling output
use a Topflash system that links TCF binding domains to reporter luciferase
introduce into cells of interest via a plasmid
measure TOpflash/Fogflash ratio
HOw do you identify proteins involved in a signalling pathway cia genome wide RNAi screens
- Use short RNA molecules (siRNA) to inhibit expression of specific genes
- Proceed by introducing all of these siRNA molecules into cells, and observing the effect on a phenotype of interest
can detect activating and inhibitory regulator
(does not provide machanistic information)
what are the limitations of using RNAi screens to study signalling pathways
does not clarify mechanisms
may be specific to the model organism used
How do protein-protein interactions differ and what kinds of things are they influenced by?
- Differing properties: Permanent/transient, Obligate/non-obligate
- Influenced by: Post-translational modifications, Sub-cellular location, Rate of synthesis and/or degradation
how do you detect interactions using yeast two-hybrid
- fuse test proteins X and Y to activating and binding domains of transcription factor to make two hybrid proteins
- the reporter gene is the one that allows yeast to grow when turned on, is only active when activation and binding domains interact
- if test proteins X and Y interact the transcription factor is reconstructed, the reporter gene is turned on and the yeast cells grow
limitations: other proteins might be needed for interactions
how do you detect interactions using yeast two-hybrid
- fuse test proteins X and Y to activating and binding domains of transcription factor to make two hybrid proteins
- the reporter gene is the one that allows yeast to grow when turned on, is only active when activation and binding domains interact
- if test proteins X and Y interact the transcription factor is reconstructed, the reporter gene is turned on and the yeast cells grow
limitations: other proteins might be needed for interactions
what is yeast two-hybrid?
4 points
- An assay performed in yeast cells to test whether two proteins interact
- Can be applied to proteins from any organism
- Can be scaled up to test 1000s of proteins
- Has been used to map 1000s of human protein-protein interactions
how is ELF3 involved in the wnt pathwya and in colorectal cancer?
ELF3 is an antagonist of Wnt/beta-catenin signalling – (more abundant in mutant cells that the wildtype cells)
Elf3 represses beta catenin and beta catenin represses ELF3
ELF3 is associated with good prognosis in colorectal cancer
