7. signalling Flashcards
what are rich in potential viral oncogenes?
signalling pathways - drivers of cancers in various settings.
what is the receptor at the top of the signalling cascade that results in cyclin D/E expression called? describe the viral version
ErbB2
vErbB3 lacks the extracellular part and the intracellular part is constituently active
name the central kinase that performs most of its phosphorylation at the membrane. describe the viral version
Akt
viral version has alterations meaning that it is permanently attached to the membrane meaning that it is constitutively active
name two viral oncogenic transcription factors that cannot be switched off?
v-fos and c-jun
what is conditioned medium?
this is media that has been exposed to cells and it will contain all their secretions
what is the difference between cell grown in non-cancerous conditioned medium and cells grown in cancerous conditioned medium? and what does this show?
in non-cancerous conditioned medium cells do not proliferate
in cancerous medium, cells proliferate
>cells have secreted something that triggers other cells to enter S phase.
what growth factor was being released from cancer cells?
TGFβ
how are cancer cells that secrete TGFβ
self-sustaining?
it can act in an autocrine manner
when an antibody that binds a RTK that recognises GF, how does this affect tumour growth?
this stops the volume of tumour increasing
how was EGF signalling first seen?
proteins harvest from salivary glands of new born mice shown to enhance development, premature opening of eyes and eruption of teeth
>after 20 years he isolated EGF
describe structure of EGF
6kDa
53 amino acids
3 disulphide bridges to help maintain structure
describe the paper that first reported the discovery of EGFR
> total cells lysate of cells that respond to EGF
purified with high affinity matrixes that bind specific glycosylated proteins
after lots of fractioning one band was seen on gel - EGFR
what did this EGFR band have the equivalent sequence? and what gave them confidence they had found the humans receptor?
oncogene v-ErbB
>the fact that they had identified a cellular homologue of this viral oncogene gave them confidence that they had found the human receptor
what antibody is used in an immunoprecipitation to show that when EGFR is activated by EGF it is phosphorylated?
anti-phosphotyrosine
describe EGFR
> single TM glycoprotein of 2010 aa
what happens when EGFR is activated on the extracellular side?
activation of EGFR causes conformational changes which result in the exposure of a single stranded beta hairpin dimerization arm that promotes the dimerization to another receptor, to form a homodimer
describe the intracellular portion of EGFR
tyrosine kinas domain followed by regulatory region bearing tyrosines to phosphorylate
what happens on the intracellular side when EGFR has dimerised?
intracellular domains are close enough and in the correct orientation for trans-autophosphorylation to occur
what are phosphor-tyrosines on inside of EGFR able to do?
transduce the signal to the rest of the cascade.
what is the next protein in the pathway after EGFR? and describe its structure
Grb2
it has three domains that fold independently of one another
what are the three domains of Grb2
1 Sh2 domain - this binds phospho-tyrosines
2 SH3 domains - these binds proline sich sequence and propagate signal downstream
describe the SH2 domain
it is symmetrical
two alpha helixes with some beta strands across the back
name a flexible interaction motif
SH3
what gives SH3 domains specificity and can also change its affinity to a certain peptide?
> loop structures coming off the beta barrel
> peptide sequence that SH3 is binding
what does Grb2 bind? and why?
Son of Sevenless
there are two proline rich regions in SOS
what is the EGFR receptor in Drosophila called?
Sevenless
what type of protein is SOS?
a Ras-GEF
where is SOS located? and what implication does this have?
in the cytoplasm
>recruited to the membrane by Grb2
>Ras is a membrane bound protein
what two mutants in Drosophila look similar?
Sevenless and SOS mutants
>EGFR pathway is used in development of eyes
>each bump normally consist of 7 cells
>in mutants they only have 6 and this results in roughened appearance
describe SOS function
it is a Ras-guanine nucleotide exchange factor
>causes GDP to dissociate from Ras and for GTP to associate to activate Ras
why is Ras found at the membrane?
it is farnesylated
how is Ras inactivated?
by hydrolysing GTP to GDP - this can occur intrinsically or accelerated with a GAP - GTPase activating protein
what is the major difference in conformational change when Ras is bound GTP?
when Ras is activated the effector loop change conformation (forms a more ordered alpha helical structure) and can interact with and regulate the function of other proteins
what is the most mutated oncogene in cancer and what implication does this have?
Ras
lots of things can occur downstream of Ras signalling e.g. cell cycle progression and cell motility
what are the three kinases downstream of Ras and what type of kinase are they?
Raf
Mek
Erk
they are serine threonine kinases that sequentially activate each other
how is raf activated?
it is recruited to the membrane by ras and this activated Raf through an allosteric mechanism
what is used to physically organise the MAP kinase module so that P occurs in the correct order and most efficiently?
scaffold proteins
what happens once Erk is activated?
it translocates into the nucleus
what does Erk P?
Jun and Fos - activates and stabilise them
what are jun and Fos?
TF that bind to cyclin D1 promoter to drive proliferation
from the stimulation of a cell by EGF to this phosphorylation of Jun and Fos takes less than 5 minutes, how long does it take to for the production of cyclin D?
up to 5 hours
what is one of the most mutated TS in cancer?
PTEN
what is present on the inner leaflet of the membrane? and what are they?
Phosphoionsitides
>they are two fatty acid chains linked to a glycerol which is attacked to an inositol
what does PTEN do?
remove phosphates added to position 3 of Phosphoionsitides by PI3kinase so that PH domain can no longer bind membrane (prevents ATK activation)
what is the regulatory subunit of PI3kinase? describe it?
p85
>it has an SH2 domains
>this recruits PI3Kinase to the membrane so that it can P Phosphoionsitides
how is PI3kinas kept unactive in non-stimulated cells?
it is not localised to the membrane
when localised to the membrane, what does PI3Kinase do??
it uses ATP to attach phosphate to the 3 position on the inositol head group of inositol group of Phosphoionsitides
>produces 3 different species that are signalling molecules: PIP, PIP2, PIP3
what levels rapidly rise after RTK stimulation with GF? and what does this imply? what levels don’t change?
PIP2 and PIP3
>they are important for downstream signalling - they are the second messengers
>PIP levels don’t change
what can PIP3 function as?
a docking site for other proteins to be activated on the membrane
what domain binds PIP3? and what do they bind? describe this interaction
PH domains
>they bind phosphorylated inositol head groups
>the loops between two beta strands have a high affinity of binding to PIP3
name two proteins that have PH domains, and what happens when they are recruited to the membrane?
PDK1 and AKT
>PDK1 is constitutively active, when brought to membrane in close proximity to AKT it P AKT and partially activates it. mTOR P ATK again to fully activate it.
ATK P lots of things, give some examples
> AKT contributes to cell cycle progression through the phosphorylation of CDK inhibitors p21 and p27
they are excluded from nucleus
p21 is then degraded
what P cyclin D and what affect does this have?
GSK3
this results in cyclin D degradation
what inhibits GSK3 and what affect does this have?
P by ATK
>this is an inhibitory phosphorylation which results in the stabilisation on cyclin D1
what TF leads to the activation of p27? and how is this regulated by ATK?
FOXO
AKT can P this causing its nuclear exclusion
what two types of affects does AKT have?
pro-proliferative and anti-apoptotic
define how ATK has anti-apoptotic affects?
inactivated Bax
upregulates Bcl2
what are mice heterozygous for PTEN like?
they are more prone to tumours in some tissues over others
what are the two types of therapeutic intervention made on EGFR and what do they do?
- monocolonal antibody prevent EGF from binding receptor
- small molecule that inhibits EGFR kinase activity
why do farnesyltransferase inhibitors inhibit that prevent Ras from being localised to the membrane have no therapeutic value?
other enzymes that can add other lipids to Ras in order to localize it to the membrane for it to have activity
how is Raf being therapeutically targeted?
there is a common mutations found in melanomas in Raf. there is a small molecule that targets this mutant and not the WT form and so does not have off target effect. this has proven clinically useful.
how is ATK being targeted therapeutically?
> it has proven hard to directly inhibit this
>blocking its recruitment to the membrane prevents activation
why is can very specifically targeting something in cancer eventually not work?
> cancer cells genomes are very unstable
>applying a selective pressure will select for cells that are resistant to drugs
