cell cycle growth and regulation Flashcards
what stage are most cells in the cell cycle *
not constantly dividing - in absence fo grwoth signals they enter G0 or quiescent phase
describe cell cycle entry *
requires external stimulus this is translated into transcription of cMyc gene which stimulates the expression of the genes and proteins that regulate the cell cycle - these are transiently stimulated
if you stimulate the receptor with GF - cause increase in transcription of cMyc - this is fast transcription and occurs just before the synthesis phase - cells then enter s phase
the GF receptor is stimulated by a ligand - these are mitogenic factors that activate the signalling cascade
describe GF stimulation of signalling pathways *
mitogenic GFs ie growth factor signals from other cells eg hepatocyte GF released after damage
this activates signalling cascades involving receptor protein tyrosine kinase and small G (GTP-binding) protein (Ras)
these are master regulators that go into nucleus and activate a variety of factors (ie regulatory gene proteins, early response genes (C-Myc), delayed response genes) - cascade will activate cell cycle control genes
intermediate early genes that are activated include c-Jun, c-Fos, c-Myc - they are TFs and activate other genes - they are upregulated in cancer
what are the key components of signalling pathways *
regulation of enzyme activity by kinases
adapter proteins
regulation by GTP-binding proteins
mechanism of signalling by peptide GF *
ligand binds to receptor = receptor dimerisation = cross phosphorylation of tyrosine kinases - activating themselves
the phosphorylation provides docking site for adapter proteins
adaptor proteins bring together kinase and substrate for protein
different phosphorylation sites = different adaptor proteins - this scaffolds cascade and results in signal being relayed
describe how cancer treatment targets signalling by peptide GF *
block cascade at beginning
herceptin binds to extracellular domain - prevent ligand binding - prevents all cell signalling
herceptin is anti-Her2 Ab
describe the function and mechanism of adaptor proteins *
tyrosine phosphorylation provides docking sites for adaptor proteins
this mediates protein-protein interactions ie protein binding, brings proteins closer together
proteins are modular and contain domains - functional and structural units that are copied in proteins
some domains are important in molecular recognition but have no enzymatic function of their own - bring other proteins together - proteins can swap domains and so swap function
describe Grb2 *
it is an adaptor protein - small protein with 2 SH3 regions and 1SH2 region
SH = Src homology regions - these are onchogenes that contain a conserved domain
SH3 recognises proline rich domain - any protein with certain proline sequence will allow SH3 binding - this is consitiuitive ie always bound
SH2 binds to tyrosine - this is inducible depending on the specific sequence context - recognition domain is tyrosine and the 4AA before and after
Grb2 binds to EGF and HER2 receptors at tyrosine phosphorylated domain
this binding activates the cascade
describe GTP binding (G) proteins ie Ras *
characterised by binding to GTP which makes it activated - this is normally transient - signal in causes GDP to be removed and bring in GTP
GTP is removed by converting it to GDP by GTPase Activating Proteins (GAP) - this hydrolysis of GTP releases inorganic phosphorus
RAS is then inactive and associated with GDP
exhange factors eg Sos activate Ras by exchanging GTP for GDP
descrive how receptor protein tyrosine kinases are linked to Ras *
ligand bind = dimerised GF receptor = phosphorylation = adaptor protein coming to tyrosine kinase at C terminus
Grb domain (adaptor protein) bind to Sos constituitively (ie always)
this occurs close to membrane = activation of Ras - Ras has to be bound to the membrane to remain activated
this results in signal transmission downstream
describe how Ras can be oncogenically activated by mutations *
V12Ras - constituitively active because glycine12 is replaced by valine - prevents GAP binding ie inactivation
L61Ras - constituitively active because glutamine61 is converted to leucine which prevents GTP hydrolysis
both mechanisms mean Ras cannot be inactivated
describe how Ras transmits downstream signals *
it activates kinases that then phosphorylate each other
the downstream path of Ras is extracellular signal-regulkated kinase (ERK) cascade generically called - Mitogen-activated protein kinase (MAPK) cascades - mitogen refers to the GF
Ras activates Raf which phosphorylates MEK, which phosphrylates ERK
significance of B-Raf *
it is an onchogene - mutationally activated in melanomas
describe the effect of the Erk pathway *
causes change in protein activity - alter cytoskeleton proteins and transcription factors
also changes gene expression - phosphorylates gene regulatory proteins a and b eg phosphorylation of c-Myc which enters the cell cycle
what are cyclin dependant kinases (Cdks) *
they are present in proliferating cells throighout the cell cycle
activity is regulated by interaction with cyclins and phosphorylation
they are cyclically activated protein kinases and they control the cell cycle
describe cyclins *
they are transiently expressed at specific points in the cell cycle
regulated at level of expression
sythesised and then degraded
up and down regulation is important for activity
describe cyclin-cdk complexes and their roles in the cell cycle *
the start kinase phsophylates cdk which inactivates the cyclin
cyclin is degraded
at the start of G1 - cdk binds to a different cyclin - forms complex that starts dna replication machinery - cyclin is then chopped up
cdk binds to another subset of cyclin - these are mitotic cyclins - cdk1 binds to mitotic cyclin B
describe the regulated expression of cyclins *
there is a gradual increase in cyclin then a sudden drop in relative concentration as they are degraded at the end of mitosis
this illustrates the transient down regulation of cyclins
describe the regulation of cdks by phosphorylation *
cdk1 binds to cyclin B - this is inactive - there needs to be another level of regulation
cdk is phosphorylated by cdk activating kinase (CAK) and inhibitory kinase (Wee1)
even though Cdk1 is bound to cyclin - it needs inhibitory kinase to be removed before it is active
phosphtase (Cdc25) clips the inhibitory site (removes the phosphate that was added by Wee1) = actuvated complex at end of interphase
describe how phosphtase activates cdk1 and mitotic cyclin B *
cd25 activates phosphtase
positive feedback reinforces activation of MPF and drives mitosis
why is cdk activatioin complicated *
to check that everything is ready for progression of the cell cycle
describe how cdk1-cycB activation fits into the cycle *
in early mitosis - cdk1/cycB is active - mitosis is put on hold - key substrates are phosphorylated
at the anaphase checkpoint the signal from the fully attached kinetochores cause cyclin B to be degraded - cdk1 is inactivated, key substrates are dephsophorylated - mitosis progresses
describe teh variation in cyclin cdk complexes *
different cyclins and cdks are required at different stages of cell cycle
in M - cdk1 bind cyc B
between g1 and S - cdk2 binds cycE
in S - cdk2 binds cyc A
cyclins target cdk to specific substrates depending on the cell cycle phase because of the substrate availability at that phase
describe how GF stimulation links to cyclins *
GF stim = activation of Ras = production of early immediate gene TFs eg c-jun, c-fos, c-myc
c-myc stimulates cyclin D1 to enter the cell cycle
cdk4/6 binds to cycD and causes cell to enter G1 - this stimulates the synthesis of cyclin E
significance of cyclin D1 *
it is an onchogene that is overexpressed in 50% of breast cancers
describe the regulated expression of cyclins/cdks *
cdks become sequentially active and stimulate synthesis of genes for next phase - eg cyclin D/cdk4/6 stimulates expression for cyclin E - this gives direction and timing to the cycle
cyclins are suseptible to degredation - hence cyclical activation
cMyc activates cyclinD4/6 - causing cell to go from G0 - G1, this degrades which causes activation of cyclin E which activates A which activates B
B sharply degrades at the anaphase checkpoint
what do activated cdk’s do *
they are phosphorylated and bind to cyclins - activated
then they phosphorylate proteins on serine or threonine to drive cell cycle progression
cdk1/cyclin B is the M-phase promoting factor and phosphorylates nuclear lamins - they are the structural component of the nuclear envelope - break down of these causes dissembly of the membrane
cdk2/cyclinE is the start kinase for synthesis - posphorylates retinoblastoma protein which os a tumour suppressor that is inactivated in many cancers
describe regulation by Rb *
pRb acts as a break on the cell cycle
cdks phosphorylate at multiple sites, and progressively inactivate pRb
Rb is a tumour suppressor
active rb is found in G0 - it binds to E2F keeping E2F inactivated
cdks phosphorylate rb = inactivate rb = activate E2F which is a TF that allows cyclin E transcription
what genes are regulated by TF E2F *
proto-oncogenes - c-myc, n-myc
cell cycle - E2f-1,2,3, pRb, cyclin a and e, cdk2 and 4
dna synth - thymidine kinase, thymidine synthetatse, dihydrofolate reductase, dna polymerase
effect when pRb is mutated*
E2f is always activated = cell cycle on full drive
transcribe and increase levels of all cyclin and cdks
summarise the regulated control of the production of cyclins *
c-myc promotes cyclinD transcription which promotes kinases that phosphorylate pRb - pRb releases E2f = transcription of cyclin E which binds to cdk2 = kinase phosphorylate pRb = transcription of A which binds to cdk2 = kinase phos prb = increase TF = transcription of cyclin b which binds to cdk1
what is deregulated in the cell cycle process in tumours *
more than 1 step - cause deregulation
what are the 2 things that regulate cyclin/cdk
phosphtases that deactivate them
inhibitors that bind to them so they cant exert their function - these are cdk inhibitors (CKI)
describe the families of CKI *
2 families separated by timing of cell cycle
INK4 family - p15INK4b p16INK4a, p18INK4c, p19INK4d
CIP/KIP family - p21CIP1/WAF1, p27KIP1 p57KIP2
INK familiy are G1 phase CKIs - they inhibit cdk4/6 by displacing cyclin d
CIP/KIP family - s phase cki - inhibit all cdks by binding to the cdk/cyclin complex (no displacement)
describe the control of CKIs *
must be degraded to allow progression of the cell cycle
INK degrade at G1 phase to allow progression of cell cycle
CIP degrade at S so can get activation of cyclin a and b
what type of cancer has a large proportion of loss of p16 *
pancreatic
what cancer has a large overproduction of cdk4 or cyclin d *
mantle cell cancer
what cancer has a loss of RB *
small lung cell cancer
and non-small, but less so
what are proto-onchogenes *
normal proteins in cell that become changed
what are the onchogenes *
EGFR/HER2 - mutationally activated/overexpressed in breast cancer - herceptin Ab is treatment for metastatic breast cancer
Ras - mutationally activated in many cancers - treated by inhibitors of membrane attachment = reduction in downstream pathways
cyclin D1 overexpressed in 50% breast cancer
B-raf mutationally activated in melanomas - treatment is kinase inhibitors in trials
c-myc is over expressed in many tumours
what are tumour suppressors &
rb inactivated in many cancers
p27kip1 underexpression - relates to poor prognosis in many malignacies
what makes pathway blocking more difficult *
blocking higher up in the pathway
what are motility circles *
when cells have metostatic potential
