cell cycle Flashcards
cell cycle molecular control: explain the principle of molecular regulation of the cell cycle, including the role of cyclins and their kinases
what does premature, aberrant mitosis result in
cell death
3 features of tumours which show relevance of appropriate cell division regulation
most solid tumours (with mutations in oncogenes and tumour suppressor genes) are aneuploid (abnormal chromosome number), various cancer cell lines show chromosome instability (+/- whole chromosomes), perturbation of protein levels of cell cycle regulators is present in different tumours
what stops cells from growing
contact inhibition (spatial limit), which is lost in tumours
what is one of the most successful anti-cancer strategies in clinical use
attacking machinery that regulates chromosome segregation
why do cells enter G0 and what happens in this phase
in absence of growth signals (not constantly dividing), become quiescent e.g. liver hepatocytes
what happens at the restriction point in G1
cell monitors its own size and external signals
what is c-Myc
ongogene overexpressed in many tumours
what does c-Myc do physiologically
acts as a transcription factor to stimulate expression of cell cycle genes (synthesised when growth factor present)
3 key components of signalling pathways
regulation of enzyme activity by protein phosphorylation (kinases), adapter proteins, regulation by GTP-binding proteins
growth factor stimulation of signalling pathway: membrane
mitogenic growth factor from other cells binds to EC part of receptor (master regulator)
growth factor stimulation of signalling pathway: cytosol
receptor protein tyrosine kinase -> small G (GTP-binding) protein (Ras) -> kinase binding
growth factor stimulation of signalling pathway: nucleus
immediate early genes (c-Jun, c-Fos, c-Myc - transcription factors) which control gene expression
how rapidly from when growth factor binds do early-response genes in nucleus become activated to activate cell cycle control system
15 minutes
how rapidly from when growth factor binds do delayed-response genes in nucleus become activated to activate cell cycle control system
> 1 hour
what does the receptor protein tyrosine kinase recruit
adaptor and signalling proteins
what 2 things are mutationally activated or overexpressed in many breast cancers
EGFR/HER2
in addition to activation, what is tyrosine phosphorylation providing
docking sites for adapter proteins (example of protein-protein interactions)
what can EC receptors be targeted with
anti-HER2 antibody in cancer therapy
example of tyrosine phosphorylation docking site protein and domains
Grb2 (domains: SH3 - SH2 - SH3)
adaptor proteins: define modular protein domains
functional and structural units that are copied in many proteins
adaptor proteins: function of some domains allowing molecular recognition
no enzymatic function of their own, but bring other proteins together
adaptor proteins: function of Grb2 SH3 domain
recognise proline-rich regions (constitutive) to bring in specific proteins with SH3 domains
adaptor proteins: function of Grb2 SH2 domain
bring in phosphorylated tyrosines (inducible, specific sequence domains)
what act as molecular switches
GTP-binding (G) proteins (not kinases)
example of an IC GTP-binding (G) protein
Ras
what activates Ras
when GTP binds: GDP dissociates, GTP binds and activates, causing Ras to bind to the plasma membrane to become activated
what provides exchange of GTP for GDP, activating Ras
exchange factors e.g. Sos, which bind to adaptor protein attached to phosphorylated tyrosine docking site
how is Ras inactivated (95% in cell normally)
GTP hydrolysed by GTPAse activating proteins (GAP)
receptor protein tyrosine kinase signal to Ras: what form is the binding growth factor, and effect on receptor
dimer, causing activation dimerisation of receptor
receptor protein tyrosine kinase signal to Ras: how is the receptor activated
tyrosine kinase phosphorylated
receptor protein tyrosine kinase signal to Ras: what adaptor protein activates the Ras-activating protein (exchange factor Sos)
Grb2 (consitutively i.e. Sos always bound to Ggrb2 via SH3 domains), which is bound to phosphorylated tyrosine on receptor
effect of Ras being oncongenetically activated by mutations
increase amount of active GTP-loaded Ras
2 methods by which a Ras mutation increases amount of active GTP-loaded Ras
V12Ras prevents GTPAse activating proteins binding (prevents inactivation), L61Ras prevents GTP hydrolysis
what does Ras activate
protein kinase cascade
specific Ras protein kinase cascade downstream
EC signal-regulated kinase (ERK) cascade (generically mitogen-activated protein kinase cascades)
what is cell cycle control based on
cyclically activated protein kinases (Cdks)
when are cyclin-dependent kinases (Ckds) present in proliferating cells
throughout cell cycle (not transient)
what is cyclin-dependent kinase activity regulated by
interaction with cyclins, phosphorylation
when are cyclins expressed
transiently at specific points of cell cycle (huge decrease at end of mitosis, before steadily climbing during interphase to peak in mitosis when M-phase-promoting factor (MPF) high, as cyclin forms MPF complex here)
where are cyclins regulated, and fate of synthesised cyclin
regulated at level of expression, and synthesised then degraded
effects of cyclin binding and activation of Cdks: G1 to S
binding of Cdk2 by G1 cyclin (e.g. cyclin E) forms start kinse (phosphorylates substrates at start), which triggers DNA replication memory (e.g. retinoblastoma protein which acts as a tumour suppressor)
effects of cyclin binding and activation of Cdks: M
binding of Cdk1 by mitotic cyclin (e.g. cyclin B) forms M-phase-promoting factor (MPF which phosphorylates substrates at mitosis), which triggers mitosis machinery (e.g. nuclear laminins which cause breakdown of nuclear envelope)
what amino acids does phosphorylation of proteins occur at by activated Cdks
serine or threonine, driving cell cycle progression
when is retinoblastoma protein (pRb) inactivated
in many cancers
what does activation of Cdks require, and why
3 stages: activating phosphorylation (Cdk-activating kinase (CAK)), removal of inactivating phosphorylation (inhibitory kinase), dephosphrylation at end of interphase (phosphatase Cdc25); complex so tight regulation for cell cycle
during mitosis, what happens when Cdk1 and cycB are active
mitosis on hold, as key substrates phosphorylated (2 reactions)
what causes cyclin B to be degraded in mitosis
signal from fully attached kinetochores
3 effects of cyclin B degradation
Cdk1 inactivated, key substrates dephosphorylated, mitosis progresses
what cyclin and Cdk is required during S phase
Cdk2, cyclin A
2 effects of cyclins
activate Cdks, alter substrate specificity (substrate accessibility changes throughout cell cycle)
stages of G0 to G1 transition
growth factor -> dimerisation and phosphorylation -> immediate early gene transcription factors (e.g. c-Myc) by Ras and ERK -> transcription of other genes (e.g. cyclin D) -> activation of Cdk4 and Cdk6 -> stimulate synthesis of cyclin E -> binds to Cdk2
what gives direction and timing to cycle
Cdk sequential activation and stimulation of synthesis of genes required for next phase (c-Myc at G0 -> cyclin D/Cdk4/6 -> expression of cyclin E -> Cdk2-cyclin E -> expression of cyclin A -> Cdk2-cyclin A -> expression of cyclin B -> Cdk1-cyclin B)
why is there cyclical activation of cyclins (appear transiently)
as cyclins are susceptible to degradation
what acts as a brake on cell cycle
pRB (Rb - retinobastoma gene - is a tumour suppressor)
how is pRb inactivated
progressively inactivated by Cdks phosphorylating at multiple sites
regulation of gene expression by Rb (retinoblastoma gene)
activated found at G0, and binds to E2F transcription factor to inactivate it -> when phosphorylated by Cdks, releases E2F which transcribes genes e.g. cyclin E
genes regulated by transcription factor E2F
many, including: oncogenes e.g. c-Myc; cell cycle e.g. cyclin A, E and pRb; DNA synthesis e.g. DNA polymerase
multiple steps to synthesise correct cyclin
E2F activated and deactivated at different times by phosphorylation of Cdks, so each phosphorylation causes it to produce next cyclin
what else regulates Cdks after cyclin binding to Cdk, and what must be done to allow cell cycle progression
Cdk inhibitors (CKIs), which bind to cyclin and Cdk; must be degraded to allow cell cycle progression
2 families of CKI
INK4, CIP/KIP
function of G1 phase CKIs (INK4)
inhibit Cdk4/6 by displacing cyclin D
function of S phase CKIs (CIP/KIP)
inhibit all Cdks by binding to Cdk/cyclin complex
when is degradation of G1 CKIs (INK4)
during G1
when is degradation of S phase CKIs (CIP/KIP)
during S phase
effect of tumours on CKIs, pRB, cyclins, c-Myc, Ras and Raf
loss of CKIs (inhibit) and pRBs (tumour suppressor), overproduction of cyclins (drive next stage; become oncogenes), c-Myc overexpressed, Ras and Raf mutationally activated
