LEC50: Cell Cycle Progression and Checkpoints Flashcards
what determines entry into S phase?
phosphorylation of pRb
what are the 3 kinds of signaling/feedback in the cell cycle?
1) the previous cyclin-CDK is responsible for activating the next step
2) negative feedback, the next step turns off the previous step - if the 2nd complex is active, it causes phosphorylation and inhibition of the previous step
3) amplifying signal: previous complex starts the next complex to go, but there are steps that cause amplification fo that 2nd complex to cause a signal
what upregulates cyclin D?
Erk pathway:
Growth factors bind to cell surface receptor
Receptor is a RTK on the membrane
It auto-phoshporylates
Activates Ras
Ras > Raf
Raf > Mek
Mek > ERK
ERK upregulates Cyclin D
what do cells need to proliferate? why?
cells need growth factors b/c they need upregulation of cyclin D, and cyclin D is what causes proliferation
what does Rb mean
retinoblastoma
how is Rb normally? what does it do?
Rb normally bound to E2F
When Rb bound to E2F, Rb acts as a repressor, prevents E2F from functioning; E2F expression is **off **in Rb presence
what is E2F?
a transcription factor that regulates gene expression
turned OFF when Rb binds it
turns ON when Rb gets phosphorylated
E2F’s target = Cyclin E
what disrupts Rb-E2F complex? resulting cascade?
Cyclin D-Cdk4 and Cyclin D-Cdk6 phosphorylate Rb
this releases Rb from E2F
E2F can now act as a transcription factor; regulates Cyclin E
Cyclin E levels increase from E2F transcription activation
Cyclin E-Cdk2 become active; Rb is further phosphoryalted as Cyclin E-Cdk2’s substarte; and E2F is totally released
what are the 2 kinds of E2F targets
1) high affinity target: low levels of E2F will turn it on
2) low affinity targets: dihydrofolate reductase, thymidylate synthase, DNA pol-Alpha
describe signal cascade of pRb, E2F, leading into S phase?
Rb bound to E2F means no gene expression that’s E2F dependent occurs
Growth factors upregulate Cyclin D
Cyclin D-Cdk4 or 6, active now, phoshporylates Rb enough that E2F turns on Cyclin E
Activated Cyciln E-Cdk2 phosphorylates Rb further
E2F now fully active
Now can enter into S phase
what regulates Cyclin A gene expression?
E2F
Cyclin A is a low affinity E2F target; so Cyclin A is only turned on when E2F is fully free of Rb
THEN Cyclin A is upregulated, and Cyclin A-Cdk2 can initiate origin firing for DNA synthesis
what triggers DNA synthesis?
Cdk phosphorylation
what are the stipulations for origin firing?
1) origin must fire for DNA synthesis to occur
2) EACH origin must fire ONLY ONCE to get a 2N to 4N DNA synthesis
what assembles to initiate DNA replication? when?
pre-replication compres (pre-RC)
occurs during G1
what activates polymerase to do DNA replication/start origin firing?
Cyclin E-Cdk2 and Cyclin A-Cdk2 phosphorylate polymerase
polymerase is then active
ONLY CAN OCCUR when the poylmrease is sitting ON THE ORIGIN - it is a** localized ability, **kinase is localized to the origin, polymerase gets phosphorylated and fires
what ensures initiation of replication occurs only 1x each round?
Cdk phosphorylation of proteins involved in DNA replication includes licensing factors which ensure initiation occurs only 1x each round
factors then are degraded
what are the dual effects of phosphorylation at the origin of replication?
1) phosphorylation fires the origin
2) phosphrylation removes the marks of an origin that’s ready to be fired- ensures firing doesn’t occur again once it already has
what mediates entry from G2 to M?
Cyclin B/Cdk1 phosphorylation
what is stimulated by mitotic Cdk-cyclin activation?
1) nuclear envelope breakdown
2) chromosome condensation
3) mitotic spindle formation
4) targeted protein degredation
what causes nuclear membrane to break down?
Loss of a pressor means Cyclin B levels increase, due to transcriptional upregulation
Cdc25 becomes phoshprylated in response to completion of DNA synthesis
this activates Cyclin B/Cdk1
a component of the nuclear membrane is a substrate for Cyclin B/Cdk1; when phosphorylated, Lamins A and C break down, thus nuclear membrane breaks down
what makes up nuclear membrane?
lamins, which polymerize w/ each other
is nuclear envelope breakdown reversible? how?
yes
When Cyclin B/Cdk1 activity decreases, phosphorylation of Lamins is reversed by a nonspecific phosphatase which removes a phosphate from Lamin, and Nuclear membrane spontaneously will reform
what is condensin?
protein that mediates condenstion of DNA into chromsome
when ACTIVE, mediates condensation of DNA into chromsomes
when / why does chromoatin condensation occur?
condensin condensation of DNA occurs as a subsrate of Cdk1/Cyclin B complex, like nuclear membrane breakdown
when nuclear membrane breaksdown due to phosphorylation of Lamins by Cdk1/Cyclin B, Condensins come together and chromatin condensation occurs
how does sister chromatid separation occur? when?
anaphase of mitosis
Cohesin holds sister chromatids together
Separase, enzyme, is bound to an inhibitor, Securin, is Inactive
when the APC is active, it Ubiquitinates Securin, making Separase active
Separase thus cleaves Cohesin, and sister chromatids can separate
how does APC become activated?
b/c it’s associated w/ a co-factor, either Cdc20 or Cdh1
when activated, Ubiquitinates and degrades Securin; this results in Separase activation
thus Sister chromatids Separate
what are APC’s dual functions?
APC responsible for sister chromatid separation
AND
active in degredation of Cyclin B, which is necessary for exit from Mitosis
G1:
Kinase?
Cyclin?
Mechanism of upregulating cyclin transcriptionally?
Substrate of the kinase?
Effect of phosphorylation?
G1-S:
Kinase?
Cyclin?
Mechanism of upregulating cyclin transcriptionally?
Substrate of the kinase?
Effect of that phoshporylation?
S phase:
Kinase?
Cyclin?
Mechanism of upregulating cyclin txnly?
Substrate of the kinase?
Effect of that phosphorylation?
what causes mitotic exit?
what events occur as result?
Cyclin B degradation -> Mitotic exit
Chromosomes decondense, nuclear membrane reforms
checkpoint?
an ordered series of events dependent on completion of a previous step
senses a problem, causes cell cycle to stop
why doesn’t cyclin de-upregulation exist?
because checkpoints do exist
what is restriction point?
mediated by pRb phoshporylation in response to growth factors
lack of growth factors causes cell to arrest in early G1 at this restriction point
provides connection btwn extracellular signaling & cell cycle control
after restriction pt, cell is committed to DNA replication
what does loss of checkpoint control cause?
uncontrolled proliferation aka cancer
how does growth factor signaling contribute to proper cell cycle vs cancerous?
growth factor signaling results in transcriptional upregulation of cyclin D
this triggers phosphorylation of pRb and release of E2F
this regulation is lost in cancer
what does DNA damage activate?
2 checkpoints: G1-S and entry into M
purpose of each DNA damage checkpt?
- prior to S phase, so cell doesn’t replicate w/ damage
- prior to M phase, so cell doesn’t undergo cell division w/ damaged chromosomes
describe the normal state of the G1 checkpoint
normally, the tumor suppressor p53 binds to Mdm2, a ubiquitin ligase that polyubiquitinates p53 and keeps p53 low in absence of DNA damage
in presence of DNA damage, describe activation of G1 checkpoint
kinase cascade is triggered, Mdm2 and p53 become modified
p53 also is acetylated
this disrupts Mdm2-p53 interaction, p53 is no longer ubiquitinated
p53 levels increase; p53 binds to DNA and turns on gene expression, mainly via p21, the CIP inhibitor
thus CELL CYCLE ARRESTS b/c p21 binds to Cyclin E/Cdk2 and prevents entry into S phase
what are p53’s effects in regulating cell cycle
cell cycle arrest and apoptosis, depending on if damage is great in cell (apoptosis) or reparable (cycle arrest)
if DNA damage is present, what prevents entry into Mitosis?
DNA damage here triggers kinase cascade resulting in inhibitory phoshprylation of Cdc25
thus, no activation of Cyclin B/Cdk1 occurs
how does unreplicated DNA prevent entry into mitosis?
Cdc25 must be phosphorylated to be active
this phosphorylation doesn’t occur until DNA synthesis is complete, and full 4N DNA is achieved
what is spindle checkpoint function?
arrests cells in mitosis until all chromosomes are properly aligned in metaphase
when spindle checkpoint is OFF, what happens?
APC is active; it ubiquitinates securin
this means separase is active
cohesin is cleaved
sister chromatids are separated
when spindle checkpoint is ON, what happens?
what causes this?
APC is inactive
do not get separation of sister chromatids
APC inactivity maintained if there’s a microtubule attached to every kinetochore; if each kinetochore has a MT, they align the sister chromatid, and those MTs are responsible for pulling the sister chromatid apart
so an unoccupied kinetochore, unbouned by a MT, can send a signal to APC to inhibit it
what determines APC activity in the spindle checkpoint?
kinetochore occupany
if there isn’t a MT at any kinetochore, then APC activation is DELAYED so checkpoint is ON
how could spindle checkpoint be exploited therapeutically
a MT disrupting agent will prevent a cell from dividing b/c it depolymerizes all of the MT in a cell, as if any MT are depolymerized, and even 1 kinetochore unoccupied, you trigger the spindle checkpoint and cause same cellular response as you would by depolymerizing all of the MTs
how can chemotherapeutic drugs use cell cycle to their advantage?
if disrupte cell cycle by disrupting DNA damage or disrupting spindle formation, or have any DNA breaks, then cell will arrest at a checkpoint
