Cell Cycle Regulation
What is SCF and what does it do?
-SCF is responsible for destroying CKIs that inhibit the S phase -SCF adds the uqibuitin to the CKI (cyclin inhibitor protein) causing it to be degraded
What does the length of G1 stage rely on ?
-Senses environment and nutrition -If cells are need (if cells are not need entered G0)
How does the cell cycle proceed through S phase?
1. Cdc 6 binds to ORC (origin recognition complex) 2. Cdt1 and Mcm helicase bind to the Cdc6-ORC complex "licensing" the pre-RC (pre-replication complex) (Cdt1 and Cdc6 are not attached at this point) 3. Cdt1 is inactivated by geminin Cdc6 and ORC are inhibited by M-Cdk These are all degraded (except ORC which is held inactive) Geminin concentration is low in G1 and M phase but increase in S phase to degrade Cdt1 4. DNA helicase is activated 5. Requirement of DNA polymerase and replication proteins --> S phase activated
What is the pathway of proto-oncogene c-myc in response to mitogens?
1. Mitogen binds receptor 2. Ras is activate 3. Activation of Ras leads to MAP kinase activation 4. MAP kinase activated myc gene 5. Mac activates (G1 cycle, G1/S cyclin, and E2F transcription factor) 6. All lead to the entry of S phase = BAD BAD BAD
What are the 4 checkpoints?
1. Restriction Point (R Point) in G1 -cells that don't get appropriate stimulation from the environment arrest here (enter G0) and may undergo apoptosis (Is the environment favorable) 2. DNA damage checkpoints in G1 and G2 -monitor the integrity of DNA -cells with incompletely replicated or damaged DNA arrest in G1 or G2 -damage is related or cell undergoes apoptosis (Is the environment favorable and are all DNA replicated) 3. Spindle Assembly Checkpoint (Metaphase/Anaphase Checkpoint) -delays onset of chromosomes segregation until all chromosomes are attached to the mitotic spindle (Are all chromosomes attached to spindle)
How is G1 maintained?
1.Cdh1 binds APC at the end of M phase and mediates the destruction of M-cdk when cdc20 is not available 2.p27 (CKI) accumulates in G1 and inactivates M-Cdk in late M and early G1 phase 3.M-cyclin transcription decreased Basically though p27 inhibiting M-Cdk activation and APC-Cdh1 destroying M-Cdk
What are 4 ways that CDK activity is regulated?
1.Combination with different cyclins (substrate specific) 2.Phosphorylation/dephosphorylation 3. Binding of small inhibitory subunits 4. Cyclical degradation of cyclins
M-Cdk
Activated at the end of S phase and inactivate during the metaphase-anaphase stage
What does p53 activate?
Activates genes for cell cycle arrest -p21 (inhibits G1/S and S Cdk DNA Repair -GADD45 Apoptosis (if gene cannot be repaired) Bax,CD95/Fas,Apaf-1
How does excess mitogenic stimulated activate p53?
Active Mdm2 binds p53 - p53 is degrated Excessive Myc production binds Arf which inactivates Mdm2 causing an activation of p53 which leads to cell cycle arrest or apoptosis
What is APC?
Anaphase promoting complex -Responsible for degrading the M-Cdk (and other regulators of mitosis) -Activated by adding cdc20 or Cdh1 APC-cdc20 - destroys M-Cdk during early M phase (is activated by CDK) APC-Cdh1 - destroys M-Cdk during late M phase (is inactivated by CDK)
What are checkpoints?
Biochemical pathways controlling transitions between cell cycle stages Point at which cell cycle can be arrested If previous events haven't been completed or conditions are not favorable Modulate the progression of cells through the cycle in response to internal or external signals Operate through NEGATIVE intracellular signals rather than removal of positive one THEREFORE THE CELL CYCLE ONLY CONTINUES WHEN ALL THE NEGATIVE SIGNALS ARE GONE
What is the G1 DNA Damage Checkpoint?
Blocks entry to S by inhibiting activation of G1/S-Cdk and S-Cdk DNA damage leads to the activation of p53 In the absence of damage, p53 is quickly degraded due to association with Mdm2 (a ubiquity ligase) DNA damage activates protein kinases that phosphorylate p53 and decreases the affinity for Mdm2 DNA DAMAGE = P53 PHOSPHORYLATED BY A KINASE (ACTIVATED) = DECREASES AFFINITY OF P53 FOR MDM2 = MDM2 DEGRADED AND INCREASE OF P53 both the amount of transcriptional activity of p53 are increase
What are CDKs?
Cyclin-dependent kinases -phosphorylated intracellular proteins that initiate or regulate major events of the cell cycle -partically activated by the combination with cyclins -must also be phosphorylated by CAK (cdk-activating kinases) to be fully active -substrate specificity comes from the CYCLIN (that provide substrate specificity)
How do survival factors affect the cell cycle?
Different cell types require different combinations of survival signals Restricts certain cell types to certain environment in the body Competition for limited amounts of survival factors cell numbers in tissues Activate signal transduction pathways that lead to inhibition of apoptosis through activation of Blc2, inactivation of Bad, and inactivation of Hid that activates IAPs
What is the positive feedback loop of E2F?
E2F increase transcription of its own gene E2F increase transcription of G1/S and S-cyclin genes Increases G1/S and S Cdk increased the destruction of Cdh1 and p27 - increasing Cdk activity
What is the purpose of the spindle assembly checkpoint?
Ensures that all chromosomes are attached to the spindle before -sister-chromatid separation occurs -depends on sensor mechanisms that monitors the state of the kinetochores -kinetochore that isn't attached to the spindle is a NEGATIVE signal this blocks APC-CDc20 activation and sister-chromatid separation failure of this checkpoint can result in segregation errors, leading to SOMATIC NON-DISUNCTION Cohesion - holds the sister chromatids together Securin - binds separase and prevents the release of separase until all the sister chromatids are connected to the mitotic spindle Separase is released from securing through active APC-cdc20 which degrades securin allowing separase to separate the sister chromatids
What is the restriction point?
Escape from G1 and entry into S phase depends on the accumulation of G1-cyclins through intracellular signals once a cell passes the R point they are committed to cell cycle progression
What is quiescence?
G0 - where cells can retain the cell cycle or terminally differentiate and never re-renter the cell cycle
What is the phase that is most variable in length?
G1
What are the 4 types of cyclins?
G1/S cyclin - binds Cdks at the end of G1 (comments cell to DNA replication) S cyclin (required to initiate DNA replication) M cyclin (Promotes events of mitosis) G1 cyclin (not present in all cells - promotes the passage through the Restriction Point in late G1)
How are MYC family proteins regulated in proliferation vs differentiation?
In cell growth and proliferation Myc-Max When cells differentiate, Mad levels increase and replace Myc therefore you have the bind of Mad-Max
What controls the entry of Mitosis?
M-Cdk accumulated gradually throughout G2 , but is kept inactive by inhibitory phosphorylation in late G2 - the phosphate is removed the this fully activated M-Cdk M-Cdk activates the phosphatase that activates it (cdc25 phosphatase) and inhibits the kinase (week1 kinase) that inhibits it This is a POSITIVE FEEDBACK LOOP
What are examples of external signals that affect the cell cycle?
Mitogens Growth factors Survival factors Cellular response to external sequences is mediated by receptors on the cell surface that send signals to the nucleus via various signal transduction pathways
What are examples of signaling molecules?
Mitogens - stimulate cell division Growth factors - stimulate cell growth (Increase in mass) Survival factors inhibit apoptosis
How is R point controlled?
RETINOBLASTOMA PROTEIN (RB) Resting cells: p16 - inhibits G1-Cdk Rb binds and inhibits transcription factor E2F (Expression of S phase inhibited) inhibits activation of genes needed for S phase (G1/S and S cyclins) When cells are stimulated by a mitogen: G1-Cdk is active Rb is phosphorylated E2F is released Genes needed for S phase are transcribed
What is S phase?
S phase is where DNA is replicated
What is G0 stage?
Senescence = is a permeant G0 like stage (cannot re-enter G0) long term G1 stage If no signal is received to promote cell proliferation, Cdk inhibition is maintained and the cell cycle is arrested in G0 Most cells in the body are in G0 can be permenant (terminal differentiation) but many cells can be stimulated to re-enter the cycle G0 cells are metabolically active
How do growth factors affect the cell cycle?
Through activation of PI-3 kinase and mTOR kinase Increased glycolysis and lipid synthesis and protein synthesis In cancer becomes abnormally activated
How is S-Cdk prevented by re-replicating?
Through multiple mechanisms S-Cdk remains high in G2 and early M phase - helps to prevent re-replication Geminin (inhibitor of Cdt1) increases in S phase (Geminin will be degraded by APC ubiquitin ligase after M phase) M-Cdk - phosphorylated ORC and Cdc6 - inactive at the end of M-phase - Mcm and ORC are de-phosphorylated by S-Cdk and the pre-RC can reassemble in G1
When is G1 phase activated?
When M-Cdk is inactive causing a destruction of APC-cdc20 and the cell accumulates M-Cdk for the next cycle but are held inactive through phosphates G1 does not occur in rapidly proliferating cells meaning that M-Cdk is quickly made during S phase In cells with G1 phase - Cdh1-APC keeps the levels of M-cyclin low
Non-disjunction
When sister chromatids are not lined up and attached to mitotic spindle the separated of the cells do not occur evenly
What is Li Fraumeni Syndrome?
a rare autosomal dominante disorder caused by the deficiency of p53 -Risk of developing a malignant tumor before the age 50 Somatic mutations in p53 are found in more than 50% of human tumors
What do mitogens control?
act in G1 to control the rate of cell division activate G1-Cdk and G1/S Cdk often activate Ras, trigger the MAPK pathway and increase synthesis of c-Myc c-Myc increases -G1-cyclin -Subunit of SCF ubiquitin ligase -E2F (transcription factor)
S-Cdk
activated at the start of G1 and inactivated at the metaphase-anaphase stage
G1/S -cyclin
activated at the start of G1 and inactivates at the end of G1
Cyclins levels _____ throughout the cell cycle
change -Cdks stay the same but cyclins are degraded in order to proceed to the next step
What is the G2 checkpoints and how is it regulated?
ensures that mitosis doesn't begin until all DNA has been replicated and any damage is repaired Sensors detect damaged or un-replicated DNA and become activated, then activate kinases Kinases inactivate cdc25 - block the activation of M-Cdk when damage is repaired the inhibitory signal is turned off and the cell cycle resumes p53 is ONLY required to prolong the arrest
What is the purpose of M-Cdk?
induces assembly of mitotic spindle and ensures that replicated chromosomes attach to it sister-chromatid separation is triggered by APC ubiquitin ligase -- activated by Cdc20 (APC-Cdc20) exit from M phase depends on the inactivated of M-Cdk via the degradation of M-cyclins, which is triggered by Cdc20 triggered by (ubiquitin dependent degradation of M-Cyclins)
Cdk phosphorylation and dephosphorylation is controlled by
inhibitory phosphorylayion at the activate site -Wee1 kinase phosphorylates to inhibit Cdk activity -Cdc25 phosphatase relives the inhibition by removing the phosphate that Wee1 adds This is important in controlling M-Cdk
The binding of p27 ____ the Cdk ____ complex
p27 is a member of CKI (CDK kinase inhibitory) inhibits the Cdk G1/S complex
What is the primary way that Cdks are regulated?
through the degradation of cyclins through proteolysis -this makes transitions irreversible -Rate limiting step is the transfer of ubiquitin, catalyzed by ubiquitin ligases
