MCB 252 Spring 2025 Topic 17 Cell Cycle Regulation, Checkpoints, and Cancer

Overview of Remaining Topics

Cdks and the APC: sequential activation and inactivation
Checkpoints
Cancer

Key Components

CDK Kinase Subunit: Cdc28
G1/S Cyclins: Cln3, Cln1, Cln2
S-phase Cyclins: Clb5, Clb6
M-phase Cyclins: Clb1, Clb2, Clb3, Clb4
Sic1: Inhibits S-phase CDKs
SCF: Ubiquitin ligase that degrades Sic1
APC/C-Cdh1: Ubiquitin ligase that degrades S and M CDKs
APC/C-Cdc20: Ubiquitin ligase that initiates anaphase
Wee1: Kinase that inhibits M CDKs
Cdc25: Phosphatase that activates M CDKs
Whi5 and Rb: Inhibit transcription of G1/S and S cyclins
Cdc14: Phosphatase required for exit from mitosis

Overview of Cell Cycle Drivers

G1 Cdks: Lead to the production of S-Cdks.
S Cdks: Drive DNA replication and other S-phase events.
M Cdks: Control the events of mitosis up to anaphase.
APC: Responsible for anaphase and later events in mitosis.

Logic of Cell Cycle Regulation

The cell cycle is regulated by a series of irreversible switches.
Once a switch is turned off, it cannot be turned back on until the next cell cycle.
Each type of CDK and the APC is switched on and off.
Each CDK & the APC activates events required for its stage of the cycle.
Each CDK & the APC triggers events that lead to the activation of the next CDK or APC-C in the sequence.
Each CDK triggers events that lead to its own inactivation.

Key Players and Their Roles

G1/S Cdks, S Cdks, M Cdks and APC/C are turned on and off at specific times in the cycle.
APC-C targets S and M CDKS for degradation.
S and M Cdks are made ahead of when they are needed but are held in an inactive form until the appropriate time when a switch is thrown and a large pool of those proteins become rapidly activated.

Detailed Breakdown of Cell Cycle Regulation

Nutrients lead to the translation and stabilization of initial G1/S cyclin.
Initial G1/S CDK leads to transcription of additional G1/S Cyclin Genes, resulting in full activation of G1/S CDKs.
The gene encoding Rb is mutated (LOF) in many cancers.
Cdc14 phosphatase “resets” Whi5 at the end of mitosis.
START:
- G1/S CDKs shut off the machinery that degrades S & M cyclins.
- S-phase cyclins are produced in G1 but held in inactive form
- As the level of G1/S CDK peaks -> START = activation of S CDKs
  1. S CDK phosphorylate G1/S cyclin -> degradation of G1/S cyclin (M CDK also targets G1/S cyclin for degradation)
  2. S CDK -> replication
  3. S CDK -> txn of M cyclins
S CDKs and M CDKs phosphorylate G1/S cyclin targeting it for degradation.
G1/S cyclins can’t accumulate again until S and M CDKs are gone.
G1/S CDKs inactivate APC which degrades S and M cyclins.
M cyclins are produced in S-phase.
S-CDKs activate transcription of genes that code for the M cyclins.
Mitotic CDKs initially held in inactive form via phosphorylation of the M-CDK by Wee1.
Initially the amount of Wee1 is greater than the amount of M-CDK.
Eventually, the amount of M-CDK becomes greater than Wee1 and M-CDK becomes active.
Activation of M-CDK activates the phosphatase and inactivates the kinase resulting in rapid activation of the entire pool of M-CDK.
M-CDKs activate APC/C-Cdc20
APC/C-Cdc20 function is required for sister chromatid separation
APC/C-Cdc20 targets S cyclins for degradation.
APC/C-Cdc20 targets a large fraction of the M cyclins for degradation
APC/C-Cdh1 targets the remainder of the M cyclins for degradation
APC = E3 ubiquitin ligase
Cdc14 is a phosphatase.
Activation of Cdc14 is required for exit from mitosis.
Cdc14 activates APC/C-Cdh1 which targets remaining M cyclins for degradation which results in activation of Sic1 via dephosphorylation, resetting Sic1 to its G1 state.
Cdc14 dephosphorylates Whi5 “resetting” Whi5 to its early G1 form.
Cdc14 leads to repression of txn of M cyclins
APC-C activity persists into early G1
G1/S CDKs shut off APC-C allowing accumulation of S cyclins
Destruction of M cyclins paves the way for accumulation of the G1/S cyclins
G1/S CDKs inactivate APC/C-Cdh1; thereby allowing accumulation of S-phase CDKs

Lecture Overview

Regulation of DNA Replication
Regulation of anaphase: sister chromatid separation
Other events in M
Checkpoints
Cancer

Cell Cycle Checkpoints

Cell Cycle Checkpoints = Inhibitors of Progression
Inhibit Downstream Stages Until the Critical Steps Completed
Discovery of the G2 DNA Damage Checkpoint: Lee Hartwell
Rad9 monitors DNA damage and halts cell cycle progression until damage is repaired

Experimental Evidence for Checkpoints

Rationale: Incompletely Replicated DNA leads to Mitosis and then Death
Hydroxyurea (HU) stalls DNA Replication, providing Evidence for an Checkpoint that Monitors Completion of DNA Replication (An S-phase Checkpoint)
Genetic Screen: Look for cells (colonies) that can grow on media lacking HU but not on media containing HU
Checkpoints and Cancer
- Loss of Checkpoints -> loss of brakes on cycle = excess proliferation, loss of cell death, increased genomic instability
Checkpoints Insure Faithful Transmission of DNA/Genetic Material
- Some act when there is a problem: DNA damage Stalled Replication Forks
- Some act to prevent cell cycle transitions until previous step is complete: Ongoing replication Spindle assembly checkpoint Spindle position checkpoint
Checkpoints block progress to the next state of the cell cycle until:
1. A Previous step is completed OR
2. Until a problem is fixed
Examples:
- Mitosis doesn’t begin until replication is completed (ongoing replication sends a signal that prevents the G2 to M transition)
- Anaphase doesn’t begin until all kinetochores are properly attached to the mitotic spindle (unattached kinetochores send a signal that prevents anaphase)
- Cells arrest in G1 or G2 until DNA damage is fixed (DNA damage sends a signal that prevents G1-S transition and the G2-M transition until DNA damage is repaired).
Spindle position checkpoint in yeast
Activation of Cdc14 and the MEN does not occur until the spindles are properly localized
Kinetochores sends signal that inhibits the APC until properly attached (under tension). A single unattached spindle sends enough of a signal to inhibit the APC.
Kinetichores not properly attached send signal that inhibits Cdc20 (and therefore the APC). [Cdc20 bound by the inhibitor Mad2]
Kinetichores properly attached -> no signal -> Cdc20 and APC become active. [Cdc20 released from Mad2]

Checkpoints and Genomic Stability

Checkpoints = “Brakes” on the Cell Cycle
Checkpoints insure Faithful Replication, Repair and Segregation of Chromosomes
Loss of Checkpoints -> Genomic Instability

Hallmarks of Cancer Cells

Unregulated Proliferation
Genomic Instability (increased mutation rate)
Metastasis (often sessile -> motile)

Different Cancers -> Different Genes Mutated
Approximately 6-7 mutations -> Cancer
Mixture of Both Dominant and Recessive Mutations
Loss of Checkpoints -> loss of brakes on cycle = excess proliferation, loss of cell death, increased genomic instability

Overview of Human Cell Cycle Regulation

DNA damage activates ATM/R, leading to Chk1/2, MK2, and p53 activation.
Chk1 inhibits Cdc25B/C, and p21 inhibits Mitotic CDKs, preventing M phase entry.
Ongoing DNA replication activates ATR, leading to Chk1 activation, inhibiting Mitotic CDKs and preventing M phase entry.
DNA damage activates ATM/R, leading to p53 activation and p21 production, inhibiting G1 CDKs and preventing S phase entry.
DNA damage activates ATM/R, leading to Chk1/2, MK2 activation, and p53 activation.
Chk1 inhibits Cdc25A, and p53 leads to p21 production, inhibiting G1/S phase and S phase CDKs, preventing S phase entry.
Replication stress activates ATR, leading to Chk1 activation, which inhibits Cdc25A, preventing S phase CDKs activation.

Connecting Signaling to Cell Cycle Progression and Cancer

Ras = GOF (Gain of Function)
Myc = GOF (Gain of Function)
Rb = LOF (Loss of Function)
E2F = GOF (Gain of Function)
Oncogenes and Tumor Suppressors