Cell Cycle Checkpoints and Regulation

Cell Cycle Regulation

• The cell cycle is a tightly regulated process with three fundamental processes: cell growth, cell division, and cell survival
• Each process is regulated by interactions between intracellular and extracellular signal molecules
• Extracellular signals include mitogens, growth factors, and survival factors
• Intracellular programs include checkpoints, cyclins, cyclin-dependent kinases (Cdks), APC, Cdk inhibitors, and tumor suppressor proteins
• These components are activated and inactivated in response to extracellular signals

Cell Cycle Checkpoints

• Checkpoints are points in the cell cycle where the cell assesses internal and external conditions to determine its ability to proceed
• Upon passing through a checkpoint, the cell commits to the subsequent cell cycle event and it cannot be reversed
• Cell cycle arrest occurs at specific checkpoints if a cell fails to meet the required conditions
• Three major checkpoints are: G1, G2, and M (Metaphase)
• DNA damage checkpoint is primarily active in G1 and G2
  • DNA damage is repaired.
  • Progression through G1 to S, and G2 to M is stopped.
• DNA replication checkpoint (G2/M- transition)
  • Check for unreplicated DNA, incomplete replication, or DNA damage
  • M-Cdk drives mitosis entry but is kept inactive until repair is complete

Cell Cycle Regulatory Proteins

• Regulatory proteins are highly conserved and similar across all eukaryotes
• Correct sequential order of events at a specific time
• One of each event per cell cycle, with an irreversible nature
• The central regulatory proteins of the cell cycle are cyclin-dependent kinases (Cdks) with changes in cyclinical activity
• Cdks alone are inactive and require binding to cyclins for activation
• Each Cdk-cyclin complex initiates specific events in the cycle
• Cdks cause cyclical changes by phosphorylating targeted proteins
• Changes in activity leads to initiation and termination of cell cycle events

Cyclins and CDKs

• Cyclins have no kinase activity, and Cdks are inactive without a tightly bound cyclin
• Cyclin levels change throughout the cell cycle, while Cdk levels remain relatively constant.
• Each Cdk-cyclin complex triggers specific cell cycle events.
• Once the cyclin role is fulfilled, it is degraded by the ubiquitin-proteasome system
• The cyclical activity of Cdks is regulated by:
• Activation by cyclins
  • Cyclins bind to Cdks activating them at specific phases of the cell cycle
  • Cyclin levels fluctuate while Cdk levels remain relatively constant
• Activation by Cdk-activating kinase (CAK)
  • Phosphorylates cyclin-bound Cdk to activate it
• Inactivation by Cdk inhibitor proteins (CKI)
  • Interferes with the binding of cyclin to Cdk.
  • More important for regulating G1/S-Cdk and S-Cdk

APC for Cyclin Degradation

• Anaphase promoting complex (APC) is a proteolytic complex
• It's a ubiquitin ligase, polyubiquitinating proteins for degradation
• APC destroys cyclins (especially M and S-cyclins)
• Activation of APC leads to:
  • Cohesion in sister chromatids being destroyed during anaphase
  • Inactivation of mitotic cyclin-dependent kinase (M-Cdk) during mitosis

Activation of Cdks by Cyclins and CAKS

• Cdks are activated by Cdk-activating kinase (CAK)
• CAK phosphorylation is needed for optimal Cdk activity
• CAK phosphorylates cyclin-bound Cdk
• Unbound Cdk active site is obscured by a protein loop.
• Binding of a cyclin exposes the active site and partially activates the protein.
• Phosphorylation of the active site by CAK leads to full activation of Cdk

Inactivation of Cdks by CKIs

• Cyclins are inactivated by Cdk inhibitor proteins (CKIs) to prevent uncontrolled cell division
• CKIs, such as p27, bind to both cyclin and Cdk, distorting the Cdk active site and rendering it inactive
• Loss or mutation of CKIs can contribute to cancer and cell cycle disorders

Types of Cyclins

• G1 cyclin (D) binds to Cdk to form a complex to regulate G1/S activities. Levels fall after metaphase.
• S cyclin (A) binds to Cdk after the restriction point to create S-Cdk. It's needed for DNA replication initiation. Levels fall in late G2
• M cyclin (B) binds to Cdk to create M-Cdk, which promotes mitosis (specifically from prophase to metaphase). Levels fall after metaphase
• G1/S cyclin(E) binds to Cdk in G1 to create G1/S-Cdk. This drives the cell through the restriction point to commit to S phase. Levels fall in S phase.

M-Cdk

• Activated at the end of G₂ phase and increases during G₂ phase
• Accumulates during G₂ phase
• Triggers events from prophase to metaphase
• DNA replication checkpoint (G2/M) checks for:
  • Unreplicated DNA
  • Incomplete replication
  • DNA damage

M-Cdk Activity at APC

• Stimulates APC activity
• Stimulates Cdc20
• Activated APC inactivates M-Cdk and other proteins at metaphase end
• Causes dephosphorylation of M-Cdk and S-Cdk targets
• Triggers the metaphase to anaphase transition in mitosis
• Separation of sister chromatids
• Inactivation of M-Cdk at anaphase, and loss of most other Cdk activity.

Cdk

• At the end of mitosis, Cdk activity is nearly zero due to cyclin gene transcription inhibition.
• APC levels remain high to degrade M-cyclin and M-Cdk.
• Increased expression of CKIs like p27
• Inactivation triggers chromosome decondensation and nuclear envelope reformation.
• Ensures proper mitotic exit and transition to G1 phase

Retinoblastoma (Rb)

• Rb is a tumor suppressor gene functioning as a brake in G1 cells
• Rb inhibits cell cycle progression
• Inhibits transcription of S-phase genes
• Rb is inactivated by G1-Cdk
• Activation of Rb is needed for cell progression to S-phase.
• Rb mutations cause cancer.

G1-Cdk

• Inactivates Rb protein by phosphorylation
• Increases gene expression of G1/S cyclins and S cyclins
• Cell progression is driven to S-phase.
• G1-Cdk is activated by favorable extracellular signals, oncogenes, and growth factors, resulting in G1-cyclin expression increase and thus G1-Cdk activity increase

p53

• p53 is activated by DNA damage in G1 and S phases.
• Inhibits G1/S-Cdk and S-Cdk
• Prevents cells from advancing through the restriction point.
• Stimulates gene transcription including p21.
• p21 binds and inhibits G1/S-Cdk and S-Cdk, blocking entry to S-phase
• At least 50% of human cancers have p53 mutations.

Cdk4/6 (G1-Cdk) Inhibitors

• Cdk4/6 inhibitors, such as palbociclib, ribociclib, and abemaciclib, are FDA-approved for treatment of breast cancer.
• Disrupt the mitogenic and hormone signals that are driving proliferation
• Inhibits growth of cancer cells by inducing cytostatic (G1-cycle arrest).
• Prevents cells from transitioning from G1 to S phase

Summary of Cell Cycle Control

• Cell cycle control is a highly coordinated process with multiple checkpoints to ensure accuracy and prevent uncontrolled cell growth.
• Regulatory proteins, including cyclins, Cdks, CKIs, and p53, precisely orchestrate the different phases of the cell cycle.

Explore cell cycle checkpoints, their functions, and regulatory molecules. This quiz covers DNA damage response, extracellular signals, and potential cancer therapy targets. The malfunction of cell cycle checkpoints can cause uncontrollable division.