🏳️‍⚧️ Lecture 14: Regulation of CDK Activity and Transitions Between Stages of Cell Cycle

Cell Cycle – Restriction/Start Point

Decision to Divide
 Occurs in late G1 at the restriction (or START) point
 Determines whether the cell will continue through the cell cycle or pause

Regulation
 Decision is tightly controlled by CDKs, cyclins, and other regulatory factors

G0 – Non-Dividing Cells
 Cells that never divide (highly differentiated cells) arrest before the START point
 Enter G0, a resting phase outside of the normal cell cycle

CDK Regulation – Phosphorylation

Activating Phosphorylation
 Phosphorylation of a threonine (T) residue near the CDK active site is required for activity
 Mediated by CDK-Activating Kinase (CAK)

Inhibitory Phosphorylation
 Wee1 kinase phosphorylates tyrosine (Y) and threonine (T) residues on CDKs
 Inhibits mitotic kinases during S-phase
 Y/T kinases can phosphorylate both tyrosine and serine/threonine residues

Activation at Mitosis
 Cdc25 phosphatase removes inhibitory Y/T phosphorylation added by Wee1
 Activates mitotic CDKs to promote entry into mitosis

Yeast Mutants – CDK Phosphorylation

Wee1 Deficient Mutants
 Fission yeast (S. pombe) lacking Wee1 kinase enter mitosis prematurely
 Shows inhibitory phosphorylation by Wee1 is important to delay mitosis until the cell is ready

Cdc25 Deficient Mutants
 Cells lacking Cdc25 phosphatase show delayed entry into mitosis
 Shows removal of inhibitory phosphorylation is required to activate mitotic CDKs

Takeaway
 Phosphorylation and dephosphorylation of CDKs are critical for proper cell cycle progression

CDK Regulation – Activating and Inhibitory Phosphorylation

Wee1 Kinase
 Adds inhibitory phosphate groups to CDKs
 Prevents premature entry into mitosis

Cdc25 Phosphatase
 Removes inhibitory phosphate groups from CDKs
 Activates mitotic CDKs so the cell can enter mitosis

MPF (Maturation Promoting Factor)
 Cyclin B-CDK complex
 Cyclin B is made in advance but only acts during mitosis
 Dephosphorylation by Cdc25 allows MPF to become active and trigger mitosis

G1–S Phase Transition *Only look at metazoans, but just know this entire thing was first discovered in yeast

Key Concept
 Decision to enter S phase is tightly controlled in metazoan cells
 Mechanisms were first discovered in yeast but are conserved across species

CDK-Cyclin Complexes
 Drive the transition from G1 to S phase
 Cyclin-CDK activity triggers DNA replication machinery

Regulation
 Inhibitory proteins can bind CDKs to prevent premature S phase entry
 Ubiquitin ligases degrade cyclins to reset the cycle
 Activating kinases and phosphatases fine-tune CDK activity

G1–S Phase Regulation – Mammals

Rb and E2F
 In early G1, genes required for S phase (Cyclin E and A) are suppressed
 Rb protein binds transcriptional activator E2F to block S-phase gene expression

Growth Factor Stimulation
 Growth factors trigger signal transduction pathways
 Stimulate expression of Cyclin D

Cyclin D/CDK Activity
 Cyclin D/CDK4 and Cyclin D/CDK6 phosphorylate Rb
 Phosphorylation causes Rb to dissociate from E2F

E2F Activation
 Free E2F stimulates transcription of Cyclins E and A
 Cyclin E-CDK2 further phosphorylates Rb
 Also activates S-phase promoting factors to drive DNA replication

S-Phase Entry – Yeast

Sic1 Inhibition
 Sic1 protein inhibits S-phase CDKs when unphosphorylated
 Prevents premature entry into S phase

Phosphorylation of Sic1
 Sic1 is phosphorylated at 6 sites
 Multiple phosphorylations make Sic1 a very effective target

Proteolysis and Activation
 Phosphorylated Sic1 is recognized by SCF ubiquitin ligase
 Sic1 is degraded by the proteasome
 Degradation releases S-phase CDKs
 S-phase cyclins and CDKs become active
 Cell can enter S phase and begin DNA replication

Fig. 19-19: Molecular Mechanisms Governing the Initiation of DNA Replication

DNA Replication – Pre-Replication Complex

Origin Recognition Complexes (ORCs)
 Bind to replication origins (specific DNA sites rich in adenine and thymine)
 Mark where DNA replication will start

Loading of MCM Helicase
 Cdc6 and Cdt1 act as loaders
 Place inactive MCM helicase onto ORC

Pre-Replication Complex (pre-RC)
 Formation of pre-RC occurs once MCM is loaded
 Prepares DNA for replication in S phase

MCM (Mini-Chromosome Maintenance) Complex
 Helicase that unwinds DNA during replication
 Essential for replication fork progression

S-Phase Initiation – Activation of Replication

S-Phase Cyclins and CDK Inhibitors
 S-phase cyclins are synthesized
 Inhibitors of S-phase CDKs are degraded
 This allows CDKs to become active

Activation of MCM Helicase
 Kinases such as DDKs and CDKs phosphorylate MCM
 Phosphorylation activates the helicase activity for DNA unwinding

Prevention of Re-Replication
 Cdc6 and Cdt1 are degraded after loading MCM
 Ensures replication occurs only once per cell cycle

Recruitment of Additional Factors
 Other proteins are recruited and phosphorylated
 This prepares the replication machinery to begin DNA duplication

DNA Replication – Elongation

Polymerase Recruitment
 DNA polymerases are recruited to the replication forks
 These enzymes synthesize new DNA strands complementary to the template

Elongation
 DNA synthesis commences
 Replication proceeds bidirectionally from the origin

Protein Degradation During Mitosis

Purpose
 Degradation of specific proteins is necessary for cell cycle progression
 Ensures proper transition from S-phase to M-phase and exit from M-phase

Mechanism
 Proteins are tagged for destruction, often by ubiquitin ligases
 Targeted degradation allows regulated activation and inactivation of cyclins and other factors

Cell Cycle Surveillance Mechanisms

Checkpoints
 Ensure next stage of cell cycle does not start before preceding stage is complete

Checkpoint Pathways
 Comprised of event sensors (detect problems), signaling pathways (relay information), and effectors (halt cell cycle and activate repair)

Functions
 Arrest cell cycle progression in response to DNA damage or improper spindle assembly
 Activate repair pathways to maintain genomic integrity

DNA Damage Response System

DNA Damage Sensors
 Detect double strand breaks, stalled replication forks, DNA mismatches, or nucleotide errors
 Most mutations occur in non-coding DNA and are harmless
 Mutations in coding DNA can disrupt proteins and lead to cancer

Key Kinases
 ATM – activated by double strand breaks
 ATR – activated by stalled replication forks and other DNA stress
 DNA-PK – activated by double strand breaks

Downstream Kinases
 Chk1, Chk2, MK2 – activated by ATM/ATR
  Cause cell cycle arrest
  Inhibit Cdc25 phosphatase
  Slow CDK activity to prevent mitosis until DNA is repaired

p53 Pathway
 Activated by DNA damage
 Can trigger apoptosis in irreparable cells
 Activates p21 which inhibits CDKs to halt cell cycle progression

Overall Function
 Facilitates DNA repair, prevents damaged cells from dividing, and removes potentially tumorigenic cells

DNA Damage Checkpoint Controls

Checkpoint Timing
 Kinases act at multiple points in the cell cycle:
  G1 – before S-phase entry
  S – during DNA replication
  G2/M – entry into mitosis

G1 Checkpoint
 ATM/ATR detect DNA damage
 Inhibit G1 CDKs
 Prevent entry into S-phase if damage is present

S-Phase Checkpoint
 Replication stress or stalled forks activate ATR and Chk1
 Inhibit Cdc25 phosphatase
 Regulate S-phase CDKs to pause DNA synthesis until problems are resolved

G2/M (Mitotic) Checkpoint
 Excess DNA damage or incomplete replication activates ATM, ATR, Chk1/2, MK2
 Inhibit Cdc25 and other activators of mitotic CDKs
 Prevent entry into mitosis until replication is complete and DNA is repaired

Overall Function
 Ensures that each phase of the cell cycle only proceeds when prior events are complete
 Prevents propagation of damaged DNA and reduces risk of cancer