Lecture #16 | Cell Cycle, Rb, & DNA Tumor Viruses

Checkpoints

Monitor the passing between cell cycle phases

Mitogens role in cell cycle

Are needed for normal cells to leave G0 and progress through G1, up to the first checkpoint (also called the Restriction point).

Cyclins

allow the cell to pass through the checkpoint and from one phase to the next.

• Cyclin D1 is one of the main downstream targets of MAPK, JNK, and NFkB pathways.

• cyclin b → d1 → e → a

Cyclin dependent kinases (CDKs)

Needed for the cell cycle to progress

• Cyclin binding is required for CDK kinase activity – positive regulator.

• Cyclins target CDKs to specific substrates.

• PO4 of CDK substrates stimulates progression through the cell cycle.

Process:

1. cyclin binds to CDK to expose active cite

Cyclin-CDK levels change during the cell cycle

what protein do CDKs phosphorylate

Rb

• Rb is phosphorylated by CDK4/6 and CDK2 which releases E2F (transcription factor).

• PO4 of RB changes during the cycle.

  • Active RB (Hypo-phosphorylated).

    • Active RB, RB binds E2F, Inactive E2F, Non-proliferating cell.

  • Inactive RB (Hyper-phosphorylated).

    • Inactive RB, RB does not bind E2F, Active E2F, Proliferating cell.

      • this is the restriction pt

  • Rb inactivates E2f and also represses it by recruiting HDAC to the promoter

Rb and E2F transcription factors

E2F is critical for cell cycle progression

– Binds DNA as heterodimer with DP1

• If E2F is over expressed, the cell does not need stimulation

by growth factors to go into S-phase

In addition to PO4, how else can RB be inactivated ?

by binding viral proteins expressed by human DNA tumor viruses

• HPV, HBV, EBV

HPV

• Human papillomavirus (HPV) – DNA tumor virus, found in >99% of human cervical cancer, a vaccine has been developed (also causes genital warts).

• HPV infection is thought to be necessary but not sufficient for cervical cancer.

• HPV E7 protein with LxCxE motif competes with HDAC for binding in the RB pocket.

Mutation in RB

• Frequently in the pocket region.

• Same effect as phosphorylation by CDK or binding viral proteins.

• Does not bind E2F.

The pocket region of RB

• A shallow groove stabilized by charge compatibility.

• Critical for txn repression and tumor activity.

• Binds highly conserved LXCXE motif in viral proteins and cyclin D.

• HDAC and viral proteins bind in the pocket.

• E2F does not bind the pocket region.

• Phosphorylation is adjacent to the pocket, not in it.

• Mutations in pocket: Viral proteins: Large T ag E1A E7 Progression into S phase

Connection between growth factors, Myc, and RB

1. MAPK pathway stimulates expression of MYC gene

2. Myc activates the expression of Cyclin D & E2F genes!

3. Cyclin D/CDK phosphorylates RB RB-PO4

4. PO4-RB releases E2F

5. E2F activates target genes involved in proliferation