Molecular Cell Biology (Bio 5068) Cell Cycle I Ron Bose, MD PhD - - PowerPoint PPT Presentation

Molecular Cell Biology (Bio 5068) Cell Cycle I Ron Bose, MD PhD

November 10, 2016

G1 M G2 S

CELL DIVISION CYCLE

DISCOVERY AND NAMING OF CYCLINS

A protein (called “cyclin”) was observed to increase as cells approached mitosis, peak in mitosis and then precipitously disappear as cells exited mitosis.

Two proteins (cyclins A and B) increased as cells approached mitosis, peaked in mitosis and precipitously disappeared as cells exited mitosis.

The cell cycle is primarily regulated by cyclically activated protein kinases

Figure 17-15, 17-16 Molecular Biology of the Cell, 4th Edition

Table 17-1. Molecular Biology of the Cell, 4th Edition

Overview of major cyclins and Cdks of vertebrates and yeast

Malumbres M, Nature Reviews Cancer 2009

Evolution of cell cycle control: from yeast to humans

Cdk activity is regulated by inhibitory phosphorylation and inhibitory proteins

Figure 17-18, 17-19. Molecular Biology of the Cell, 4th Edition

Why is cell cycle progression governed primarily by inhibitory regulation?

Figure 17-20. Molecular Biology of the Cell, 4th Edition

Cell cycle control depends on cyclical proteolysis

UBIQUITIN-MEDIATED PROTEOLYIS

E1 (Ubiquitin activating enzyme) Binds to Ubiquitin in an ATP-dependent manner Passes Ubiquitin to E2 E2(Ubiquitin conjugating enzyme or UBC) At least 12 in yeast some are specific to a given target E3 (ubiquitin protein ligase) Large complex in both clam (cyclosome) and in frog (APC = anaphase promoting complex). Final transfer of ubiquitin to substrate can be mediated by E2 alone or E2 acting in concert with E3 Proteosome (26S complex) Structure from archaebacterium solved.

Deshaies RJ and Joazeiro CA. Annu Rev Biochem. 2009

UBIQUITIN-MEDIATED PROTEOLYSIS

APC PC = A na napha hase e P romoti ting ng C

• mple

plex Re Required f for

• r d

degrad radat ation of

• f sub

substr strates es at t Meta etapha hase to to Ana napha hase tr transi nsiti tion ( n ( ie ie : : B-type e Cyclins ns a and nd sec secur urin) Cdc dc20 : : ta targets ets cycl cyclin A a and nd B B-type e Cyclins ns, sec , secur urin Cdh1 h1 / Hc Hct1 t1 : : ta targets ets Pl Plk1 a and nd B-typ ype cycl cyclins sub substr strate Ring ng f fing nger er UBC BC ( E2 E2 ) for

• r

ub ubiqui uiti tina nati tion Ap Apc10 10 Cdc dc20 Apc pcx Apc pc4 Apc pc5 Apc pc7 Cdc dc27 Apc pc3 Apc pc1 / Bi BimE Cdc dc23 Apc pc8 Cdc dc16 Apc pc6 Hav ave D D or

• r

KEN KEN Bo Box Cullin llin = = Apc pc2 Ap Apc11 11

• r
• r

Cdh1 h1 / Hc Hct1 t1 WD rep epea eat- containi ning ng protei teins ns

Ubiq iq Ubiq iq Ubiq iq Ubiq iq

Components: F Box: adapter Brings substrate to E3 ligase. F-box binds to Skp1 Additional protein interaction domains (PID: WD repeat, leucine-rich repeat) binds to substrate E2:

• UBIQ. Conjugating enzyme (transfers UB to substrate)

Skp1: Bridges F-box to cullin Cullin: Organizes and activates E3 complex Recruits E2-UBIQ conjugating enzyme Ring finger protein Participates in E2 binding and catalysis

SCF Ubiquitin Ligases

O'Connell BC, Harper JW. Curr Opin Cell Biol. 2007

SCF E3 Ubiquitin Ligases

CELL CYCLE REGULATION OF Cdc2

Phosphatase(s) P T161 P P P T14 Y15 T161 Cyclin B Cdc2 Inhibitory kinase(s) Activating Kinase(s) Cyclin B Cdc2 Cyclin B Cdc2 Cdc2 Cyclin B Cdc2

INACTIVE INACTIVE INACTIVE INACTIVE ACTIVE

Protein-protein interactions Reversible phosphorylation

Ubiquitin- mediated proteolyis

Cyclin-dependent Kinase Inhibitor Proteins (CKI’s)

• 1. CIP/KIP family (p21Cip1, p27Kip1, p57Kip2):

a. Binds to Cdk2 and inhibits activity. b. Binds Cdk4/6 and helps assemble complexes with cyclins.

• 2. INK4 family (p16, p15, p18, p19).

a. Specific for Cdk4 and Cdk6. b. Binds Cdk subunit alone and prevents cyclin binding c. Bind and inhibit Cdk4/6-Cyclin D heterodimers.

G1 Control

Cdk 4 & 6 Cyclin D1, 2, 3 Cdk2 Cyclin E G1 M G2 S Cip/Kip proteins (p21, p27, p57) INK4a proteins (p15,16, 18, 19) Assembly & Sequestration

Mechanisms controlling G1/S-phase transition

Figure 17-30. Molecular Biology of the Cell, 4th Edition MITOGENIC

• r

HORMONAL SIGNALS CYCLIN D Stability and CYCLIN D-dependent Kinases (Cdk4/Cdk6)

Rb1 E2F Transcription factor

MITOGENIC SIGNALS CYCLIN D STABILITY CYCLIN D-DEPENDENT KINASES (Cdk4/Cdk6) RB E2F E2F CYCLIN E CYCLIN E CDK2 CYCLIN A & S-PHASE GENES S-Phase S-PHASE GENES Relief of Rb- mediated transcriptional repression S-Phase

G1 Control

MITOGENIC SIGNALS CYCLIN D STABILITY CYCLIN D-DEPENDENT KINASES (Cdk4/Cdk6) RB E2F E2F CYCLIN E CYCLIN E CDK2 CYCLIN A & S-PHASE GENES p27KIP1 p27KIP1-Phosphorylation Ubiq-Mediated proteolysis Assembly & Sequestration S-Phase S-PHASE GENES Relief of Rb- mediated transcriptional repression S-Phase

G1 Control

Checkpoints

What are they? How were they defined? How does their derailment contribute to cancer?

CHECKPOINTS

G1 G1 M M G2 G2 S S DNA DAMAGE DNA DAMAGE UNREPLICATED DNA IMPROPER SPINDLE ASSEMBLY

STOP!

Checkpoints: intracellular signaling pathways that determine if previous steps are complete before proceeding onto the next stage (complete DNA synthesis before entering mitosis; spindles must be assembled before exiting metaphase and entering into anaphase) and whether there has been any damage to the DNA. DNA damage checkpoint: integrity of DNA DNA damage is repaired before entering S, completing S or entering M. DNA replication checkpoint: replication state of DNA Complete DNA synthesis before mitosis. Spindle assembly checkpoint: integrity of spindle spindles must be assembled before exiting metaphase into anaphase.

G1 M G2 S

G1-PHASE CHECKPOINT S-PHASE CHECKPOINT G2-PHASE CHECKPOINT

DNA DAMAGE RESPONSE PATHWAY

STOP!

CELLULAR RESPONSES TO CHECKPOINT ACTIVATION (IR, etoposide, HU, gemcitibine, irinotecan, carboplatin…) G1 S G2 M CHECKPOINTS APOPTOSIS SENESCENCE TEMPORARY CELL CYCLE ARREST

& activation of DNA repair pathways

IR Etoposide Gemcitabine Cytarabine 5-Fluorouracil Irinotecan Topotecan Cisplatin Carboplatin ATM ATR CHK2 p53 CHK1 RAD51 FAND2 FANCE p21 14-3-3σ BAX PUMA CDC25A Apoptosis Cell Cycle Arrest DNA Repair

Signaling Cascade Cellular Response Chemo- & Radio-therapy Signal

• Fig. 2

Senescence

DNA DAMAGE CHECKPOINTS

Chk1 Cyclin E / Cdk2 G1 S G2

G1-checkpoint S-phase checkpoint G2 checkpoint

M

DEATH

Cdc25A DNA DSBs ssDNA ATM Mdm2 p53 p21, 14-3-3σ

IR/VP16 replication stress

ATR Chk2 Cyclin B/Cdk1 Overproduced in certain cancers. Inactivated in certain cancers.

DNA damage leads to cell cycle arrest in G1

Figure 17-33. Molecular Biology of the Cell, 4th Edition

Figure 17-41. Molecular Biology of the Cell, 4th Edition

Mitogens stimulate cell division

Figure 17-42. Molecular Biology of the Cell, 4th Edition

Excessive stimulation of mitogenic pathways can lead to cell cycle arrest or cell death

Figure 17-47. Molecular Biology of the Cell, 4th Edition

Extracellular Survival Factors Suppress Apoptosis

Malumbres M, Nature Reviews Cancer 2001

Cell cycle regulators are frequently disrupted in cancer

O’Leary et al., Nature Reviews Clinical Oncology 2016

Overview of CDK inhibitors in clinical development for cancer therapy

Turner et al, NEJM 2015

NEJM Nov. 3, 2016