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 14, 2017

CELL DIVISION CYCLE M G2 G1 S

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

Overview of major cyclins and Cdks of vertebrates and yeast Table 17-1. Molecular Biology of the Cell, 4th Edition

Evolution of cell cycle control: from yeast to humans Malumbres M, Nature Reviews Cancer 2009

Cdk activity is regulated by inhibitory phosphorylation and inhibitory proteins Why is cell cycle progression governed primarily by inhibitory regulation? Figure 17-18, 17-19. Molecular Biology of the Cell, 4th Edition

Cell cycle control depends on cyclical proteolysis Figure 17-20. Molecular Biology of the Cell, 4th Edition

UBIQUITIN-MEDIATED PROTEOLYSIS Deshaies RJ and Joazeiro CA. Annu Rev Biochem. 2009 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 UBIQUITIN-MEDIATED PROTEOLYIS = 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.

APC PC = A na napha hase e P romoti ting ng C omple plex Re Required f for or d degrad radat ation of of 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) Hav ave D D or or WD rep epea eat- KEN KEN Bo Box Ubiq iq Ubiq iq containi ning ng Ubiq iq sub substr strate protei teins ns for or ub ubiqui uiti tina nati tion Ubiq iq UBC BC Cdc dc20 or or ( E2 E2 ) Apc pcx Ring ng f fing nger er Cdh1 h1 / Hc Hct1 t1 Ap Apc11 11 Apc pc1 / Apc pc4 BimE Bi Cullin llin = = Apc pc2 Apc pc5 Cdc dc23 Apc10 Ap 10 Cdc dc27 Apc pc8 Apc pc3 Apc pc7 Cdc dc16 Apc pc6 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

SCF Ubiquitin Ligases 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 E3 Ubiquitin Ligases O'Connell BC, Harper JW. Curr Opin Cell Biol. 2007

CELL CYCLE REGULATION OF Cdc2 Reversible phosphorylation Inhibitory kinase(s) Phosphatase(s) Protein-protein interactions T14 Y15 T161 T161 P P P P Cdc2 Cdc2 Cdc2 Cdc2 Cdc2 Cyclin B Cyclin B Cyclin B INACTIVE INACTIVE INACTIVE INACTIVE ACTIVE Ubiquitin- Cyclin B mediated Activating Kinase(s) 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 M Cdk 4 & 6 INK4a proteins Cyclin D1, 2, 3 (p15,16, 18, 19) G2 Assembly & Sequestration G1 S Cdk2 Cip/Kip proteins Cyclin E (p21, p27, p57)

Mechanisms controlling G1/S-phase transition MITOGENIC E2F CYCLIN D Stability and or Transcription Rb1 CYCLIN D-dependent Kinases HORMONAL (Cdk4/Cdk6) factor SIGNALS Figure 17-30. Molecular Biology of the Cell, 4th Edition

G1 Control CYCLIN E CDK2 RB CYCLIN E S-Phase CYCLIN D STABILITY MITOGENIC E2F CYCLIN D-DEPENDENT KINASES SIGNALS (Cdk4/Cdk6) E2F Relief of Rb- mediated CYCLIN A & transcriptional repression S-PHASE GENES S-PHASE GENES S-Phase

G1 Control p27KIP1-Phosphorylation p27KIP1 Ubiq-Mediated proteolysis Assembly & Sequestration CYCLIN E CDK2 RB CYCLIN E S-Phase CYCLIN D STABILITY MITOGENIC E2F CYCLIN D-DEPENDENT KINASES SIGNALS (Cdk4/Cdk6) E2F Relief of Rb- mediated CYCLIN A & transcriptional repression S-PHASE GENES S-PHASE GENES S-Phase

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

CHECKPOINTS IMPROPER SPINDLE ASSEMBLY M M DNA DAMAGE UNREPLICATED DNA STOP! G2 G2 G1 G1 S S DNA DAMAGE

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.

DNA DAMAGE RESPONSE PATHWAY M G2-PHASE CHECKPOINT STOP! G2 G1 S G1-PHASE CHECKPOINT S-PHASE CHECKPOINT

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

Fig. 2 Gemcitabine Chemo- & Cytarabine IR Etoposide Irinotecan Cisplatin Radio-therapy 5-Fluorouracil Topotecan Carboplatin Signal ATM ATR Signaling CHK2 p53 CHK1 Cascade BAX p21 CDC25A RAD51 PUMA 14-3-3 σ FAND2 FANCE Cellular Senescence Apoptosis Cell Cycle Arrest DNA Repair Response

DNA DAMAGE CHECKPOINTS IR/VP16 replication stress DNA DSBs ssDNA ATR ATM Chk2 Mdm2 Chk1 p53 Cdc25A p21, 14-3-3 σ Cyclin B/Cdk1 Cyclin E / Cdk2 S G1 G2 M DEATH G1-checkpoint S-phase checkpoint G2 checkpoint 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

Mitogens stimulate cell division Figure 17-41. Molecular Biology of the Cell, 4th Edition

Excessive stimulation of mitogenic pathways can lead to cell cycle arrest or cell death Figure 17-42. Molecular Biology of the Cell, 4th Edition

Extracellular Survival Factors Suppress Apoptosis Figure 17-47. Molecular Biology of the Cell, 4th Edition

Cell cycle regulators are frequently disrupted in cancer Malumbres M, Nature Reviews Cancer 2001

Overview of CDK inhibitors in clinical development for cancer therapy O’Leary et al., Nature Reviews Clinical Oncology 2016

Turner et al, NEJM 2015

NEJM Nov. 3, 2016

Conclusions 1. The cell cycle is a coordinated and tightly organized process to ensure the successful replication of the cell. 2. Activity of CDK-Cyclins is determined by: 1. Synthesis of Cyclins. 2. Reversible phosphorylation/dephosphorylation of stimulatory and inhibitory sites on CDK. 3. Ubiquitin mediated degradation of Cyclins. 4. CDK inhibitors – INK4 and CIP/KIP families. 3. Checkpoints can halt the cell cycle if all steps have not been properly completed. 4. Cancers have many alterations in cell cycle proteins and selective CDK4/6 inhibitors are now used in cancer treatment.