Molecular Cell Biology (Bio 5068) Cell Cycle I Ron Bose, MD PhD November 10, 2016

CELL DIVISION CYCLE MITOSIS G1 M G2 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 = 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. UBIQUITIN-MEDIATED PROTEOLYIS

Required for degradation of substrates at Metaphase to APC = A naphase P romoting C omplex Required for degradation of substrates at Metaphase to Anaphase transition ( ie : B-type Cyclins and securin) substrate Ring finger UBC ( E2 ) for ubiquitination Apc10 Cdc20 Apcx Apc4 Apc5 Apc7 Cdc27 Apc3 Apc1 / BimE Cdc23 Apc8 Cdc16 Apc6 Have D or KEN Box Cullin = Apc2 Apc11 or Cdh1 Hct1 WD repeat- containing proteins Ubiq Ubiq Ubiq Ubiq Cdc20 : targets cyclin A and B-type Cyclins, securin Cdh1 / Hct1 : targets Plk1 and B-type 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 Cyclin B Ubiquitin-mediated proteolyis Activating Kinase(s)

Cyclin-dependent Kinase Inhibitor Proteins (CKI’s) CIP/KIP family (p21Cip1, p27Kip1, p57Kip2): Binds to Cdk2 and inhibits activity. Binds Cdk4/6 and helps assemble complexes with cyclins. INK4 family (p16, p15, p18, p19). Specific for Cdk4 and Cdk6. Binds Cdk subunit alone and prevents cyclin binding Bind and inhibit Cdk4/6-Cyclin D heterodimers.

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

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

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

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

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

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

DNA damage checkpoint: integrity of DNA 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 G2-PHASE CHECKPOINT STOP! G2 G1 S G1-PHASE CHECKPOINT S-PHASE CHECKPOINT

& activation of DNA repair pathways 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

Chemo- & Radio-therapy Signal Signaling Cascade Cellular Response Fig. 2 Gemcitabine Cytarabine 5-Fluorouracil Chemo- & Radio-therapy IR Etoposide Irinotecan Topotecan Cisplatin Carboplatin DSBs Stalled replication forks SSBs Signal RPA-ssDNA ATM ATR Signaling Cascade CHK2 p53 CHK1 BAX PUMA p21 14-3-3s CDC25A RAD51 FAND2 FANCE Cellular Response Senescence Apoptosis Cell Cycle Arrest DNA Repair 26

DNA DAMAGE CHECKPOINTS IR/VP16 replication stress DNA DSBs ssDNA ATM ATR Chk2 p53 Mdm2 Chk1 Cdc25A p21, 14-3-3s Cyclin B/Cdk1 Cyclin E / Cdk2 G1 S 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 The cell cycle is a coordinated and tightly organized process to ensure the successful replication of the cell. Activity of CDK-Cyclins is determined by: Synthesis of Cyclins. Reversible phosphorylation/dephosphorylation of stimulatory and inhibitory sites on CDK. Ubiquitin mediated degradation of Cyclins. CDK inhibitors – INK4 and CIP/KIP families. Checkpoints can halt the cell cycle if all steps have not been properly completed. Cancers have many alterations in cell cycle proteins and selective CDK4/6 inhibitors are now used in cancer treatment.