Molecular Cell Biology (Bio 5068) Cell Cycle I Ron Bose, MD PhD November 13, 2018
CELL DIVISION CYCLE M G2 G1 S
DISCOVERY AND NAMING OF CYCLINS MARINE INVERTEBRATES ( sea urchin, clam & starfish) 1983: Evans & Hunt. sea urchin egg (completed meiosis and arrested in G1) 35S-methionine fertilize remove samples every 10 min. examine for protein synthesis in first few cell cycles of sea urchin egg A protein (called “ cyclin ” ) was observed to increase as Saw 55kDa protein= sea urchin cyclin (periodic protein synthesized throughout the cell cells approached mitosis, peak in mitosis and then cycle but degraded at the end of mitosis: 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 =Anaphase Promoting Complex Required for degradation of substrates at Metaphase to Anaphase transition ( ie: B-type Cyclins and securin) Have D or WDrepeat- Ubiq KEN Box Ubiq containing Ubiq substrate proteins for ubiquitination Ubiq UBC Cdc20 or (E2) Apcx Ring finger Cdh1 / Hct1 Apc11 Apc1/ Apc4 BimE Cullin = Apc2 Apc5 Cdc23 Apc10 Cdc27 Apc8 Apc3 Apc7 Cdc16 Apc6 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 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
Rb binds to histone deacetylases (HDACs) and ATP-dependent nucleosome remodeling complexes (hSWI/SNF) thereby promoting nucleosome assembly and inhibiting transcription. Phosphorylation by cyclin D/Cdk4 and Cyclin E/Cdk2 displace HDACs and hSWI/SNF from Rb, respectively. HDACs hSWI/SNF X RETINOBLASTOMA PROTEIN E2F1 CELL CYCLE GENE
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 s FAND2 FANCE Cellular Senescence Apoptosis Cell Cycle Arrest DNA Repair Response
DNA DAMAGE CHECKPOINTS IR/VP16 replication stress DNA DSBs ssDNA ATR ATM Mdm2 Chk1 p53 Chk2 Cdc25A p21, 14-3-3 s Cyclin B/Cdk1 CyclinE / 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. CDK inhibitors – INK4 and CIP/KIP families. 4. 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.
Question Which 3 non-mammalian species were instrumental in our understanding of the cell cycle?
Prize and Question
Recommend
More recommend
