1. THE CELL CYCLE April 17, 2015 2. Objectives Review the basic principles of mammalian cell division and the mechanisms that have evolved to safeguard the integrity of the process Discuss the normal control mechanisms of cell division. 3. Introduction Cell division is a process that must be carried out with absolute fidelity. Organisms have evolved cell duplication that include redundant safeguards to prevent errors or, if error occurs, to correct them. 4. Nevertheless, errors do occur at a measurable frequency, and mutations accumulated over time can weaken protective mechanism, rendering the genome increasingly vulnerable to challenges. 5. The resulting loss of genetic and genomic stability has serious implications for survival in that it is a major contributing factor to the development of malignancy. 6. Cell Cycle The cell cycle is the ordered series of events required for the faithful duplication of one eukaryotic cells into two genetically identical daughter cells. 7. Phases of Cell Cycle Gap1 (G1) Preceding phase before cell division Cellular contents (except chromosomes) are duplicated 8. Synthetic phase (S-phase) Each of the 46 chromosome is duplicated by the cell 9. Gap2 (G2) The subsequent phase before cell division Double checking of the duplicated chromosome for error and repair 10. Mitosis (M-phase) Most visible dynamic interval of the cell cycle Divided into 5 phases 11. Phases of Mitosis 1.Prophase: Most of the internal membranous compartments of the cell, including the nucleus, are disassembled and dispersed. Replicated chromosomes are condensed in paired compact rods and a bipolar microtubule spindle is assembled. Biosynthesis of proteins largely ceases 12. 2.Prometaphase: Chromosomes form bivalent attachment to the spindle, driving them to the cellular equator 3.Metaphase: There is proper alignment of paired chromatids on the spindle 13. 4.Anaphase: The paired sister chromatids lose cohesion and microtubule forces separate the chromatids and pull them to opposite poles of the cell 14. 5.Telophase The nuclei and other membrane structures reaassembles, the chromosome decondense, and protein synthesis resumes 15. Cytokenesis Happens after mitosis The process where two daughter cells pull apart and separates 16. The events that together make up the cell division process do NOT all occurs continuously Growth and protein synthesis occur constantly while synthesis of DNA occur only during a discreet interval 17. Cyclin Dependent Kinases (CDKs) Are binary, proline-directed, serine- threonine-specific protein kinases that consist of catalytic subunit that has little if any intrinsic enzymatic activity and a requisite positive regulatory subunit known as cyclin 18. CDKs Consists of 2 globular domain (N-lobe and C-lobe) N-lobe contains the ATP binding site The functional intervals of CDKs are determined by the accumulation and disappearance of cyclin 19. Cyclins Substrate docking function Realigns critical active site residues into a catalytic permissive configuration Open the catalytic cleft to accommodate substrates 20. Once bound to cyclin, the CDK active site is configured similarly to other protein kinases that do not require cyclin binding 21. CDKs are expressed at constant level throughout the cell cycle Cyclin accumulation is dynamic, regulated at the level of biosynthesis and degradation 22. D Type Cyclins Partially redundant D-type cyclins (D1, D2, D3) activates two partially redundant CDKs (CDK4 and CDK6) It is not expressed with high periodicity in cycling cells; their activities occurs in mid to late G1 to direct phosphorylation of the cell cycle inhibitor pRb, p107, p130. 23. Phosphorylation of these active proteins by Cyclin D/CDK4/6 inactivates their negative regulatory functions, allowing progression to S- phase 24. E Type Cyclins (E1 and E2) are expressed with high cell cycle periodicity, accumulating in late G1 and declining during S- phase Activates CDK2 25. Cyclin A Accumulates initially in G1/S phase boundary and persist until prometaphase. Best characterize as activator of CDK2 CDK2 (activated by Cyclin A and E) promotes cell cycle progression from G1/S boundary through the G2 26. B Type Cyclins In conjunction with CDK1 are responsible for getting cells into and through mitosis. Only Cyclin B1 appears to be essential (accumulates through S- phase and G2, then degraded in metaphase-anaphase transition) 27. Modes of CDK Regulation CDKs are highly regulated Exhibits a distinct temporal program of accumulation and degradation- to determine the price window of CDK activation 28. Cyclin D D Cyclins are strongly regulated as cells exits the cell cycle into a non- proliferative state, and then resynthesized in response to mitogen activation and cell cycle re- entry 29. Cyclin E Genes encoding Cyclin E1 and E2 coupled with ubiquitin mediated proteolysis of Cyclin E (to active form E-CDK2) creates a window of Cyclin E accumulation from late G1 to mid S- phase 30. Cyclin A Accumulation is determined by periodic transcription Cyclin A remains stable in active CDK2 complexes Timing of ubiquitin-mediated- proteolysis is determined by anaphase promoting complex/cyclosome (APC/C) in prometaphase 31. The window of accumulation of Cyclin A accumulation is from G1 to S-phase transition until early mitosis 32. B Cyclin Accumulation is linked to transcription (late S-phase to G2) Targeted for ubiquitin mediated proteolysis during mitosis 33. Modes of CDK Regulation 1.) Negative regulation 2.) Phosphorylation 3.) Action of inhibitory proteins that can form either binary complexes with cyclin- CDK dimer 4.) Control of nuclear import/export (Cyclin B-CDK1 complexes) 34. Families of Inhibitory Proteins INK4 family (p15, p16, p18, p19) -specifically targets CDK4 and CDK6 Cip/Kip family (p21cip1, p27kip1, p57kip2) -potent inhibitor of CDK2 and CDK1 Two members of pRb proteins (p107, p130) 35. Modes of CDK Regulation 36. Induction of Cell Cycle Phase Transition Two action phases: S-phase and M- phase -genetic material is duplicated and component of mother cell is divided into 2 daughter cells Intervening phase: G1 and G2 -allow time for cell growth and for regulatory inputs 37. Cell proliferation is controlled operationally at two key transitions 1.) between G1 and S-phase 2.) between G2 and M-phase Characteristics: initiated based on integration of regulatory signals, executed decisively to maintain genetic and genomic integrity 38. Ubiquitin Mediated Proteolysis 76 amino acid polypeptide that can be covalently linked to lysine of other protein via the formaation of isopeptide bond with C-terminal carboxylate Can be attached to lysine of already conjugated ubiquitin to form polyubiquitin chains 39. Polyubiquitin- are usually targeted for rapid proteolysis by large multisubunit protease known as proteosome Protein Ubiquitin Ligases- are enzymes that transfer ubiquitin to target proteins 40. Families of Ubiquitin SCF (Skp1-cullin-F box protein), target proteins that are marked for destruction by phosphorylation APC/C- target of substrate is determined by ligase activation rather than substrate activation 41. Important mitotic targets such as Cyclin A, Cyclin B and securin- are degraded during mitosis and prevented from accumulating during the subsequent G1 interval. 42. Quiescence & Differentiation Quiescence- state of reversible cell cycle exit Largely mediated by growth factors and mitogens that interact with cell surface receptors > linked to intracellular signalling cascades that up-regulate rate of protein synthesis as well as promote proliferation 43. Antimitogenic Signals Mitogen is a chemical substance that encourages a cell to commence cell division, triggering mitosis. Mitogenesis is the induction of mitosis, typically via a mitogen. Mitogens trigger signal transduction pathways in which mitogen-activated protein kinase (MAPK) is involved. 44. Classic example: effect of TGF beta on epithelial cells 45. Checkpoints Two points in cell cycle where cells are susceptible to harmful effects of DNA damage: S-phase M-phase 46. Checkpoints In instances in which cell cycle progression would be harmful or catastrophic before repair of damage, further mechanism have evolved to delay progression pending repair called cell cycle check points 47. DNA Damage Checkoints Cell cycle progression is blocked at 3 points. Before S-phase entry (G1 DNA damage checkpoint) During S-phase (intra S-phase DNA damage checkpoint) Before M-phase entry (G2 damage checkpoint) 48. DNA damage in various form is first detected by DNA bound protein complexes that serves as sensor ATM and ATR are two primary signal transducers that are activated by DNA damage at all points in the cell cycle 49. P53-is activated and stabilized leading to increased level in response to cell damage. 50. Replication Checkpoints Under normal circumstances, DNA replication is complete well before the time when the accumulation and activation of Cyclin B-CDK1 would drive cell into mitosis There are points where through the action of toxins although rare, replication extends beyond normal time 51. The replication checkpoint ultimately functions like G2 DNA damage checkpoints in that mitotic entry is blocked by inhibiting CDC25C- thus preventing action of CDK1 52. Spindle Integrity Checkpoint Assembling a mitotic spindle and attaching chromosome to it is an extensively monitored process. The mechanism of delay at prometaphase or metaphase in response to spindle defects or improper chromosome attachment is the spindle point integrity. 53. The sensor of this checkpoint are number of proteins which resides in the kinetochore, which is also a site of spindle microtubule attachment 54. Senescence Replicative senescence- cell approach end of their proliferative capacity Programmed senescence is not known, but it has been largely elucidated that restricting cells to finite number of division is protective againts malignant growth 55. It is characterized by accumulation of high levels of CDK inhibitors and ultimately G1 arrest.