Cell Cycle Control PDF

Cell Cycle Control Prof. Dr. Selma Yılmazer Department of Medical Biology In each mitotic division cycle Parent cell divides and produces a pair of genetically identical daughter cell Chromosome complement of the daughter cells is equal to that of the parent cell  In each division...

Cell Cycle Control Prof. Dr. Selma Yılmazer Department of Medical Biology In each mitotic division cycle Parent cell divides and produces a pair of genetically identical daughter cell Chromosome complement of the daughter cells is equal to that of the parent cell  In each division-cycle To produce a pair of genetically identical daughter cell A cell must replicate its DNA and must segregate duplicated chromosomes to daughter cells Most cells also grow and duplicate all of their cytoplasmic contents  Phases of the Cell Cycle I- M phase (Mitosis) II-Interphase – G1 phase (Gap1) Cell Growth – S phase (DNA synthesis) – G2 phase (Gap2) Preparation for mitosis  Cell division Must be carefully regulated and coordinated with both CELL GROWTH DNA REPLİCATİON How this coordination is achieved? Our understanding of the cell cycle regulation has undergone a revolution in recent years Recent experiments have shown the presence of a Cell Cycle Control System What is the nature of the cell cycle control system? How is proliferation in mammalian cells regulated? What are the extracellular signals that determine whether the cell will grow and divide?  Cell-Cycle Control System Cyclically operating biochemical mechanism Constructed from a set of interacting proteins  Cell Cycle Control System regulated by brakes that can stop the cycle at specific check points. 3.  Control System regulated by; – extracellular signals from the environment – intracellular signals coordinate cell growth, DNA replication, mitosis For the cells of multicellular animals In order to divide a cell must receive positive signals from other cells, many of these signals are protein Growth Factors  Extracellular Signals Protein Growth Factors bind to their receptors in the plasma membrane to stimulate cell proliferation Regulation of cell cycle by growth factors In the presence of growth factors cell pass the restriction point and enter S phase. If GF’s are not present in G1 cell enters to Gο (resting stage)  Cell Cycle Check Points 1. G1 check point (late in G1 just before entry into S phase) Cell size, environmental signals, DNA damage ? 2. S-phase check point (controls incomplete replication,DNA damage 3. G2 check point (at the entry to mitosis) DNA replication is completed or not and DNA damage? (For some cells; cell size, environmental signals(i.e.oocytes) 4. Metaphase check point (at metaphase-anaphase boundary) (Alignment of chromosomes on mitotic spindle)  G1 check point (restriction point) the major point of decision. When a cell passes G1 check point It will complete S phase Proceed through G2 and Divide  The Cell Cycle Control System is Based On Two key families of proteins: 1- Cyclin-dependent protein kinases (Cdk) Phosphorylate selected proteins (induce downstream processes) 2- Cyclins (activating proteins) – Bind to Cdk ; form Cyclin-Cdk complex – Control their ability to phosphorylate target proteins – Cylically synthesized and degraded Cdk-Cyclin complex  Events driving the cell cycle Cyclic synthesis of cyclins → assembly→ Cyclin- Cdk complex ↓ activation (by phosphorylation and ↓ dephosphorylation) Active Cyclin- Cdk ( phosphorylation of selected proteins disassembly(Breakdown of cyclin) ↓ inactivation of cdk  Two major Cdk + Cyclin Complexes (in yeast) 1-Cdk + mitotic cyclin = MPF (Mitosis Promoting Factor) required for entry into mitosis Mitotic cyclins Bind to Cdk during G2 2-Cdk + G1 cyclin= Cdk/ G1 cyclin complex =Start kinase G1 cyclins Bind to Cdk during G1 required for passage through G1 check-point and entry into S phase  In Higher Eukaryotes Cell Cycle controlled not only by multiple cyclins (A,B,C,D,E) but also multiple Cdk’s (Cdk1-Cdk 8) Complexes of Cyclins and Cyclin-dependent Kinases 1-Cdk4,6/ cyclin D ;passage through G1 checkpoint 2-Cdk 2/ cyclin E required for G1-S transition and initiation of DNA synthesis 3-Cdk 2/ cyclin A required for progression through S phase 4-Cdk1/ cyclin B (MPF) drive G2 to M phase transition (Cdk1)  MPF Phosphorylates a set of target proteins including; Lamins (leads to nuclear envelope breakdown) Condensins and H1 histone (chromosome condensation) Microtubule associated proteins (MAP s) (formation of mitotic spindle) , Phosphorylation of condensins Pore complex protein GM130 Phosphorylation of MAPs Interphase Early prophase Late prophase Prometaphase Metaphase Early anaphase Late anaphase Telophase  Mechanism of Cdk Regulation Association with cyclins (Formation of Cdk/cyclin complexes) Cyclin synthesis and degradation Activating phosphorylation by CAK (Cdk activating enzyme) Inhibitory phosphorylation by Wee 1 (protein kinase) Activating dephosphorylation by Cdc25 protein phosphatase Association with Cdk inhibitors CKIs  Two Families of Cdk inhibitors CKIs Cip/Kip family (p21, p27, p57) inhibits Cdk4, 6/Cyclin D G1- S transition Cdk 2/Cyclin E G1-S transition Cdk 2/Cyclin A progression through S phae Ink 4 family (p15, p16, p18, p19) inhibits Cdk 4,6/Cyclin D passage through G1 checkpoint Mitosis Promoting Factor (MPF) MPF ;cytoplasmic regulator which controls the entry into mitosis MPF first discovered in mature Xenopus oocyte Xenopus oocyte so big→ 1 mm in diameter Easy to inject substances Cytoplasm from M phase oocyte was injected into G2 oocyte → G2 oocyte entered into M phase Oocyte maturation Maturation-Promoting Factor (MPF) Discovery of MPF Experiments byY.Masui and C. Markert in 1971 Cytoplasm from M phase Xenopus oocyte was injected into G2 oocyte → G2 oocyte entered into M phase in the absence of progesteron Result:A cytoplasmic factor is responsible for G2 → M phase transition.This factor is named as MPF  Cell fusion experiments M + G1 →Nucleus of G1 cell enters into mitosis M + S → Nucleus of S cell enters into mitosis M + G2→ Nucleus of G2 cell enters into mitosis Result;Cytoplasm of M phase cell contains a potent factor (MPF) MPF is sufficient to induce entry into mitosis.. Cell fusion experiments Result: A cytoplasmic factor (MPF) is sufficient to induce entry into mitosis  MPF Xenopus mature egg is a good source to purify MPF MPF is of universal importance to eukaryotic cells Highly preserved during evolution (mammals,sea urchin,yeast) Passage through G1 check- point t.fi #II  What are the target proteins of Cdk4,6/CyclinD It has been shown that Retinoblastoma protein is the target protein of Cdk4,6/cyclin D complex Rb is phosphorylated by Cdk4,6/cyclin D complex  Rb protein Retinoblastoma is a rare inherited childhood eye tumor Rb gene is mutated in retinoblastoma Rb is also frequently mutated in wide variety of human tumors Inactivation of Rb leads to tumor development Rb is a tumor supressor gene  Retinoblastoma Rb Protein Rb is Key Substrate protein of Cdk 4, 6/Cyclin D (G1-Cdk) Activity of Rb is regulated by phosphorylation In underphosphorylated form Rb binds to E2F family of Transcription factors (Repress transcription) Cdk 4, 6/cyclin D  Phosphorylates Rb  Dissociation from E2F  Activation of transcription of target genes (S phase) In underphosphorylated form Rb binds to E2F family of Transcription factors Cdk 4, 6/cyclin D Phosphorylates Rb Dissociation from E2F E2F activates transcription of target genes (S phase genes G 1/S-cyclin (cyclin E), S-cyclin (cyclin A) Active S-Cdk entry into S phase (replication)  Start Kinase in G1 Synthesis of G1 cyclins (cyclin D) binding to Cdk → (Cdk4,6 cyclinD) (Protein phosphorilations) Rb phosphorilation E2F activates transcription of target genes G1/S-cyclin (cyclin E), S-cyclin (cyclin A)Active S-Cdk Cell start DNA replication) ↓  Cell fusion experiments: S + G1 Fusion G1 nucleus starts DNA replication S phase cell contains a cytoplasmic factor that starts DNA replication = Start Kinase Result; G1 cell is ready for replication but an S phase cytoplasmic factor absent  DNA damage control points Several cell cycle check points function to ensure that incomplete or damaged chromosomes are not replicated and passed on to daughter cells.  DNA damage The cell-cycle control system can detect DNA damage and arrest the cycle at one of two checkpoints- 1-one at Start in late G1, which prevents entry into the cell cycle and into S phase, 2- and the other one at the G2/M checkpoint, which prevents entry into mitosis  DNA damage arrests cell cycle at G1 checkpoint allows time to repair the damaged DNA before S phase Arrest in G1 is mediated by the action of a protein known as p53 The gene encoding p53 is mutated in most of the human cancers p53 is a tumor supressor gene At G1 phase DNA damage ↓ Increase in p53 protein ↓ arrest at G1 checkpoint p53 protein is a transcription factor and progresses the transcription of target proteins (i.e p21) that arrest cell division at G1. p21 is a Cdk inhibitor  DNA damage control Some of the proteins recognize DNA damage and bind to damaged DNA Target of these proteins are Protein Kinases called ATM and ATR ATM and ATR are activated and Phosphorylate → Chk2 and Chk1 Kinases  DNA damage initiates a signaling pathway by activating a pair of related protein kinases called ATM and ATR, which associate with the site of damage and phosphorylate various target proteins, including two other protein kinases called Chk1 and Chk2. these kinases phosphorylate other target proteins that lead to cell-cycle arrest. A major target is the gene regulatory protein p53,  ATM and Chk2 Phosphorylate → p53 protein Phosphorylated p53 protein accumulates within the cell p53 protein is a transcription factor and stimulates the transcription of target proteins (p21) that arrest cell division at G1  Target of damaged DNA binding proteins are Protein Kinases called Damaged or unreplicated DNA ATM and Chk2 damaged DNA binding Phospholylates p53 proteins p53 accumulates, stabilized ATM ATR and stimulates expression of CKI (p21) p21 arrest cell division at Chk1 Chk2 G1 check point Cell cycle arrest  Regulatory protein p53, stimulates transcription of the gene encoding a CKI protein called p21; this protein binds to G1/S-Cdk and S-Cdk complexes and inhibits their activities, block entry into the cell cycle DNA damage activates p53 by an indirect mechanism. In undamaged cells, p53 is highly unstable and is present at very low concentrations. P53 has been described as "the guardian of the genome", In 1979 Professor Sir David Lane Discovery of the p53 cancer protein has been a revolution in our understanding of cell division control? ( including cancer cells) p53 was the first ‘tumour suppressor’ found within our cells – that usually acts to protect us from cancer. In 1993, p53 protein has been voted molecule of the year by the Science magazine  p53 mutations (loss of function) ↓ prevents G1 arrest in response to DNA damage ↓ damaged DNA is replicated and passed on to daughter cells ↓ Inheritance of damaged DNA results in increased frequency of mutations and genomic instability ↓ cancer development P53 mutations are the most common genetic alterations in human cancers  loss of p53 function allows the cancer cell to accumulate mutations Similarly, a rare genetic disease known as ataxia telangiectasia is caused by a defect in ATM, one of the protein kinases that is activated in response to x-ray-induced DNA damage;  patients with ataxia telangiectasia disease are very sensitive to x-rays and suffer from increased rates of cancer. What happens if DNA damage is so severe that repair is not possible  Proteolysis and inactivation of MPF Ubiquitin-mediated proteolysis of M-cyclin by activation of Ubiquitin-ligase called Anaphase-promoting complex (APC) Activation of APC; 1- proteolysis of M-cyclin 2- proteolysis of securin →activation of separase, → proteolysis of cohesins→sister chromatid separation Control of proteolysis by APC  Ubiquitin-ligase; SCF 1- proteolysis of G1/S –cyclins 2- proteolysis of some CKI’s Phosphorylation of CKI →recognition by SCF → Ubiquitinylated CKI →degradation Control of proteolysis by SCF