Buněčný cyklus - principy regulace buněčného růstu a buněčného dělení Mitöza (e) Anaphase {ft Telophase (g) Interphase (Gi) Dr. B. Duronio, The University of North Carolina at Chapel Hill Průběh mitózy v buněčné kultuře fibroblast MetaphasB Anaphase Dr. B. Duronio, The University of North Carolina at Chapel Hill Buněčný cyklus Figure 17-12. Molecular Biology of the Cell, 4th Edition. Dr. B. Duronio, The University of North Carolina at Chapel Hill Kinázy závislé na cyklinech kontrolují buněčný cyklus Dr. B. Duronio, The University of North Carolina at Chapel Hill Kontrola vstupu do mitózy - výsledek využití různých buněčných modelů Experimental Systems Important for Cell Cycle Studies Schizosaccharomyces pombe Xenopus laevis Dr. B. Duronio, The University of North Carolina at Chapel Hill Cyclin was Discovered in Sea Urchin Embryos can stimulate to lay lots of eggs 1 Protein Level cyclin A yn cyclin B r'-Time Dr. B. Duromo, The University of North Carolina at Chapel Hill Frog life cycle OOCYTE GROWS WITHOUT DIVIDING FERTILIZED EGG DIVIDES WITHOUT GROWING (MONTHS) FERTILIZATION (HOURS) The Maturation of Frog Eggs (metaphase) An Assay for Maturation Promoting Factor (MPF) Egg arr-esied in rnetaphdse II Yoshio Masui, 1971 MPF Activity Peaks Before Each Cell Division A Progesterone Fertilization G? arrest Meiosis I Meiosis II rnetaphase arrest Time First Second embryonic efrtbryonic mitosis mitosis Moreover, MPF has kinase activity Proc. Natl. Acad. Sci. USA Vol, 85s pp. 3009-3013, May 1988 Cell Biology Purification of maturation-promoting factor 9 an intracellular regulator of early mitotic events (cell cycle/mitosis/protein phosphorylation) Manfred J. Lohka*, Marianne K. Ha yes t, and James L. Maller Department of Pharmacology, University of Colorado School of Medicine, Denver, CO 80262 Communicated by Raymond L. Erikson, December 22, 1987 (received for review October 10, 1987) ABSTRACT Maturation-promoting factor causes germinal vesicle breakdown when injected into Xenopus oocytes and can induce metaphase in a cell-free system. The cell-free assay was used to monitor maturation-promoting factor during its purification from unfertilized Xenopus eggs. Ammonium sulfate precipitation and six chromatographic procedures resulted in a preparation purified >3000-fold that could induce germinal vesicle breakdown within 2 hr when injected into cyclo-heximide-treated oocytes. Proteins of 45 kDa and 32 kDa were correlated with fractions of highest activity in both assays. These fractions contained a protein kinase activity able to phosphorylate the endogenous 45-kDa protein, as well as histone HI, phosphatase inhibitor 1, and casein. The highly purified preparations described here should help to identify the mechanism of action of maturation-promoting factor and to elucidate the role of protein kinases in the induction of metaphase. Dr. B. Duronio, The University of North Carolina at Chapel Hill Dr. B. Duronio, The University of North Carolina at Chapel Hill Purification of MPF: The Birth of Cyclin Dependent Kinases B This is cdc2+\\ (Cdc28 in S. cerevisiae) NE BD (units) 0 GVBD(%) ( 0 ■ 1 1 0 0 50 85 1 I ■ m Fraction Number 12 15 12 15 Fig. 2. Polyacrylamide gel analysis of fractions eluting from the Mono S column. A 45-ji.l aliquot of fractions 5-16 was incubated with [y-32P]ATP and electrophoresed through a 10% NaDodS04/poly-acrylamide gel. (A) Silver-stained Polyacrylamide gel of purified MPF. The activity of the fractions in the cell-free assay (NEBD) and in the oocyte microinjection assay (GVBD) is shown below the gel. NEBD is expressed in units/50 /xl. GVBD is expressed as the percentage of oocytes that underwent GVBD during a 2-hr incubation in cycloheximide (0,5 jxg/ml). (B) Autoradiograph of the silver-stained gel shown in A. (C) HI kinase activity of purified MPF. Mono S fractions 6-15 were assayed for HI kinase activity. The autoradiograph of the region of the gel with histone 1 is shown. Fraction 12 had a specific activity of -270 nmol-min" '-mg-1. This is cyclin!! Which = cdcl3+ in S. pombe Fission yeast: Schizosaccharomyces pombe Chromosome condensation Dr. B. Duromo, The University of North Carolina at Chapel Hill Budding Yeast Saccharomyces cerevisiae Dr. B. Duronio, The University of North Carolina at Chapel Hill Cdc Mutants Arrest at the Same Cell Cycle Phase Dr. B. Duronio, The University of North Carolina at Chapel Hill Cdc Genes Encode Proteins Needed for the G2-M Transition: Studies in S. pombe cdc2+ encodes a kinase Moreover = cdc28 in S. cerevisiael Dr. B. Duronio, The University of North Carolina at Chapel Hill Phosphorylation of CDK Targets Changes Their Activity Dr. B. Duronio, The University of North Carolina at Chapel Hill Jak j sou CDK regulovány? 1. prostřednictvím syntézy a odbourávání cyklinů 2. fosforylací 3. pomocí CDK inhibitory proteins (CKIs) trigger mitosis machinery t M-Cdk S-Cdk I ................._........ trigger DNA replication machinery Figure 17-16. Molecular Biology of the Cell, 4th Edition. Cyclin Destruction is Controlled by Ubiquitination (a) Mitotic cyclin destruction box H2N - |— COOH Cyclin A Arg—Thr—Val-Leu-Gty-Val—lle-Gly-Asp Cyclin B1 Arg— Thr-Ala— Leu — Gly — Asp — lie — Gly — Asn Cyclin B2 Arg-Ala -Ala-Leu-Gty-Glu — He - Giy -Asn (b) Polyubiquitinmion of mitotic cyclin APC (E3) n E2-0 T n E2 Proteasome Degraded cyclin peptides n E1 ■n El Ubiquitin Dr. B. Duronio, The University of North Carolina at Chapel Hill Představují cykliny jediný způsob regulace CDK? Dr. B. Duronio, The University of North Carolina at Chapel Hill CDK jsou regulovány fosforylací Cdk activating phosphate ACTIVE INACTIVE Figure 17-18. Molecular Biology of the Cell, 4th Edition. Conformational Changes Associated with CDK Phosphorylation Free CDK CDK + Cyclin Tl61 phosphorylation The T-loop blocks Binding of cyclin Phosporylation moves substrate access moves the T-loop the T-loop more Cyclin Dependent Kinase Inhibitors (CKIs) Figure 17-19. Molecular Biology of the Cell, 4th Edition. Dr. B. Duronio, The University of North Carolina at Chapel Hill The p21 Family of CDK inhibitors (p21CIP1/WAF1, p27KIP1, p57KIP2) active Broil 2 Jeffrey et al. (1995) Nature 376:313 inactive CDF A Russo et al. (1996) Nature 382:325 Dr. B. Duronio, The University of North Carolina at Chapel Hill The INK4 Family of CDK inhibitors (p!6INK4a, pl5INK4b, p!8INK4c, pl9INK4d) active inactive CKIs Regulate the Gl-S Transition Dr. B. Duronio, The University of North Carolina at Chapel Hill Figure 1 | Regulation of G1 and the G1/S transition. In quiescent, GO cells, E2F-DP transcription factors are bound to p 130, the principal pocket protein In these cells, which keeps thern inactive. In G1, however, RB-E2F-DPcomplexes predominate. Mitogenic signalling results In cyclln D (Cyc) synthesis, formation of active CDK4/S-cyclin-D complexes and initial phosphorylation of RB. Partially phosphorated RB still binds to E2F-DP, but the transcription factor is still able to transcribe some genes, such as cyclln E, presumably due to impaired repression. Cyclin E binds to and activates CDK2. It is generally accepted that CDK2-dependent phosphorylation of RB results in its complete Inactlvatlon, which allo^/s Induction of the E2F-responsive genes that are needed to drive cells through the G1/S transition and to initiate DNA replication. INK4 and WAF1/KIP proteins can inhibit CDK4/6 or CDK2 kinases, respectively, following specific antimitogenic signals. The CDK4/6 complexes can also bind WAF1/KIP inhibitors, while remaining active. This sequesters them from GDK2, which facilitates its full activation. R represents the restriction point that separates the mltogen-dependent early G1 phase from the mltogen-independent lateGI phase. Nature Reviews Cancer 2001, 1:222 Cell cycle regulated phosphorylation of the retinoblastoma protein Dr. B. Duronio, The University of North Carolina at Chapel Hill pRB Binds to the E2F Transcription Factor Transactivation E2F target genes on Dr. B. Duronio, The University of North Carolina at Chapel Hill Active repression E2F target genes off Gl Cyclin CDKs Phosphorylate pRB ^^^^ ^flSSH^^ IQ |-V^ §Q i—- TTTCGCGC TTTCGCGC Active repression E2F target genes off Transactivation E2F target genes on Dr. B. Duronio, The University of North Carolina at Chapel Hill p 16 Regulates pRB Phosphorylation! Proliferation Signals Cyclin D1 cdk4 or cdk6 pRB Repression of Transcription GA Phase peätino phase Activation of E2F-Dependent DNA Transcription CELL CYCLE PROGRESSION Dr. B. Duronio, The University of North Carolina at Chapel Hill Figure 1 | Regulation ot G1 and the G1/S transition. In quiescent, GO cells, E2F-DP transcription factors are bound to p 130, the principal pocket protein In these cells, which keeps thern inactive. In G1, however; RB-E2F-DPcomplexes predominate. Mitogenic signalling results In cyclln D (Cyc) synthesis, formation of active CDK4/S-cyclin-D complexes and initial phosphorylation of RB. Partially phosphorated RB still binds to E2F-DP, but the transcription factor is still able to transcribe some genes, such as cyclln E, presumably due to impaired repression. Cyclin E binds to and activates CDK2. It is generally accepted that CDK2-dependent phosphorylation of RB results in its complete Inactlvatlon, which allo^/s Induction of the E2F-responsive genes that are needed to drive cells through the G1/S transition and to initiate DNA replication. INK4 and WAF1/KIP proteins can inhibit CDK4/6 or CDK2 kinases, respectively, following specific antimitogenic signals. The CDK4/6 complexes can also bind WAF1/KIP inhibitors, while remaining active. This sequesters them from GDK2, which facilitates its full activation. R represents the restriction point that separates the mltogen-dependent early G1 phase from the mltogen-independent lateGI phase. Nature Reviews Cancer 2001, 1:222 Kontrola buněčného cyklu úzce souvisí s: ► kontrolou buněčného růstu; ► přítomností růstových faktorů a dalších růstových stimulů a živin; ► působením ostatních buněk populace a mezibuněčné hmoty. The Difference Between Growth and Cell Division Growth with No Cell Division o o o o A A /V /V oooo oooo Cell Division No Growth Cell Division + Growth Proliferation! Growth with No Cell Division: A Differentiated Neuron neuron 0 lymphocyte Dr. B. Duronio, The University of North Carolina at Chapel Hill Cell Division with No Growth: Early Development Dr. B. Duronio, The University of North Carolina at Chapel Hill Box 1 I Cell growth versus cell division Cell growth (the Increase in cell size and protein mass) is a term that has frequently been misused to mean cell proliferation. In fact, both processes are highly coordinated. Only in certain biological systems — such as oocytes, neurons and muscle cells, where cell growth might exist without cell division, and in fertilized eggs, where cell divisions might occur without cell growth — can these processes function in an independent, or even complementary, fashion. In most cells, however, cdl division without concurrent cell growth would generate smaller daughter cells, which would affect their viability. Ri bo so me biosynthesis is a key process for cell growth. Before entering the cycle, cells need to accumulate sufficient translational machinery, mainly ribosomes, to ensure the rapid processing of transcripts through the cycle. This is accomplished, at least in part, by phosphorylation of the ribosomal S6 protein by S6 kinase (S6K) (see REE for a review). Once the appropriate pool of ribosomes has been achieved, the system is desensitized, either by negative regulators of S6Kor by the size of the ribosomal pool (.see figure). S6Kis regulated by mitogenk stimuli mediated through the insulin receptor (IRi/TR substrate (IRS)/phosphatidylinositol-3 kinase (Pl3K)/PDKl pathway. S6K is also regulated directly by TOR, a member of the PI3K-related kinase family86,87. TOR is thought to be important in cell growth and a mi no-acid sensing37, but its upstream activators and mechanism of activation are unknown. TOR controls several growth-related readouts, including act in organization, transcription and ri bo some biosynthesis TOR at so affects translation of key regulators of cell proliferation, such as cyclin D and MYC, by phosphoryluting 4E-BPI (a transitional inhibitor that is also targeted by AKT/PklJj and causing its dissociation from the initiation factor elF-lE. Mitogen-activated protein kinases such as l.'Kk phosphorylate and activate MN'Kl, which in turn is able Ui phiHsphorylak i J F4 E REI S w.*1-.' i. The RA5/ERK cascade is alio known to signal to cell-cycle regulators such as cyclin Dor KIP] to induce pnigressii »n through C I i KIT 121. Several i »f these proteins, such as PI ?K, AKT, MYC and RAS, caji be ac t i vu t ed a s on cogenes, w h ic h i II u st ra t e s t h e intimate connections between cell growth and cell proliferation. Growth factors O O Cell-cycle progression Nature Reviews Cancer 2001, 1:222 Growth Factors Induce Cell Cycle Progression Dr. B. Duronio, The University of North Carolina at Chapel Hill Box 2 I The Restriction Point The term1 Rest fiction Point1 was coined in ] LJ74 by Aft hut Pardee00 to define a specific eve tit in C I after which cultured cells could proliferate independently of mitogenic stimuli. Briefly, cultured mammalian cells that had undergone mitosis within the previous 3 hours could be prevented from progressing through the cell cycle by growth-factor starvation or moderate inhibition of protein synthesis. These cells then re-entered the cell cycle after re-stimulation with growth factors. However, if the cells had undergone cell division more than 4 hours before, they did not respond to mitogen deprivation and advanced through the cell cycle with the same kinetics as un starved cells. It was postulated that the latter cells had * passed* the restriction point (R). Today, R is often used to divide the early and late C I phases. R does not represent a checkpoint as originally defined in yeast2,01. In culture cells, R occurs 3-4 hoars after mitosis f see figure J. However, entry into S phase is usually initiated 5-13 hours after mitosis. This variability is characteristic of the late C I phase and accounts for most of the observed differences in the length of the cell cycle. Indeed, the differential kinetics of these two transitions indicates distinct control mechanisms. The molecular events that allow cells to pass R have not been well defined. However, members of the RB family are likely to be important, as ablation of this gene fa mi Ly eli mi nates R°i,t)3. It has been postulated that loss of regulation of R is critical in cancer. R normally prevents cells from entering the cycle until they have accumulated a certain threshold of mi tog en-induced events, so loosening of R control due to mutations in C1 regulators or other, as yet unidentified., genes would allow cells to enter the cycle even in the absence of adequate mitogenic signalling, leading to unscheduled proliferation. Validation of this model will require definition of the molecular players that regulate R. Mitogens EariyGI Remove gm.lh factors Early Gl Gra-Arth-factor -+ removal Late Gl Nature Reviews Cancer 2001, 1:222 Dr. B. Duronio, The University of North Carolina at Chapel Hill 1 hr 8 hr pm = post mitotic ps = pre-synthetic Fig. 7. Schematic model of cell cycle in Swiss 3T3 cells. During the first 3.5-hr after mitosis (dpm), the cell makes the decision whether or not to progress through the cell cycle. This decision depends on the presence of growth factors (gf). If the cell senses a lack of growth factors (-gf) in Gxpm, it will leave the cell cycle within 15-60 min and enter a state of quiescence (G0) from which it takes 8 hr to reenter the cycle after the growth factor level in the environment again becomes optimal (+gf) for proliferation. Once the cell has entered Gips, it will eventually initiate DNA synthesis. However, Gips is highly variable in length and in fact responsible for most of the variability in the duration of Gi and of the whole cell cycle. Zetterberg and Larsson, PNAS 82:5365 (1985) Growth Factors Induce Gene Expression Delayed-response genes Early-response genes 0 1 2 4 8 12 f Timel &ene Transcrjpttort NUCLEUS Sherr and McCormick, Cancer Cell, Vol 2, 103-112 (2002) Mitogen Induced Cell Cycle Progression in Cell Culture G0 G1 S GrM G1 S GrM G0 Dr. B. Duronio, The University of North Carolina at Chapel Hill Is all DNA replicated? i environment favorable? G2 CHECKPOINT Are all chromosomes attached to the spindle? METAPHASE CHECKPOINT ENTER S Gl CHECKPOINT Is environment favorable? Figure 17-14. Molecular Biology of the Cell, 4th Edition. Cell Cycle Checkpoints [2 unfavorable excess chromosome Z extracellular DNA mitogenic unreplicated DNA unattached to O environment damage stimulation DNA damage spindle u u p53 Hrt1 Gl-Cdk Gi/S-Cdk—I i 11 lu Gt/S-cyclin synthesis O u S-cycMn synthesis S-Cdk -^Cdc25—- IVhCdk APC DNA rereplication S M Figure 17-34. Molecular Biology of the Cell, 4th Edition. Cell Cycle Checkpoints Improve Cell Viability Fig. 3. Rapid loss of viability in cells defective both for DNA ligase and for the RAD9 gene. cdc9 (•) and cdc9-rad9 (O) cells growing at23°C were shifted to the restrictive temperature and viability was determined by plating for viable colonies at the permissive temperature (23°C)J The cell viability reported is relative to viability at the time of temperature shift. Results were reproducible in separate experiments and with other congenic strains. Time (hours) at 36" Weinert and Hartwell, Science 246:629 (1989) How do Cell Cycle Checkpoints Work? DNA Replication damage '—> stress fT^JT^^vraT SIGNALS i SENSORS . TRANSDUCERS T Cell cycle Apoptosis Transcription DNA repair transitions Zhou and Elledge Nature 408, 433 - 439 (2000) D N A d am age Replicationstress arrest Figure 2 Organization of the mammalian DNA damage response pathway. Arrowheads represent positively acting steps while perpendicular ends represents inhibitory steps. Gene names ane shown at the approximate positions where their encoded proteins function in the pathway. Although the general organization of the pathway is correct some details are omitted, especially concerning the relationship between the ATR/ATM and Hus1/Rad17/Rad9/Rad1 proteins, which may participate in mutual regulation. Zhou and Elledge Nature 408, 433 - 439 (2000) Genotoxic Stress (i.e. IR or UV) Signal Sensor Cytoplasmic sequestration Inactive G2 Arrest cyclin B Active Mitosis Transducer Effector Dr. B. Duronio, The University of North Carolina at Chapel Hill Metaphase in a mammalian cell The Metaphase to Anaphase Transition G2 Prophase Metaphase Anaphase Telophase G The Spindle Assembly Checkpoint Check point activated CENP-E BiibR1/BuP3 Bubi/Bub3 ? Mad2 Madl Mad2* Securin degradation (Up -dependent) :£n£ckpoin<:. silenced Cohesin cteavage Anaphase onset Checkpoint proteins displaced ProducLion of Mad2* ceases {'fl Cohesin phosphorylation ? poškození DNA poruchy buň. cyklu poruchy buň. dělení nedostatek živin a růstových faktorů kontaktní inhibice vliv ECM \ dostatek živin a růstových faktorů mitogeny zástava buň.cyklu (restriction point, checkpoints) setrvání v G0 fázi apoptóza proliferace