Buněčný cyklus - principy regulace buněčného růstu a buněčného dělení Mitóza (aí Interpha&e (Gj) (b} Early prophage- [c] Late prophage- id) Metaphase .Centrüsomes .Spindle poles •Kinetochore Sister chromatids (e) Anaphase {f> Telophase (g) Interphase (Gi) Cleavage furruw Dr. B. Duronio, The University of North Carolina at Chapel Hill Průběh mitózy v buněčné kultuře fibroblastů Eariy prophase Late prophase MetaphflSB Anaphase Dr. B. Duronio, The University of North Carolina at Chapel Hill Buněčný cyklus o ÚJ E C cells in Gl phase cells in G2 and M phases n cells in S phase A Li i^ft 0 1 2 relative amount of DNA per cell (arbitrary units) Figure 17-12. Molecular Biology of the Cell, 4th Edition. Dr. B. Duronio, The University of North Carolina at Chapel Hill Kinázy závislé na cy klínech kontrolují buněčný cyklus Dr. B. Duronio, The University of North Carolina at Chapel Hill Kontrola vstupu do mitózy - výsledek využití různých buněčných modelu Experimental Systems Important for Cell Cycle Studies , .wtaflüüfil • f' • t - * ■ "'í y ■• s 'J' ! ». -■'„ ^Ä ^»»-.WEÍWffl""*» Saccharomyces cerevisiae Arbacia punctulata 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 Protein Level cyclin A^i cyclin B p^Time Dr. B. Duronio, The University of North Carolina at Chapel Hill Frog life cycle OOCYTE GROWS WITHOUT DIVIDING FERTILIZED EGG DIVIDES WITHOUT GROWING (MONTHS) FERTILIZATION (HOURS) O -0-0-0- 1 mm sperm tadpole feeds, grows and becomes an adult frog j The Maturation of Frog Eggs (a} Oocyte maturation in vitro An Assay for Maturation Promoting Factor (MPF) Yoshio Masui, 1971 MPF Activity Peaks Before Each Cell Division a > H-» (J co Progesterone Fertilization G?arrest Meiosis I Meiosis II metaphase arrest First Second embryonic embryonic mitosis mitosis Time Moreover, MPF has kinase activity Proc. Natl. Acad. Sei. USA Vol. 85, pp. 3009-3013, May 1988 Cell Biology Purification of maturation-promoting factor, 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) 30- l i i NE BD (units) 0 0 11 2 2 GVBD (%) 0 0 0 0 50 85 Fraction 6 9 12 Number 15 12 15 Fig. 2. Polyacrylamide gel analysis of fractions eluting from the Mono S column. A 45-/li1 aliquot of fractions 5-16 was incubated with [?-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 ju.1. GVBD is expressed as the percentage of oocytes that underwent GVBD during a 2-hr incubation in cycloheximide (0.5 fxg/ml). (B) Autoradiograph of the silver-stained gel shown in A. (C)H1 kinase activity of purified MPF. Mono S fractions 6-15 were assayed for HI kinase activity. The autora-diograph of the region of the gel with histone 1 is shown. Fraction 12 had a specific activity of -270 nmo!*min"1,mg"1. This is cyclin!! Which = cdcl3" in S. pombe Fission yeast: Schizosaccharomyces pombe Chromosome condensation Spindle pole body duplication DNA replication START Growth Spindle formation Chromosome segregation Nuclear division Dr. B. Duronio, The University of North Carolina at Chapel Hill Budding Yeast Saccharomyces cerevisiae Nuclear migration Spindle formation DNA replication O Bud ' emergence Spindle pole body duplication START Chromosome segregation; \ nuclear division Cytokinesis Dr. B. Duronio, The University of North Carolina at Chapel Hill Cdc Mutants Arrest at the Same Cell Cycle Phase Permissive (low) temperature Restrictive (high) temperature 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. cerevisiae! Dr. B. Duronio, The University of North Carolina at Chapel Hill Phosphorylation of CDK Targets Changes Their Activity Cyclin- CDK binding Now perfonns a cell cycle function Target-protein binding Phosphorylation of targel protein 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-cyclin 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- |—COQH Cyclin A Arg—Thr—Val —Leu-Gly-Val—lle^Gly—Asp Cyclin B1 Arg—Thr—Ala—Leu —Gly—Asp —He —Gly—Asn Cyclin B2 Arg-Ala-Ala-Leu-Giy-Glu —He-Gly-Asn {b} Polyubiquitination of mitotic cyclin APC (E3) Prótea so me nE2-0 => 4> Degraded cyclin peptides nE2 nE1-Q^ n El Ubiquitin Dr. B. Duronio, The University of North Carolina at Chapel Hill Představují cykliny jediný způsob regulace CDK? Exprese cyklinu v jednotlivých fázích BC Cdkl-cyclin B Cdkl-cyclin A CdkS-cydin A Cdk4-cyclín D Cdk6-cyclin D Cdk2-cyclín E Dr. B. Duronio, The University of North Carolina at Chapel Hill CDK jsou regulovány fosforylací cyclin inhibitory phosphate 'i v /^a weei >^r^\ rx j ^=- Ékj 1 >pV. Cdc25 ■ >pV V^_3^ phosphatase ^^^^^ 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 T161 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) Cdk cyclin J active cyclin-Cdk complex inactive p27-cyc!in-Cdk complex 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 Cvcli inactive Jeffrey et al. (1995) Nature 376:313 Russo et al. (1996) Nature 382:325 Dr. B. Duronio, The University of North Carolina at Chapel Hill The INK4 Family of CDK inhibitors (pl6INK4a, pl5INK4b, pl8INK4c, p 19^) active CDK4/6 Cyclin D inactive Russo et al. (1998) Nature 395:237 Brotherton et al. (1998) Nature 395:244 CKIs Regulate the Gl-S Transition Cdkl-cyclin B Cdkl'CycIín A Cdk2-cyclin A i«|w;(pi6) 1 Cdk4-cyclin D Cdk6-cyciin D (p21,p27) Dr. B. Duronio, The University of North Carolina at Chapel Hill Figur© 1 I 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 them Inactive. In G1, however, RE-E2F-DP complexes predominate, Mltogenlc signalling results In cyclin D (Cyc) synthesis, formation of active CDK4/6-cyclin-D complexes and Initial phosphorylation of RB. Partially phosphorylated RB still binds to E2F-DR but the transcription factor Is still able to transcribe some genes, such as cyclin E, presumably due to Impaired repression. Cyclin E binds to and activates CDK2. It Is generally accepted that GDK2-dependent phosphorylation of R B results In Its complete inactivation, which allows 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, vjhile remaining active. This sequesters them from CDK2, which facilitates its full activation. R represents the restriction point that separates the mitogen-dependent early G1 phase from the mitogen-independent late G1 phase. Nature Reviews Cancer 2001,1:222 Dr. B. Duronio, The University of North Carolina at Chapel Hill pRB Binds to the E2F Transcription Factor pRb TTTCGCGC Transactivation E2F target genes on TTTCGCGC Active repression E2F target genes off Dr. B. Duronio, The University of North Carolina at Chapel Hill Gl Cyclin CDKs Phosphorylate pRB , cyclin D cdk4/Ej pRb j^ ■ e2f; dp |^^9 [cdk2 J cyclin E TTTCGCGC 1 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 Inhibits Cyclin D1 cdk4 or cdk6 Repression of Transcription t- phase Activation of E2F-Dependent DNA Transcription ^ % %> ^ / CELL CYCLE PROGRESSION Dr. B. Duronio, The University of North Carolina at Chapel Hill Figur© 1 I 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 them Inactive. In G1, however, RE-E2F-DP complexes predominate, Mltogenlc signalling results In cyclin D (Cyc) synthesis, formation of active CDK4/6-cyclin-D complexes and Initial phosphorylation of RB. Partially phosphorylated RB still binds to E2F-DR but the transcription factor Is still able to transcribe some genes, such as cyclin E, presumably due to Impaired repression. Cyclin E binds to and activates CDK2. It Is generally accepted that GDK2-dependent phosphorylation of R B results In Its complete inactivation, which allows 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, vjhile remaining active. This sequesters them from CDK2, which facilitates its full activation. R represents the restriction point that separates the mitogen-dependent early G1 phase from the mitogen-independent late G1 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 A A oooo oooo Cell Division No Growth / \ / \ / \ Cell Division + Growth Proliferation! Ä -^S r-^^^gV /.. Growth with No Cell Division: / — f 25 [im A Differentiated Neuron neuron 0 lymphocyte Dr. B. Duronio , The University of North Carolina at < ľhapel 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 h,is frequently been misused to mean cell proliferation. In fact, both processes are highly coordinated. Only in certain biologic»] 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, faslm m. In must cells however, cell division without concurrent cell growth would generate smaller daughter cells, which would affect their viability. Ribosome biosynthesis is a key process for cell growth. Before entering the cycle, cells need to accumulate sufficient translations! machinery, mainly ribosomes, to ensure the rapid processing of transcripts through the cycle. This is accomplished, at least En part, by phosphorylation of the ribosomal Sb protein by So kinase (S6K) (see REE 05 for a review). Once the appropriate pool of r i bosom es has been achieved, the system is desensitized, either by negative regulators of 56 K or by the size of the ribosomal pool (see rig u re). S6K is regulated by mitogenit stimuli mediated through the insulin receptor ([Rj/IR substrate (IRSVphosphatidylinositol-3 kinase (Pl3K)/PDKl pathway. SoK isalso regulated directly by TOR» a member of the Pl3K-retated kinase family86-87. TOR is thought to be important in cell growth and a mi no-acid sensing47, but Its upstream activators and mechanism of activation are unknown. TOR controls several g row t h-related readouts, including actiti organization, transcription and ribosome biosynthesis. TOR also affects translation of key regulators of cell proliferation, such as cyclin D and MYC, by phosphor y titling 4E-BP1 (a translational inhibitor ih i L is also targeted by AKT'! T B) and causing its dissociation from the initiation factor eIF4E. Mitogen-activated protein kinases .such as ERK phosphorytate and activate MNKl, which in turn is able to phosphor y late eIF4E iREFS 68, S9). The RAS/ERK cascade is also known to signal to cell-cycle regulators such as cyclin Dor KIPl to induce progression through Gl (REP, 12). Several of these proteins, such as PI3K. AKT, MYC and RAS, can be activated as oncogenes, which illustrates the intimate connections between celt growth and cell proliferation. Growth factois O O Cell-cycle progression Nature Reviews Cancer 2001,1:222 Growth Factors Induce Cell Cycle Progression Growth Factors act at the Restriction Point Dr. B. Duronio, The University of North Carolina at Chapel Hill Box 2 I The Restriction Point The term 'Restriction Point1 was coined in 1974 by Arthur Pardee™ to define a specific event in G t after which cultured cells could proliferate independently of mitogeuic stimuli, Briefly, cultured mammalian celts 1 ha t had undertime mitosis wit hin the previous } hours could be prevented from progressing through the cell cycle b v growth-fa c to r starvation or moderate inhibition of protein synthesis. These cells then re-entered the cell cycle after re-stimuhition with growth factors. However, if the cells had undergone cell division more than -1 hours before, they did not respond to mitogen deprivation and advanced through the cell cycle with the same kinetics as u n starved cells. Et was postulated that the latter cells had 'passed' the restriction point (R). Today; R is often used to divide the early and late 0I phases. R does not represent a checkpoint as origináli}' defined in yeast2,01. In culture cells, R occurs 3-4 hours after mitosis í see figure). However, entry into S phase is usually initiated 5-13 hours after mitosis. This variability is characteristic of the Lite C I phase and accounts for most * >f 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 family eliminates R92,93. It has been postulated that loss of regulation of R is critical in cancer. R normáli)' prevents cells from entering the cycle until they have accumulated a certain threshold of mitogen-induced events, so loosening of R control due to mutations En G1 regulators or other, as yet unidentified, genes would a How cells to enter the cycle even in the absence of adequate autogenic signalling, leading to unscheduled proliferation. Validation h if this model will require definition of the molecular players that regulate R. MitogQns Early G1 Growth-factcr -------------* removal Remaws growth Factors Early Gl LateGI Mr M G2 *oP oV Nature Reviews Cancer 2001,1:222 Dr. B. Duronio, The University of North Carolina at Chapel Hill 3.5 hr variable ------------------1 I--------------------1 H Gi pm +gf -gf TV 1 hr 8 hr 1 i +9f © 8.5 hr Gi ps S + G2 + M +/-gf + /-gf 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 (Gipm), 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 (a) Addition of serum (b) Addition of serum + inhibitors of protein synthesis Delayed-response genes Early-response genes ] 4 Time (h) Early-response genes Delayed-response genes do not turn on ______I___________L 4 Time (h) 8 12 12 Dr. B. Duronio, The University of North Carolina at Chapel Hill Growth Factors Induce Cyclin Dl Expression \ MEK \ ERK API Receptor Activation -------- Ros --------------»- ra< ♦ AKT 1 ■*- Holoenzyme Assembly G5K-3H Dl * CdkA + p27 Protease mal Degradation NUCLEUS Dl CdkA Sherr and McCormick, Cancer Cell, Vol 2, 103-112 (2002) Mitogen Induced Cell Cycle Progression in Cell Culture Dr. B. Duronio, The University of North Carolina at Chapel Hill Is all DNA replicated? s environment favorable? G2 CHECKPOINT Are all chromosomes attached to the spindle? METAPHASE CHECKPOINT Gl CHECKPOINT Is environment favorable? Figure 17-14. Molecular Biology of the Cell, 4th Edition. Cell Cycle Checkpoints {5 unfavorable excess chromosome Z extracellular DNA mitogenic unrepticated DNA unattached to O environment damage stimulation DNA damage spindle o LU o p53 Gl-Cdk GVS-Cdk—|"Ji—I S-Cdk -^Cdc25—^ M-Cdk —* APC i 11 CKI Lu Gi/S"Cyclin synthesis S-cyclin synthesis' DNA rereplication M |üt 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 RÁD9 gene. cdc9 (•) and cdc9-rad9 (O) cells growing at 23°C were shifted to the restrictive temperature and viability was determined by plating for viable colonies at the permissive temperature (23°C), 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 SB* Weinert and Hartwell, Science 246:629 (1989) How do Cell Cycle Checkpoints Work? DNA damage 4 Replication ^ stress SIGNALS SENSORS TRANSDUCERS EFFECTORS ^^ ^ ■1 1£hé Apo ptosis Tran script i c \^r ' S^^^^^^V** —^^^^^^| . ~^r |STOP^^| Cell cycle transitions >n DNA repair Zhou and Elledge Nature 408,< 433 - 439 (2000) DNA damage Replication stress Ochkl Ochk2 Mdm2 H p53 BRCA1 1 Cdc25 I Cdk Nbs1 oAbl Cell cycle Apoptosis arrest Transcription DNA repair Figure 2 Organization of the mammalian DNA damage response pathway. Arrowheads represent positively acting steps while perpendicular ends represents inhibitory steps. Gene names are 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) G en ot ox i c Stress (i.e. IR or UV) I ATM A"m Signal Sensor Cytoplasmic sequestration Chk1/2 Inactive G2 Arrest Ť ® Cdc25Cj (P)(p)g) A © ^ ÜU V Jtj^ 1 Cdc2 ] ^------------- r Cdc2 1 cyclln B cyclln B Active Mitosis Transducer Effector Dr. B. Duronio, The University of North Carolina at Chapel Hill ssDNA DSB ATR ATRI fRIP^ G1 Dr. B. Duronio, The University of North Carolina at Chapel Hill Metaphase in a mammalian cell (b) Zone of interdigitation Kinetachore MT\ / Kinetochare Astral MT Pole {centrosome} Polar MTs (+> Chromosome Aster ->k- Spindle >k- Aster The Metaphase to Anaphase Transition APC ™> APC $ Anaphase inhibitor " a □ 0^(§ Prophase Metaphase Anaphase Telophase oft, oo The Spindle Assembly Checkpoint Checkpoint activated CENP-E * BubR1/Bub3 Bub'1/8ijb3? Mad2 Madi t mú2' —|APC/CCdcřů Checkpoint silenced Checkpoint proteins displaced Production of Mad2' ceases (?) Cohesin cteavage Securin 'degradation (U h -dependent) .11- Anaphase onset ii 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