cell cycle control (mammalian) - 國立臺灣大學ntur.lib.ntu.edu.tw/bitstream/246246/... · apr....
TRANSCRIPT
Apr. 21, 2005
Cell cycle control (mammalian)Basic mechanisms & protein componentsCheckpoints
Chap. 21, by Lodish et al.,5th ed. 2004Chap. 17, by Alberts et al.,4th ed. 2002
鍾明怡
Advanced readings (not all required)
By mechanismSCF
Nat Rev MCB 2004;5:739-751.Nat Rev MCB 2005;6:9-20.
APCOncogene 2005;24:314-325.Oncogene 2005; Ralph Wasch
& Dirk Engelbert (publishedonline ahead of print).
Polo-like kinasesNat Rev MCB 2004; 5:429-
440.Oncogene 2005; 24:277-286.
Role of translationNat Rev MCB 2005; 6:69-78.
By checkpointDNA damage checkpoint
Nat Rev MCB 2004; 5:792-804.Nat Rev MCB 2005;6:44-55.
Spindle checkpointChromosome Research
2004;12:599-616.
OthersConnecting proliferation and apoptosis
in development and disease. Nat RevMCB 2004; 5:805-815.
Cancer genes and the pathways theycontrol. Nat Med 2004; 10:789-799.
Haploinsufficiency for tumor suppressorgenes: when you don’t need to goall the way. Biochimica et BiophysicaActa 2004;1654:105-122.
Mammalian cell cycle control
The basic organization of cellcycle and its control systemare essentially the same in alleukaryotic cells.
Why discuss mammalsseparately?Differences between yeastand mammals.
Cell division in multicellularorganisms
In addition to availability of nutrients, itmust also receive stimulatoryextracellular signals for a multicellularorganism to divide.Mitogens vs. growth factorsProliferation vs. growthe.g. PDGF
Cell cycle control and human diseases.
Cell growth andcell cycle control
Only cycle control
both
N/C ratio↑
N/C ratio↓↓
A simplified modelof one way thatmitogensstimulate celldivision.
•••
Study of cell cycle inmammalian systems
Cell-culture based. Normal tissues, e.g. fibroblast. Complication:
replicative cell senescence after 25-40 divisions. Tumor tissues
Reveal characteristics of normal cells. Diploid. Anchorage dependence. Contact inhibition. Replicative cell senescence.
Cell cycle controlwhy & how?
Basic programs: timed events in thecorrect order; triggered only once ineach cycle; irreversible; must completeprior to initiation of the next event.Sensor.Adaptability: adjust to cell type,environment, …etc.Robustness: backup mechanisms.
Cell cycle checkpoints
The two most important things in a cell cycle:1.2.
Mechanisms of cell cycle control (1)
Generally operate through negativeintracellular signals. Default setting: “STOP”. Gradual decrease in the
negative signal to zero results in “GO”. Inactivation of checkpoints results in progression of
cell cycle without surveillance.
Based on cyclically activated protein kinases,cdks. Cyclin-dependent kinase (cdk). Regulated by a bunch of enzymes and proteins, e.g.
cyclin.
Cyclins and cdks in vertebrates
Cyclins activate cdks; alsodirect their targets.
Another level of regulation:accessibility of the substrate,or target site on the substrate.
B-type cyclinsTarget of APC
Mechanisms of cell cycle control (2)
Transcriptional regulation.Translational regulation.Post-translational regulation. Activating and inhibitory phosphorylation. Inhibitory proteins.Protein degradation. SCF mediated. APC mediated.Subcellular localization.
Transcriptional regulation
Level of cyclin isregulated at the level oftranscription,translation anddegradation. In embryonic cell cycles, M-cyclin
is synthesized at a constant rate;M-cyclin accumulates due todecreased degradation.
In most cells, however, M-cyclinincreases during G2 and M byincreasing transcription.
Will be discussed later.
G0 G1
Translational regulation←Basal cellhyperplasia inprostate
Normal basal cell
Webpathology.comhttp://www.webpathology.com/case.cfm?case=18
JBC 1998;273:29864-29872.
eIF4
cyclins and cdks
(Cdk7-cyclin H-Mat1)
amountvs.
activity
Cannot activate oneself via autophosphorylation
Cdk7/cyclin HMat1
Cdk7(Ser170 Thr176)/cyclin HCdk7/cyclin H/Mat1
“Active CAK”Cdk2/cyclin A Cdk2 (Thr160) /cyclin A
Cdk2 (Tyr15 Thr160) /cyclin A
Cdc25AWee1
Activating and inhibitoryphosphorylations of M-Cdk
Cyclin A/Cdk2 Cyclin A/Cdk2
Cyclin B/Cdk1 Cyclin B/Cdk1
Cdc25A
Cdc25B
Cdk inhibitory proteins (CKIs)
Two classes of Cdk inhibitorsCdk Inhibitory proteins (CIP/KIP)Inhibit all Cdk1,2,4,6 complexes.p21, p27, p57.
Inhibitors of kinase 4 (INK4)Inhibit Cdk4&6.p16, p15.
Proteolysis in cell cycle control (1)–SCF mediated
Named after its three subunits.Regulation of G1/S-cyclins and CKI levels.SCF activity is constant throughout cell cycle (?).Controlled by phosphorylation of the targetprotein.
p27 p27Cyclin ECdk2
SCF ubiquitin ligases
Skp1-Cul1-F box (Skp2) = SCFSkp2
E2
Substrateinteraction
E2/E3interaction
Nat Rev Mol Cell Biol 2004;5:739-751.
Proteolysis andCDK activity
3
Nat Rev Mol Cell Biol 2004;5:739-751.
Proteolysis in cell cycle control (2)–APC mediated
Anaphase-promoting complex.Degrades M-cyclins and otherproteins at mitosis.
Oncogene (2005)24, 314–325
APC/C (cyclosome)APC/C is a multisubunit E3 ubiquitin ligase with atleast two alternative forms, one activated byCdc20 (APC/CCDC20) and the other by Cdh1(APC/CCDH1). Cdh1=Hct1.The homolog of the largest subunit of APC, Apc1,in fission yeast has also been described as aconstituent of the 20S cyclosome.Required for Entry into anaphase (APC/CCDC20). Exit from mitosis (APC/CCDH1). Maintenance of low Cdk activity in early G1 (APC/CCDH1).
Subunits of APC
Oncogene (2005) 24, 314–325
Role ofAPC incell cycle
Oncogene (2005)24, 314–325
active inactive inactive inactive active active
4
Checkpoints(by cell-cycle phases; by specific aims)
Molecular signaling cascades that promote cell-cycle delay or arrest in response tounfavorable conditions, e.g. DNA damage,thereby providing more time to overcome theunfavorable condition or to pursue analternative.
-- Adapted from Nat Rev Mol Cell Biol 2004;5:792-804.
Lodish, et al. 5th ed.2
Passage through “restriction point”:G1 progression & initiation of S phase
Growth factor → cyclin D (↑txc) =>active G1-Cdk
(cyclin D:cdk4/6)
(cyclin E:cdk2)
Restriction point
Intracellular control of cell cycleRe-replication block
Cells at S phasecontain signals forDNA replication andinitiate DNA synthesisin G1 cell after fusion.
S + G2 => no DNAreplication in G2 =>DNA can be replicatedonly once in a cycle.
G2 + G1 => cannotinduce G1 cells into Sphase.
Control of entry into S phase (1)
Origin of replication.ORC. Associated with Ori throughout
the cell cycle.
Regulatory proteins Cdc6.
Transient in early G1. Mcm (Minichromosome
maintenance ) proteins, … etc. Form “pre-replicative complex”.
Ready for DNA replication.
Control of entry into S phase (2)Activation of cyclin A/Cdk2in late G1 triggers S phase. Phosphorylation of ORC =>
initiate DNA replication. phosphorylation of cdc6 =>
degradation by SCF. Phosphorylation of Mcm =>
export from the nucleus. “geminin”occupies Ori,
cannot re-initiate untilcycle is completed.
cannot reassemble pre-RC.
How to initiate DNA synthesisin the next cycle?
DNA damagecheckpoint
Cdc25ASer126
(inactive)(degradation)
G1, G2 arrest
G1/Sblock
DNA repairApoptosis
ActivatedATM Chk2
(active)
ATM= ataxiatelangiectasiamutated.
ATR= ataxiatelangiectasiaand RADrelated
CHK1/2=checkpointkinase 1/2
Nat Rev Mol Cell Biol2005;6:44-55.
DNA damagecheckpoint
Replicationcheckpoint
DSB, or
Cdc25C(inactive)
G2 arrest
Role of p53
Overexpression of a mutated form of thep53 tumor suppressor gene in ahepatic metastasis in colorectalcancer. The brown stain(immunohistochemistry, PAP)shows that the mutated p53localizes to the nuclei of the cancercells where it fails to transduce theapoptotic signal. This renderscancer cells refractory to classicalradio- or chemotherapy.
http://www.rrk-berlin.de/igtlab/eu_network/application.html
Both Rb and p53 are “tumorsuppressor genes”and show“dominant”inheritance in affectedfamilies. What are the mechanismsfor these phenomena?
How about p27Kip1?
DNA replication checkpoint
Late S/G2.Sensor mechanisms are not fully understood. Unreplicated DNA (?). Unfinished replication fork (?). Final target: the pathway for M-Cdk activation.
Inhibits DNA synthesis
high doses
DNA replication checkpoint
RPA = replicationprotein A
Cdc25C (inactive)
G2 arrestp53p21CIPG2 arrest Repair
Apoptosis
Nat Rev Mol Cell Biol2005;6:44-55.
Summary ofS phasecheckpoints
Nat Rev MCB2004;5:792.
Nat Rev MCB2004;5:792.
Control of entry into M-phase
MitosisProphasePro-metaphaseMetaphaseAnaphaseTelophase
Cytokinesis
S
M
~30min/cyclefairly synchronous
Control of M-Cyclin quantity
G1 allows Cell growth Regulation by extracellular signals.
M-cyclin transcript
(transcriptand protein)
Control of M-Cdk activity
Polokinase
Cyclin Bconcentration
The roles of M-CdkChromosome condensation (condensin complex;phosphorylated by M-cyclin => change DNAcoiling => initiating condensation.; MCB).Induces the assembly of mitotic spindle.Ensures attachment of chromosomes to mitoticspindle.Nuclear envelop breakdown.Actin rearrangement.Re-organization of Golgi and ER.
Details of these events are not all fullyunderstood.
Cohesion of sister chromatidsSister chromatids are “joined”together by “cohesins”,deposited along the each sister chromatid as DNA isreplicated.Cohesion of sister chromatids are broken at anaphase.
Chrosome condensationM-Cdk phosphorylates condensin subunits, triggeringthe assembly of condensin complex and theprogressive condensation of the chromosomes.
Blue: DNARed: anti-condensin
Prometaphase Metaphase
Nuclear envelope breakdownin mitosis
Assembly of mitotic spindle
M-Cdk phosphorylates MAP andpromotes stabilization of microtubule.
Sister chromatid separation
APC-mediated degradation of M-cyclin=> inactivate M-Cdk => initiation ofanaphase.
txc
-
Unattached kinetochore
Spindlecheckpoint
Spindle checkpoint
Sensor monitors the status of kinetochore.Mechanism not yet clear. Mad2 at unattached kinetochore sends out (-)
signal; (-) APC activation. Defective spindle checkpoint => earlier anaphase
initiation. Spindle checkpoint is not required in frog embryo
and yeast; some other mechanism(s) is controllingthe timing of anaphase.
Mad2- tubulinDNA
(A) Just after nuclearenvelope breakdown.(B) Early prometaphase.(C) Mid-prometaphase.(D) Late prometaphase.(E) Metaphase.(F) Anaphase.
Mad2 and spindle checkpoint
Chromosome Research 2004;12:599-616.
Chromosome Research 2004;12:599-616.
Non-disjunction
Cytokinesis
Contractile ring: composed of actin andmyosin filaments, formed around theequator of the cell.Control mechanisms to assure thatmitosis occurs prior to cytokinesis.
Exit from mitosis
Degradation of M-cyclin => inactivationof M-Cdk => decrease of kinases activation of phosphatases.EventsDisassembly of mitotic spindle.Decondensation of chromosomes. Formation of nuclear envelope.
S
M
~30min/cyclefairly synchronous
Control of M-cyclin quantity
G1 allows Cell growth Regulation by extracellular signals.
M-cyclin transcript
(transcriptand protein)
Suppressed Cdk activities in early G1CKI: Sic1 in yeast; p21/p27/p57 in mammals;transcription.G1 cyclins are not inactivated by APC/CCDH1 and CKI.
S-cyclinTxc
w/ cdc20
InactiveTxc factor
activeTxc factor
CKI
Cdh1 Cdh1 Cdh1
CKI CKI CKI
CKI
CKI Cdh1
Cdh1
(1)
(2)
1
Try to give an organized generaloverview of mammalian cell cyclecontrol with updated information.Some of the details are not yet fullyexplored.Please do not be misled by the cartoons.