Cell cycle and its regulation - uniba.sk · Cell cycle and its regulation introduction. Cell cycle...
Transcript of Cell cycle and its regulation - uniba.sk · Cell cycle and its regulation introduction. Cell cycle...
Cell cycle and its regulation
introduction
Cell cycle(CC) is composed of steps proceeding one after anotherand causing :
- cell growth
- division of the cell and origin of two daughter cells
- its differentiation
Cell cycle is composed from interphaseand M-phase
Principal charakteristics of CCPrincipal charakteristics of CC
-- It is ultimate process It is ultimate process in life of a cellin life of a cell
-- Their principles are identical in all Their principles are identical in all eukaryotic eukaryotic cells cells -- fromfrom yeasts yeasts toto mammaliamammalia
-- Its basic mechanisms are Its basic mechanisms are in evoluion in evoluion markedly markedly conserved conserved (developmentaly old)(developmentaly old)
Cel cycle regulationCel cycle regulationis permited by molecules, having origin:is permited by molecules, having origin:
1.1. exogenous: exogenous: by regulatory moleculesby regulatory molecules
2. 2. endogenous:endogenous:with proteins coded by with proteins coded by regulatory genes in cell:regulatory genes in cell:
a) protoa) proto--onkogenes onkogenes ((PrOPrO))
b) tumorb) tumor --supressor genes supressor genes ((TSGTSG))
Types of regulation by molecules regulation by molecules :
- endocrine = signal molecule is hormone transported inbody by blood and impacting only on cellshaving relevant receptor on their surface(for CC regulation – e.g. growth hormone)
- paracrine = signal molecule is substance released fromparticular cell and acting on cells in surroundings (to 1 mm distance) (for CC regulation – e.g. growth factors)
- autocrine = expression of certain genes is appointed in timeand is blocked = inside the cell have to besynthesized particular signal molecule, permitting expression of these genes(for CC regulation – e.g. cyclins D and E)
(to revise)
PrOPrOTSGTSG
- inhibit apoptosis
Protoonkogenes are genes and by them codedproteins, that:
- stimululate progress in CC
- inhibit differentiation
Tumor-supressor genes are genes and by them coded proteins, that:
- stimulate apoptosis
- inhibite progress in CC
- stimulate differentiation
Regulation of cell cycle = balance betweenTSG and PrO proteins
products products of tumorof tumor--suppresor genessuppresor genes
products of protooncogenesproducts of protooncogenes
pproductsroducts of of tumortumor --suppresor suppresor
genesgenes
products of products of protooncogenesprotooncogenes
TSG „slow down “progress of CC)
PrO „lead to“progress in CC
GG00 phase is at outset of G1 phasephase is at outset of G1 phase
= = the cell grows, differentiates and the cell grows, differentiates and
works for tissue (organism)works for tissue (organism)
= = then itthen itreturnsreturnsback back totoG1 phase.G1 phase.
First check First check pointpoint
cycline cycline BB ––Cdk1 Cdk1
cycline cycline AA ––Cdk1Cdk1
cycline cycline AA ––Cdk2Cdk2
cycline cycline EE ––Cdk2Cdk2
cycline cycline DD ––Cdk4 Cdk4
cycline cycline DD ––Cdk6Cdk6
Scheme of CC progress and regulationScheme of CC progress and regulation
Regulatory enzymesRegulatory enzymeshere are here are cyclin depencyclin depen--dent kinases dent kinases (cdk(cdk//cdc).cdc).Their activators areTheir activators arecyclinescyclines..
For each CCFor each CCphase are diversephase are diversecdk cdk a other a other cyclinscyclins, in , in variable variable combinationscombinations..
In time In time correctcorrectattendanceattendanceofofcyclines cyclines isisensured byensured bycontrol control ofofexpression expression of of them them codingcodinggenes or genes or theirtheirdisposaldisposal..
CyclinCyclin--dependent kinases (Cdkdependent kinases (Cdk’’ s) are presents) are presentin nucleus during whole cell cycle! in nucleus during whole cell cycle!
CdkCdk’’ s are inactive, till bind with cycline!s are inactive, till bind with cycline!
Note!Note!CdkCdk’’ s s can have manycan have manydifferent names different names in regard in regard
to organism (and researcherto organism (and researchers will) !s will) !
Activity of complex cyclinActivity of complex cyclin--cdk iscdk is furtherfurtherregulatedregulated by phophorylation, dephosphorylation by phophorylation, dephosphorylation and binding (releasing) of inhibitorand binding (releasing) of inhibitor!!
CdkCdk’’ s are enzymes adding phosphate to others are enzymes adding phosphate to other((““ targettarget““ ) proteins to regulate their activity!) proteins to regulate their activity!
Regulation of cyclin-cdk complex activation and inhibition
1.1. ccomplexomplexcyclin-cdkoriginorigin
2. InhibInhib itingitingphphososphphoryloryl ationationof threonine 14 and tyrosione 15 3. AAcctivtiv atingating
phphososphphoryloryl ationationof threonine 160
4. InhibiInhibi titi onon ofcdk-cycline complexby binding of its inhibinhib itit oror (CKI)
5. DegradationDegradationof cyclinand/or cki byubiquitinationubiquitination
CDK
Cyclin
CKI
T-Loop
CKI’s can be specific for particular CDK complexes because their binding can depend upon a specific cyclin
(STOP)
(GO)
** p27 slows downp27 slows downactivity of complex activity of complex cyclin E+cdk2cyclin E+cdk2 = = to give enough to give enough timetime for mutations reparation in end of G1 phasefor mutations reparation in end of G1 phase
** p21 blocksp21 blocksactivity of complex activity of complex cyclin D+cdk6cyclin D+cdk6 = when are too many= when are too manymutations mutations = repair or apoptosis= repair or apoptosis(in end of G1 phase)(in end of G1 phase)
Two classes of E3Ubiquitin Ligasesare important for cell cycle control:
APC/C and SCF:Regulate Major Cell-Cycle Transitions
Importantfor mitoticcheckpoint
Important for a cell’sentry intoS-phase
Ubiquitinated Proteins are Degraded by the ProteosomeUbiquitinated Proteins are Degraded by the Proteosome
Binds to Ubiquitinated Proteins,Cleaves and Recycles Ubiquitin
(Isopeptidase)
Destroys Target Proteins(Peptidase)
DNA damage is detected by one of the DNA checkpointssystems.
- Different checkpoint proteins respond to types of DNA damage.
- Cells monitor DNA integrity at all stages of the cell cycle:
- All DNA checkpoint systems ultimately lead to an arrest in cell cycle progression, initiation of repair , and if the problem is bad enough, apoptosis.
“DNA structure” checkin g
Generalized checkpoint response pathway(signal transduction)
signal
sensor
adaptor
effector
DNA repairCell cycle arrestapoptosisapoptosis
DNA damage
ATM, ATR kinases
Claspin/BRCA1
Chk1, Chk2 kinases
“9-1-1” complexM-R-N complex
Answer Answer type dependsdependson timetime--durationduration and
intensity intensity of alarm signals from reporter molecules about DNA
damage!
Nyberg, et al., 2002.
G1 Checkpoint= licence to start S phase= licence to start S phase
= licence to synthesize all proteins= licence to synthesize all proteinsrequired for replication of DNA required for replication of DNA
= time to repair mutations in DNA= time to repair mutations in DNAor destroy the cell by apoptosisor destroy the cell by apoptosis
Exogenous regulation of CC by gene expressioncontrol:
mitogen (e.g. growth factor)receptor
ras (G-protein)
MAP -kinase
activaton of gene for regulatory protein
cytoplasm
nucleusmyc (expression of protooncogene)
myc= myc= specificspecific transcription factor that control expression of othertranscription factor that control expression of other genesgenes
Continue ....
cyclin Dcyclin Dsubunit of
SCFSCF
= expression of genes for:= expression of genes for:
cyclin Dcyclin D
activation of complexactivation of complexcyklcyklíín D n D –– cdk4cdk4
phosphorylationphosphorylationof of protein rbprotein rb
activation of complexactivation of complex
cyclin E cyclin E –– cdk2cdk2
degradation ofdegradation of
protein p27protein p27
increase of activity increase of activity of of family E2Ffamily E2F
transition to Stransition to S--phasephase
increase of transcription increase of transcription of of family E2Ffamily E2F
transcription factors family
E2FE2F
Passage through G1 check point depends on activation of E2F specific transcription factors family
by intense phosphorylation of rb1 repressor:
Mammalian cyclinMammalian cyclin--kinase inhibitors also contribute kinase inhibitors also contribute to cell cycle controlto cell cycle control!!
Regulation cascade of expression of genes for cyclins required for transition G1/S
Check-kinase chk1phosphorylates p53
I takes certain timeto synthetize p21 andreturn it back to nucleus= it is delayed answer= arrest of CC progress
If continue DNA damagesignals flow = another p53 makes expression of p73(78)
genes and proteins start mitochondrial
pathway of apoptosis
G1 Checkpoint
- Delays cell cycle prior to S phase.
- Allows time for DNA repair & prevents replication of it with mistakes fromdamage = mutations!
G1 Checkpoint
Two different types of „answer“ to DNA damage signals:
First : response occurs within minutes, but only lasts a few hours.p53-independentATM activates CHK1 or 2→ inhibits cycl. D + E with cdkcoplexes → prevents S-entry
Second: response takes several hours, but may be irreversible (?apoptosis?)ATM/ATR activate CHK 1→ activates p53→ it manages synthesis of p21 → p21 p21 inhibits complex cycline D/cdk
Nyberg, et al., 2002
rapid, rapid, transienttransientresponseresponse
slow, slow, sustainedsustainedresponseresponse
G1 � S
Activate the inhibitor Inhibit the activator
S phase DNA checkpoint
1. Functional telomere 1. Functional telomere = to start= to start
Two main controlTwo main control of Sof S--phasephasebeginningbeginning::
2. 2. „„ LicencingLicencing““ factors factors „„ arroundarround ““ ORCORC= to start in each ORI and continue= to start in each ORI and continue
-- free motif TTAGGG is needed to bind primerfree motif TTAGGG is needed to bind primeron beginning of leadin strandon beginning of leadin strand= if not → apoptosis
3. Self-proof-reading of DNA polymerase ∆
Replication at each ORI is initiated only once during the cell cycle= in S phase !
after replication is eachafter replication is eachORI ORI „„ lockedlocked““ in ORC andin ORC and
„„ licncing factorslicncing factors““
Initiation of DNA replicationInitiation of DNA replicationS
TE
P 1
ST
EP
2
Inhibition of STEP 1
Cdc6
ORC ORC ORC
ORC
ORC
ORC
MCM Proteins
Cdc28
Cdc7
Cdc28
Replication Factors
Origin Assembly / Activation
preRC
Origin Firing Origin Inactivation
Elongating Repl. Fork
ActvieCohesin
Cdt1Cdt1Cdt1Cdt1
Cdt1Cdt1
prepre--replication complexreplication complex
After replication After replication is is each ORI each ORI (origin of replication)(origin of replication) „„ lockedlocked““ in ORC (ORI in ORC (ORI recognizing complex) and recognizing complex) and „„ licncing factorslicncing factors““ : MCM proteins, cdc6 and : MCM proteins, cdc6 and cdt1 = cdt1 = prepre--replication complex replication complex appears.appears.
Since it is not specifically disassembled Since it is not specifically disassembled = = replication can not start replication can not start ! !
Initiation and elongation of replicationelongation of replicationS
TE
P 1
ST
EP
2
Inhibition of STEP 1
Cdc6
ORC ORC ORC
ORC
ORC
ORC
MCM Proteins
Cdc28
Cdc7
Cdc28
Replication Factors
Origin Assembly / Activation
preRC
Origin Firing Origin Inactivation
Elongating Repl. Fork
ActvieCohesin
Cdt1Cdt1Cdt1Cdt1
Cdt1Cdt1
BEFORE replication can start BEFORE replication can start must be must be each preeach pre--replication complex replication complex activatedactivatedby phosphorylation.by phosphorylation.Kinases cdc28 and cdc7 cause Kinases cdc28 and cdc7 cause releasing of licencing factorsreleasing of licencing factorscdc6 and cdt1 cdc6 and cdt1 and MCM protein octamer has to be loosen (relaxed).and MCM protein octamer has to be loosen (relaxed).Than Than „„ replication factorsreplication factors““ (e.g. helicase, primase ...) can open dsDNA in (e.g. helicase, primase ...) can open dsDNA in ORI, replicate it and make replication ORI, replicate it and make replication bubblebubble..Immediately after each ORI replication Immediately after each ORI replication –– it is it is „„ lockedlocked““ by ORC and two by ORC and two semiconservative way replicated new DNA molecules are separated semiconservative way replicated new DNA molecules are separated by by cohesin cohesin bridges. bridges.
G2/M phase DNA checkpoint
GG22 phase phase M phase M phase
Cell cycle regultion Cell cycle regultion -- G2/MG2/M transitiontransition
ubiquitinationubiquitination
inactiveinactive activeactive
rremoveemovessinhibitoryinhibitory
phosphatesphosphates
Principle is activation of Principle is activation of cycline B/cdc2 after cycline B/cdc2 after successful passing successful passing through G2 checkpointthrough G2 checkpoint
G2 G2 DNA ccheckpointheckpoint
- primary target of pathway is CDC25, and its regulation of CDK1 to prevent entry into mitosis.
- Both ATM and ATR are activated and then activate CHK1 & CHK2.
- CHK1 & CHK2 inhibit CDC25 directly.
- CHK1 activates Wee1, which antagonizes CDC25 activity.
- CHK1 also activates 14-3-3- proteins, which inhibit CDC25 & CDC2,and promotes Wee1 activity.
- Polo-like kinases (PKL1 & 3) inhibit CDC25, and may be activatedby ATM/ATR.
Nyberg, et al, 2002G2 � M
Polo-like kinases
G2 G2 ccheckpointheckpoint
Mitotic
checkpoint
Adapted from Shah and Cleveland (2000) Cell 103:997
Mad2 can be activated to Mad2p*; in this state it works with Cdc20p to turn offturn off APC/C activity
Mad2p is inactive until it has visited a free kinetochore.
There it becomes activated by *P and is released toinhibit APC/C.
Its activity is short-lived, so it always has to be reactivated by return to the Kinetochore.
Adapted from Shah and Cleveland (2000) Cell 103:997
When a kinetochore becomes When a kinetochore becomes fully fully occupied by MTs,occupied by MTs,
securin
it loses its ability to activate Mad2.APC/C then becomes active and it degrades:1) ssecurinecurin = separasecan cut all cohesines =sister chromatides areseparated,2) cycline Bcycline B = is not more needed,3) chromokinesinechromokinesine = an inhibitor of motoric proteins = dyneins andkinesin can help intransfer of daughter chromosomes to poles =aanaphasenaphasecan can startstart!
*** if fails ubiquitination *** if fails ubiquitination ofof ::a) a) securinsecurin= sister chromatids stay connected = = sister chromatids stay connected = nondisjunctionnondisjunctionappearsappearsa) a) chromokinesinechromokinesine= daughter chromosomes are not = daughter chromosomes are not „„ on timeon time““ = = anaphase laganaphase lagappears appears