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