Intracellular Control of Cell-Cycle

1Molecular Biology of Molecular Biology of CancerCancer

Intracellular Control of Cell-Intracellular Control of Cell-CycleCycle

FirstFirstAt the replication of DNA during S phase

SecondSecondAt the chromosome segregation and cell

division of M phase.


In yeastIn yeastS-phase cyclin-Cdk complexes (S-Cdks)

initiate DNA replication once per cycleDNA replication begins at origins of origins of

replicationreplication, which are scattered at various locations in the chromosome

Origin recognition complex (ORC)Origin recognition complex (ORC) is a multiprotein complex that binds to the yeast replication origin

ORCORC bind to replication origins throughout the cell cycle and serve as landing pads for several additional regulatory proteins


The assembly of the pre-replicative The assembly of the pre-replicative complex (pre-RC)complex (pre-RC)

In late mitosis and early G1, the proteins cdc6 and cdt1 bind to the ORC at origins

This leads to the binding of the Mcm proteinsMcm proteins complexcomplex which is composed of a group of 6 closely related proteins

The resulting large complex is the pre-RC, and the origin is now licensed for replication.


The activation of S-S-CdkCdk in late G1 initiates DNA replication:S-cdk phosphorylates

Cdc6 which is then degraded

S-cdk trigger the assembly preinitiation complex

S-cdk with help of additional kinase collaborate to phosphorylate ORC.

DNA synthesis begins .


Several cyclin-Cdk complexes cooperate Several cyclin-Cdk complexes cooperate to restrain pre-RC assembly and prevent to restrain pre-RC assembly and prevent

DNA rereplication after S phaseDNA rereplication after S phase S-CdkS-Cdk1. It phosphorylates Cdc6Cdc6

It causes the Cdc6Cdc6 protein to dissociate from ORCORC after an origin has fired

thus triggering its ubiquitylation by the SCFSCF enzyme complex and thus its degradation

2. It also phosphorylates excess McmMcm proteins the McmMcm protein complex cannot bind to a

replication origin this triggers their export from the nucleus


several cyclin-Cdk complexes cooperate to several cyclin-Cdk complexes cooperate to restrain pre-RC assembly and prevent DNA restrain pre-RC assembly and prevent DNA

rereplication after S phaserereplication after S phaseThis results in:

the disassembly of the pre-RCpre-RC, which prevents replication from occurring again at the same origin

Preventing the Cdc6Cdc6 and McmMcm proteins from reassembling at any origin

S-CdkS-Cdk activity remains high during G2 and early mitosispreventing rereplication from occurring after the

completion of S phase.


several cyclin-Cdk complexes cooperate to several cyclin-Cdk complexes cooperate to restrain pre-RC assembly and prevent DNA restrain pre-RC assembly and prevent DNA

rereplication after S phaserereplication after S phase M-CdkM-Cdkhelps ensure that rereplication does not

occur during mitosis by: phosphorylating the Cdc6Cdc6 and McmMcm proteins.

G1/S-CdksG1/S-CdksEnsures that excess McmMcm proteins unbound

to origins in late G1 are taken out of action before replication begins by: inducing McmMcm export from the nucleus,


2.2. M-Phase Cyclin-Cdk Complexes M-Phase Cyclin-Cdk Complexes ((M-CdksM-Cdks) Trigger Entry into ) Trigger Entry into MitosisMitosis

After S phase the G2 cell is left with two sister chromatidssister chromatids glued together along their length.

In M phase the duplicated chromosomes and other cell contents are distributed equally to the two daughter cells.

The events of mitosis are triggered by M-CdkM-Cdk, which is activated after S phase is complete.


Accumulation of M-cyclinM-cyclin(cyclin B(cyclin B in vertebrate cells)

After S phaseS phase is complete Increase in M-cyclin gene

transcription during G2 and Mdecreased degradation of

M-cyclin

2.2.Positive feedbackPositive feedback phosphorylates and activates Cdc25Cdc25

2.2.Positive feedback Positive feedback Phosphorylates and inhibits Wee1Wee1.

1.1. Polo kinasePolo kinase, phosphorylates and partial activates Cdc25 Cdc25 leads to

partial activation of a subpopulation of M-Cdk

complexes

The crucial event in late G2


What, then, triggers the activation of What, then, triggers the activation of the M-Cdk stockpile?the M-Cdk stockpile?

The positive feedback loop converts a gradual increase in M-cyclin levels into a switch-likeswitch-like, abrupt rise in M-Cdk activity.

Similar molecular switches operate at various points in the cell cycle to ensure that events such as entry into mitosis occur in an all-or-none all-or-none fashionfashion.

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Entry into Mitosis Is Blocked by Incomplete Entry into Mitosis Is Blocked by Incomplete DNA Replication:DNA Replication:

The DNA Replication CheckpointThe DNA Replication CheckpointEnsures that the initiation of mitosis cannot

occur until the last nucleotide in the genome has been copiedNo broken or incomplete sets of chromosomes

are passed to the daughter cells.Sensor mechanisms, of unknown molecular

nature, detect either:the unreplicated DNAor the corresponding unfinished replication forks

It send a negative signalIt send a negative signal to the cell-cycle control system, blocking the activation of M-CdkM-Cdk.


Accumulation of M-cyclinM-cyclin

(cyclin B(cyclin B in vertebrate cells)

Positive feedbackPositive feedback phosphorylates

and activates Cdc25Cdc25

Positive feedbackPositive feedback Phosphorylates and inhibits Wee1Wee1.

Polo kinasePolo kinase, phosphorylates and partial activates Cdc25 Cdc25 leads to partial activation of a subpopulation of M-Cdk complexes

Protein kinase

• If DNA is unreplicated or the corresponding replication forks isunfinished, a sensor a sensor mechanismsmechanisms, of unknown molecular nature sends a Negative signal

• The final targets of the negative checkpoint signal are the enzymes that control M-CdkM-Cdk activation


Sister Chromatid Separation Is Sister Chromatid Separation Is Triggered by ProteolysisTriggered by Proteolysis

The sister-chromatid cohesion depends on the cohesin complexcohesin complex, that is deposited along the chromosomes as they are duplicated in S phase.

Separation of the sister chromatids occurs at the metaphase-to-anaphase transition.Anaphase begins with a sudden disruption of the

cohesion between sister chromatidsM-CdkM-Cdk activity sets the stage for this eventThe anaphase-promoting complex (APC)anaphase-promoting complex (APC) throws

the switch that initiates sister-chromatid separation.


Cdc20 synthesis increases as the cell approaches mitosis, owing to an increase in the transcription of its gene. complex.

Phosphorylation of the APCAPC helps Cdc20Cdc20 bind to the APC, thereby helping to create an active

Phosphorylation of cohesincohesin just before start of anaphase mediated by Polo kinasePolo kinase provides an additional control on the timing of the metaphase-to-anaphase transition.

Kinases???

It is not clear what kinases phosphorylate

and activate the Cdc20-APC complex. M-Cdk

activity is required for the activity of these

kinases


Unattached Chromosomes Block Sister-Unattached Chromosomes Block Sister-Chromatid SeparationChromatid Separation

The Spindle-Attachment CheckpointThe Spindle-Attachment CheckpointIn most cell types, before sister-chromatid

separation occurs all chromosomes must be properly attached to the spindle.

The checkpoint depends on a sensor mechanism that monitors the state of the kinetochorekinetochore, the specialized region of the chromosome that attaches to microtubules of the spindle.Any kinetochore that is not properly attached to the

spindle sends out a negative signal to the cell-cycle control system, blocking Cdc20-APC activation and sister-chromatid separation.


Thus, sister-chromatid separation cannot Thus, sister-chromatid separation cannot occur until the last kinetochore is attached.occur until the last kinetochore is attached. Several proteins, including Mad2Mad2, are recruited to

unattached kinetochoreskinetochores and are required for the spindle-attachment checkpoint to function.

Even a single unattached kinetochore in the cell results in Mad2 binding and the inhibition of Cdc20-APCCdc20-APC activity and SecurinSecurin destruction


The G1 PhaseThe G1 Phase Is Is a State of Stable a State of Stable CdkCdk InactivityInactivity

In early animal embryos the inactivation of M-M-CdkCdk in late mitosis is due almost entirely to the action of Cdc20-APCCdc20-APC.

Recall, however, that M-Cdk stimulates Cdc20-Cdc20-APCAPC activityThus, the destruction of M-cyclinM-cyclin in late mitosis soon

leads to the inactivation of all APCAPC activity in an embryonic cell.

This is a useful arrangement in rapid embryonic cell cycles, as APC inactivation immediately after mitosis allows the cell to quickly begin accumulating new M-M-cyclincyclin for the next cycle


A. Embryonic cells with no G1 phase


Rapid cyclin accumulation immediately Rapid cyclin accumulation immediately after mitosis is not useful, after mitosis is not useful,

In cell cycles containing a G1 phase, progression into the next S phase is delayed in G1 to allow for:cell growthand for the cycle to be regulated by extracellular

signals.Thus, most cells employ several mechanisms

to ensure that CdkCdk reactivation is prevented after mitosis.


How does the cell escape from this How does the cell escape from this stable G1 state to initiate S phase?stable G1 state to initiate S phase?

Escape usually occurs through the accumulation of G1-cyclinsG1-cyclins.

In animal cells:the accumulation of G1-cyclinsG1-cyclins is

stimulated by the extracellular signals that promote cell proliferation

In budding yeast, G1-CdkG1-Cdk activity triggers the transcription of G1/S-G1/S-cyclincyclin genes, leading to:increased synthesis of G1/S-cyclinsG1/S-cyclins


The Rb Protein Acts as a Brake in The Rb Protein Acts as a Brake in Mammalian G1 CellsMammalian G1 Cells

E2F transcription factor E2F transcription factor binds to specific DNA sequences in the promoters of many genes that encode proteins required for S-phase entry, including G1/S-cyclinsG1/S-cyclins and S-S-cyclinscyclins.

E2FE2F function is controlled primarily by an interaction with the retinoblastoma protein retinoblastoma protein (Rb),(Rb), an inhibitor of cell-cycle progression.


Extracellular Extracellular SignalsSignals

PP PP

Active E2F protein

Inactivated Rb protein

DNA DNA replicationreplication



Cell-Cycle Progression is Blocked by DNA Cell-Cycle Progression is Blocked by DNA Damage and p53:Damage and p53:

DNA Damage CheckpointsDNA Damage Checkpoints Damaged chromosomes must be repaired

before replicating or segregating them. Most cells have at least Most cells have at least two two DNA DNA

Damage CheckpointsDamage Checkpoints1. In late G1, which prevents entry into S phase

by inhibiting the activation of G1/S-Cdk and S-Cdk complexes

2. in late G2, which prevents entry into mitosis: damaged DNA sends a signal to a series of protein protein

kinaseskinases that phosphorylate and inactivate the phosphatase Cdc25 (phosphatase Cdc25 (discussed earlierdiscussed earlier))


The G1The G1 DNA Damage CheckpointsDNA Damage Checkpoints

In mammalian cells, for example, DNA damage leads to the activation of the gene regulatory protein p53p53P53 stimulates the transcription of several

genes.One of these genes encodes a CKICKI protein

called p21p21, which binds to G1/S-CdkG1/S-Cdk and S-S-CdkCdk and inhibits their activities

If DNA is repaired p53 effect is reversed.


DNA damage activates p53 DNA damage activates p53 by an indirect mechanismby an indirect mechanism


Inactive DNA damage checkpoint:Inactive DNA damage checkpoint:

p53p53 Loss of function mutations occur in at least half of all human cancers and allow the cancer cell to accumulate mutations more readily

Ataxia telangiectasia: Ataxia telangiectasia: a rare genetic disease is caused by a defect in one of the protein kinasesprotein kinases that phosphorylates and activates p53p53 in response to x-ray-induced DNA damagepatients are very sensitive to x-rays and

consequently suffer from increased rates of cancer.


If DNA damage is so severe that repair is If DNA damage is so severe that repair is not possible:not possible:

Unicellular organisms such as budding yeast: life with mutations is better than no life at all.The cycle resumes despite any damage.

In multicellular organisms: the health of the organism takes precedence over the life of an individual cell.Animal cells with severe DNA damage commit suicide by undergoing programmed cell death (apoptosis)The decision to die in this way also depends on the activation of p53p53, and it is this function of p53 that is apparently most important in protecting us against cancer.


the general structure of the cell-the general structure of the cell-cycle control system cycle control system


Extracellular Control of Cell Extracellular Control of Cell Division, Cell Growth, and Division, Cell Growth, and

ApoptosisApoptosis


Control of organ and body sizeControl of organ and body size

Number of cellsNumber of cells Size of cellsSize of cells

2.2. Amount of cell divisionAmount of cell division 1.1. Cell growthCell growth

Total Cell MassTotal Cell Mass

Organ size or Body sizeOrgan size or Body size

3.3. Amount of cell deathAmount of cell death


The factors that promote organ or organism The factors that promote organ or organism growth can be operationally divided into growth can be operationally divided into

three major classes:three major classes:1.1.Mitogens:Mitogens:

Stimulate cell division, primarily by: relieving intracellular negative controls that

otherwise block progress through the cell cycle. 2.2.Growth factors:Growth factors:

Stimulate cell growth by: promoting the synthesis of proteins and other

macromolecules and inhibiting their degradation.

3.3.Survival factorsSurvival factors: Promote cell survival by:

suppressing apoptosis.


Mitogens Stimulate Cell Division Mitogens Stimulate Cell Division Cell DevisionCell Devision

Multicellular animalMulticellular animal Unicellular organismUnicellular organism

Availability of nutrients in the environment

Overcome intracellular braking mechanisms that block progress

through the cell cycle

Mitogensfrom other cells, usually its neighbors

More cells are needed


Over 50 proteins are known to act Over 50 proteins are known to act as mitogensas mitogens

Broad specificity factorsBroad specificity factors Most of mitogens, like: PDGF:PDGF: platelet derived growth factor can stimulate many

types of cells to divide including: Fibroblasts smooth muscle cells neuroglial cells

EGFEGF epidermal growth factor epidermal growth factor acts on many cell types including: epidermal cells Epithelial cells nonepithelial cells

Narrow specificity factorsNarrow specificity factors such as: Erythropoietin,Erythropoietin, which induces the proliferation of red blood

cell precursors only.


Cells Can Delay Division by Entering Cells Can Delay Division by Entering a Specialized Nondividing Statea Specialized Nondividing State

In the absence of a mitogenic signal to proliferate:CdkCdk inhibition in G1 is maintained, and the

cell cycle arrests.In some cases, cells partly disassemble

their cell-cycle control system and exit from the cycle to a specialized, nondividing state called G 0G 0 .Most cells in our body are in G0


The molecular basis and reversibility of this The molecular basis and reversibility of this state vary in different cell typesstate vary in different cell types

Neurons and skeletal muscle cells, are in a terminally differentiated G0 stateterminally differentiated G0 state the expression of the genes encoding various CdksCdks and cyclinscyclins

are permanently turned off cell division never occurs

Other cell types withdraw from the cell cycle only transientlytransiently Most liver cellsMost liver cells, for example, are in G0G0, but they can be

stimulated to divide if the liver is damaged. They retain the ability to reassemble the cell-cycle control

system quickly and reenter the cycle Still other types of cells,, withdraw from and re-enter

the cell cycle repeatedlyrepeatedly throughout their lifetime. including some lymphocyteslymphocytes


Mitogens Stimulate G1-Mitogens Stimulate G1-Cdk and G1/S-Cdk Cdk and G1/S-Cdk

ActivitiesActivities An early step is often the

activation of the small GTPase GTPase RasRas

This leads to the activation of a MAP kinase cascadeMAP kinase cascade.

This leads to increased levels of the gene regulatory protein MycMyc


MycMyc increases the transcription of genes that encode G1 cyclins (D cyclinsD cyclins) This increases G1-CdkG1-Cdk

(cyclin D-Cdk4cyclin D-Cdk4) activity

MycMyc increases the transcription of a gene for a component of the SCFSCF This promotes the

degradation of the CKICKI protein p27p27

leading to increased G1/S-CdkG1/S-Cdk (cyclin E-cyclin E-Cdk2Cdk2) activity.

MycMyc may also stimulate the transcription of the gene encoding E2FE2F


Growth Factors Stimulate Cell Growth Factors Stimulate Cell GrowthGrowth

One of the most important intracellular signaling pathways activated by growth factor receptors involves the enzyme PI 3-PI 3-kinasekinase

It adds a phosphate from ATP to the 3-position of inositol phospholipids in the plasma membrane


The activation of PI 3-kinasePI 3-kinase leads to the activation of several protein kinases, including S6 kinaseS6 kinase.

The S6 kinaseS6 kinase phosphorylates ribosomal protein S6ribosomal protein S6, increasing the ability of ribosomes to translate a subset of mRNAs, most of which encode ribosomal components Protein synthesis therefore

increases.


Growth factors also stimulate cell Growth factors also stimulate cell metabolismmetabolism

Growth factor stimulation also leads to increased production of the gene regulatory protein MycMycMycMyc increases the transcription of genes

for proteins involved in cell metabolism and macromolecular synthesis.


MitogenMitogenGrowth FactorGrowth Factor

MAP-kinase MAP-kinase pathwaypathway

PI3-kinase PI3-kinase pathwaypathway

cell-cycle progression

cell growth MycMyc

RasRas

Cells maintain their appropriate size as they proliferate

Extracellular signal proteins, can regulate Extracellular signal proteins, can regulate growth and division independentlygrowth and division independently

or act as both growth factors and mitogensor act as both growth factors and mitogens

For Example:For Example:


Independent control may be particularly Independent control may be particularly importantimportant

During embryonic development dramatic changes in the size of certain cell types can occur.

In adult animals, growth factors can stimulate cell growth without affecting cell divisionThe size of a sympathetic neuron, depends

on the amount of nerve growth factor nerve growth factor (NGF)(NGF) secreted by the target cells it innervates.


Extracellular Survival Factors Extracellular Survival Factors Suppress ApoptosisSuppress Apoptosis

If deprived of survival factorssurvival factors, animal cells activate apoptosis. Example: Nerve cells are produced in excess in the developing

nervous system They compete for limited amounts of survival factors that

are secreted by the target cells they contact.


Bcl-2 familyBcl-2 familyApoptosis-suppressing Apoptosis-suppressing

membersmembers

ApoptosisApoptosis

Cell Survival

Bcl-2 familyBcl-2 familyApoptosis-promoting Apoptosis-promoting

membersmembers

Survival factors Survival factors signaling pathwayssignaling pathways


The The Bcl-2 familyBcl-2 family of intracellular proteins of intracellular proteins helps regulate the activation of helps regulate the activation of

procaspases.procaspases.Bcl-2Bcl-2 Bcl-XLBcl-XL

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Intracellular Control of Cell-Cycle