Cell cycle regulation

Dr. Riddhi Datta

The Eukaryotic Cell Cycle

• A typical eukaryotic cell cycle is illustrated by human cells in culture, which divide approximately every 24 hours.

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• The cell cycle is divided into two basic parts:
• mitosis (or meiosis)
• interphase.

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• Mitosis and cytokinesis last only about an hour, so approximately 95% of the cell cycle is spent in interphase-the period between mitoses.

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• However, interphase is the time during which both cell growth and DNA replication occur in an orderly manner in preparation for cell division.

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Dr. Riddhi Datta

• The timing of DNA synthesis thus divides the cycle of eukaryotic cells into four discrete phases:
• The M phase of the cycle corresponds to mitosis, which is usually followed by cytokinesis.
• This phase is followed by the G1 phase (gap 1), which corresponds to the interval (gap) between mitosis and initiation of DNA replication. During G1 the cell is metabolically active.
• G1 is followed by S phase (synthesis) dur­ing which DNA replication takes place.
• The comple­tion of DNA synthesis is followed by the G2 phase (gap 2) during which cell growth continues and pro­teins are synthesized in preparation for mitosis.

Dr. Riddhi Datta

The Eukaryotic Cell Cycle

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• Some cells in adult animals cease division altogether (e.g., nerve cells) and many other cells divide only occasionally (e.g. skin fibroblasts).

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• These cells exit G1 to enter a quiescent stage of the cycle called G0 phase, where they remain metabolically active but no longer proliferate unless called on to do so by appropriate intracellular signals.

Dr. Riddhi Datta

The Eukaryotic Cell Cycle

G0

Regulation of the Cell Cycle by extracellular signals

• The progression of cells through the division cycle is regulated by extracellular signals from the environment, as well as by internal signals that mon­itor and coordinate the various processes that take place during different cell cycle phases.

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• This is accomplished by some control points that regulate progression through the cell cycle:
• START
• Restriction point
• Control of G2 to M transition

Dr. Riddhi Datta

START

• A major cell cycle regulatory point in many types of cells occurs late in G1 and controls progression from G1 to S.

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• This regulatory point was first defined by studies of budding yeast (Saccharomyces cerevisiae), where it is known as START.

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• Once cells have passed START, they are com­mitted to entering S phase and undergoing one cell division cycle.

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• The importance of this regulation is particu­larly evident in budding yeasts in which cell division produces progeny cells of very different sizes: a large mother cell and a small daughter cell.

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Dr. Riddhi Datta

START

• Some of the activities monitored at this check point are:

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• Optimum cell size: In order for yeast cells to maintain a constant size, the small daughter cell, after one division, must grow more than the large mother cell does before they divide again. This regulation is accomplished by a control mecha­nism that requires each cell to reach a minimum size before it can pass START.

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Dr. Riddhi Datta

• Nutrient status: If yeasts are faced with a shortage of nutrients, they arrest their cell cycle at START and enter a rest­ing state rather than proceeding to S phase. Thus START represents a deci­sion point at which the cell determines whether sufficient nutrients are available to support progression through the rest of the division cycle.

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• Polypeptide factors that signal yeast mating also arrest the cell cycle at START, allowing haploid yeast cells to fuse with one another instead of pro­gressing to S phase.

Restriction point

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• Such arrested cells then enter a quiescent stage, called G0, in which they can remain for long periods of time without proliferating. G0 cells are metabolically active, although they cease growth and have reduced rates of protein synthesis.

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• For example, skin fibroblasts are arrested in G0 until they are stimu­lated to divide as required to repair damage resulting from a wound. The proliferation of these cells is triggered by platelet-derived growth factor, which is released from blood platelets during clotting and signals the pro­liferation of fibroblasts in the vicinity of the injured tissue.

Dr. Riddhi Datta

• In animals, a decision point in late G1, called restriction point, functions analogously to START in yeasts.

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• In contrast to yeasts, however, the passage of animal cells through the cell cycle is regulated primarily by the extracellular growth fac­tors that signal cell proliferation, rather than by the availability of nutrients.

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• In the presence of the appropriate growth factors, cells pass the restriction point and enter S phase. Once it has passed through the restriction point, the cell is committed to proceed through S phase and the rest of the cell cycle. On the other hand, if appropriate growth factors are not available in G1 progression through the cell cycle stops at the restriction point.

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G2 to M transition

• Although the proliferation of most cells is regulated primarily in G1 some cell cycles are instead controlled principally in G2.

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• In fission yeast Schizosaccharomyces pombe the cell cycle is regulated prima­rily by control of the transition from G2 to M, which is the principal point at which cell size and nutrient availability are monitored.

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• Vertebrate oocytes can remain arrested in G 2 for long periods of time (several decades in humans) until their progression to M phase is triggered by hormonal stimulation. Extracellular signals can thus control cell proliferation by regu­lating progression from the G2 to M phase of the cell cycle.

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• In addition, some haploid mosses use G2 control point.

Dr. Riddhi Datta

Cell Cycle Checkpoints

• The events that take place during different stages of the cell cycle must be coordinated with one another so that they occur in the appropriate order.

• This coordination between different phases of the cell cycle is dependent on a series of cell cycle checkpoints that prevent entry into the next phase of the cell cycle until the events of the preceding phase have been completed.

Dr. Riddhi Datta

Characterization of MPF

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• MPF, a conserved regulator of the cell cycle is composed of two key subunits:
• Cdkl
• cyclin B

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• Cyclin B is a regulatory subunit required for catalytic activity of the Cdkl protein kinase.

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• MPF activity is controlled by periodic accumulation and degradation of cyclin B during cell cycle progression.

Dr. Riddhi Datta

MPF regulation

• Cdkl forms complexes with cyclin B during G2.

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• CdkI is then phosphorylated on threonine-161 by MO15, which is required for Cdkl activity.

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• It is then phosphorylated on tyrosine-15 (and threonine-14 in vertebrate cells) by a protein kinase called Wee1, which inhibits Cdkl activity and leads to the accumulation of inactive Cdkl/cyclin B complexes throughout G2 phase.

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• Dephosphorylation of Thr14 and Tyr15 by a protein phosphatase called Cdc25C activates MPF at the G2 to M transition.

Dr. Riddhi Datta

• Activated Cdkl protein kinase phosphorylates a variety of target proteins that initiate the M phase.

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• MPF activity is terminated at the end of mitosis by degradation of cyclin B.

Regulation of CDK activity

The activity of Cdk's during cell cycle progression is regulated by four molecular mechanisms:

Dr. Riddhi Datta

Activities of CdkI/cyclin B complexes can also be regulated by binding of inhibitory proteins called CKI’s (Cdk inhibitors). In mammals 2 families of CKI’s are present:

• Ink4 family: binds to Cdk4 & Cdk6; inhibits G1 to S progression
• Kip/Cip family: binds to Cdk1 & Cdk2; inhibits various phases of cell cycle progression

Families of Cyclins and Cyclin-Dependent Kinases

• In yeast, passage through START is controlled by Cdkl in association with G1 cyclins (Cln1 , Cln2, and Cln3).
• Complexes of Cdkl with distinct B-type cyclins (Clb’s) then regulate progression through S phase and entry into mitosis.

Dr. Riddhi Datta

• In animal cells, progression through the G1 restriction point is controlled by complexes of Cdk4 and Cdk6 with D-type cyclins.
• Cdk2/cyclin E complexes function later in G1 and are required for the G1 to S transition.
• Cdk2/ cyclin A complexes are then required for progression through S phase.
• CdkI/cyclin A regulates progression to G2 Cdkl/cyclin B complexes drive the G2 to M transition.

Growth factors & regulation of G1 to S transition

• Growth factors regulate cell cycle progression through the G1 restriction point by inducing synthesis of D-type cyclins via the Ras/Raf/MEK/ERK signaling pathway.

Dr. Riddhi Datta

• As long as growth factors are present through G1, complexes of Cdk4, 6/ cyclin D1 drive cells through the restriction point.

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• If growth factors are removed prior to this key regulatory point, the levels of cyclin D1 rapidly fall and cells are unable to progress through G1 to S, instead becoming quiescent and entering G0.

Dr. Riddhi Datta

Growth factors

Ras/Raf/ ERK/MEK

Synthesis of Cyclin D

Cdk4,6/CycD

Rb

E2F

Rb

E2F

P

Transcriptional activation

Cyclin E

Growth factors

p27

MCM helicase

ATM

ATR

Double strand break

Single strand break

CHK1

Cdc25C

Cdc25A

Cdk2

Cdk1

P

P

G1 PHASE

G1 TO S TRANSITION

M PHASE

G1, S, G2 PHASES

p53

p21

CHK2

Unattached kinetochore

Mad

Bub

Mad

Cdc20

APC

All kinetochores attached

Bub

Bub

Mad

Cyclin B degraded

Cdk1 inactive

Securin degraded

Separase active

Anaphase

Growth factors & regulation of G1 to S transition

• While cyclin D and oncogene proteins like Ras drive cell proliferation, proteins encoded by many tumor suppressor genes (like Rb and Ink4 Cdk inhibitors ) act as brakes that slows down cell cycle progression.

Dr. Riddhi Datta

• In its under-phosphorylated form, Rb binds to members of the E2F family (transcription factors that initiates transcription of genes required for initiation of S phase). This represses transcription of E2F-regulated genes.

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• Phosphorylation of Rb by Cdk4,6/cyclin D complexes results in its dissociation from E2F in late G1.

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• E2F then stimulates expression of its target genes, which encode proteins required for cell cycle progression (Cyclin E).

Dr. Riddhi Datta

Growth factors

Ras/Raf/ ERK/MEK

Synthesis of Cyclin D

Cdk4,6/CycD

Rb

E2F

Rb

E2F

P

Transcriptional activation

Cyclin E

Growth factors

p27

MCM helicase

ATM

ATR

Double strand break

Single strand break

CHK1

Cdc25C

Cdc25A

Cdk2

Cdk1

P

P

G1 PHASE

G1 TO S TRANSITION

M PHASE

G1, S, G2 PHASES

p53

p21

CHK2

Unattached kinetochore

Mad

Bub

Mad

Cdc20

APC

All kinetochores attached

Bub

Bub

Mad

Cyclin B degraded

Cdk1 inactive

Securin degraded

Separase active

Anaphase

Growth factors & regulation of G1 to S transition

• Progression through the restriction point and entry into S phase is mediated by the activation of Cdk2/cyclin E complexes.
• Passage through the restriction point induces the synthesis of cyclin E via activation of E2F. However, Cdk2/cyclin E complexes are inhibited by the Cdk inhibitor p27.
• Growth factor signaling reduces the levels of p27 by inhibiting its transcription and translation. The resulting activation of Cdk2/ cyclin E leads to activation of the MCM helicase and initiation of DNA replication (i.e. S phase).

Dr. Riddhi Datta

Restricting DNA Replication to Once per Cell Cycle

• DNA replication is restricted to once per cell cycle by the MCM helicase proteins that bind to origins of replication together with ORC (origin recognition complex) proteins and are required for the initiation of DNA replication.

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• MCM proteins are only able to bind to DNA in G1, allowing DNA replication to initiate in S phase.

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• Once initiation has occurred, the MCM proteins are displaced so that replication cannot initiate again until after mitosis.

Dr. Riddhi Datta

Dr. Riddhi Datta

Growth factors

Ras/Raf/ ERK/MEK

Synthesis of Cyclin D

Cdk4,6/CycD

Rb

E2F

Rb

E2F

P

Transcriptional activation

Cyclin E

Growth factors

p27

MCM helicase

ATM

ATR

Double strand break

Single strand break

CHK1

Cdc25C

Cdc25A

Cdk2

Cdk1

P

P

G1 PHASE

G1 TO S TRANSITION

M PHASE

G1, S, G2 PHASES

p53

p21

CHK2

Unattached kinetochore

Mad

Bub

Mad

Cdc20

APC

All kinetochores attached

Bub

Bub

Mad

Cyclin B degraded

Cdk1 inactive

Securin degraded

Separase active

Anaphase

DNA Damage Checkpoints

• DNA damage check­points arrests cell cycle progression in response to damaged or incompletely replicated DNA.
• Cell cycle arrest at the DNA damage checkpoints is initiated by the ATM or ATR protein kinases, which are components of protein complexes that recognize damaged or un-replicated DNA.
• ATM is activated principally by double-strand breaks, while ATR is activated by single-­stranded or un-replicated DNA.
• ATM and ATR then phosphorylate and activate the CHK2 and CHKI protein kinases, respectively. CHKI and CHK2 phosphorylate and inhibit the Cdc25A and Cdc25C protein phosphatases.
• Cdc25A and Cdc25C are required to activate Cdk2 and Cdkl, respectively, so their inhibition leads to arrest at the DNA damage checkpoints in G1, S, and G2.

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Dr. Riddhi Datta

DNA Damage Checkpoints

• In mammalian cells, arrest at the G1 checkpoint is also mediated by the action of an additional protein known as p53, which is phosphorylated by both ATM and CHK2.
• Phosphorylation by ATM and CHK2 stabilize p53, resulting in rapid increases in p53 levels in response to DNA damage.
• The protein p53 then activates transcription of the gene encoding the Cdk inhibitor p21, leading to inhibition of Cdk2/cyclin E complexes and cell cycle arrest.

Dr. Riddhi Datta

Dr. Riddhi Datta

Growth factors

Ras/Raf/ ERK/MEK

Synthesis of Cyclin D

Cdk4,6/CycD

Rb

E2F

Rb

E2F

P

Transcriptional activation

Cyclin E

Growth factors

p27

MCM helicase

ATM

ATR

Double strand break

Single strand break

CHK1

Cdc25C

Cdc25A

Cdk2

Cdk1

P

P

G1 PHASE

G1 TO S TRANSITION

M PHASE

G1, S, G2 PHASES

p53

p21

CHK2

Unattached kinetochore

Mad

Bub

Mad

Cdc20

APC

All kinetochores attached

Bub

Bub

Mad

Cyclin B degraded

Cdk1 inactive

Securin degraded

Separase active

Anaphase

Cdkl/Cyclin 8 and Progression to Metaphase

• Mitosis involves dramatic changes in multiple cellular components, leading to a major reorganization of the entire structure of the cell.
• These events are initiated by activation of the Cdkl/cyclin B protein kinase (MPF).
• The Cdkl/cydin B complex induces multiple nuclear and cytoplasmic changes at the onset of M phase both by activating other protein kinases and by phosphorylating proteins such as condensins, components of the nuclear envelope, Golgi matrix proteins, and proteins associated with centrosomes and microtubules.

Dr. Riddhi Datta

The spindle assembly checkpoint

• The spindle assembly checkpoint monitors the alignment of chromosomes on the metaphase spindle.
• The progression from metaphase to anaphase results from ubiquitin-mediated proteolysis of key regulatory proteins, called the anaphase-promoting complex (APC).
• The APC remains inhibited until the cell passes the spindle assembly checkpoint, after which activation of ubiquitin degradation system brings about the transition from metaphase to anaphase and progression through the rest of mitosis.

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Dr. Riddhi Datta

The spindle assembly checkpoint

• Unattached kinetochores lead to assembly of Mad/Bub protein complex that bind to Cdc20-a required component of the APC. Mad proteins are activated in this complex, and then released in active form that inhibits Cdc20, maintaining the APC in inactive state.
• Once all chromosomes are aligned on the spindle, the Mad/Bub complex dissociates, relieving inhibition of Cdc20 and leading to APC activation.

Dr. Riddhi Datta

The spindle assembly checkpoint

• Activation of APC results in ubiquitination and degradation of two key target proteins.
• APC ubiquitinates cyclin B, leading to its degradation and inactivation of Cdkl.
• APC also ubiquitinates securin, leading to activation of separase.
• Separase degrades a subunit of cohesin, breaking the link between sister chromatids and initiating anaphase.

Dr. Riddhi Datta

Dr. Riddhi Datta

Growth factors

Ras/Raf/ ERK/MEK

Synthesis of Cyclin D

Cdk4,6/CycD

Rb

E2F

Rb

E2F

P

Transcriptional activation

Cyclin E

Growth factors

p27

MCM helicase

ATM

ATR

Double strand break

Single strand break

CHK1

Cdc25C

Cdc25A

Cdk2

Cdk1

P

P

G1 PHASE

G1 TO S TRANSITION

M PHASE

G1, S, G2 PHASES

p53

p21

CHK2

Unattached kinetochore

Mad

Bub

Mad

Cdc20

APC

All kinetochores attached

Bub

Bub

Mad

Cyclin B degraded

Cdk1 inactive

Securin degraded

Separase active

Anaphase

Phases of mitosis

Dr. Riddhi Datta

Check points

Dr. Riddhi Datta

Cdk/Cyclins

Dr. Riddhi Datta

Thank you

Dr. Riddhi Datta