Cell Cycle Regulation

Cell Cycle Regulation
LO1: The 4 phases of the cell cycle and the principle event(s) of each phase
phase
time
DNA
Events
G1 (Gap 1)
9-10 hours
2N
S (synthesis)
6-8 hours
2n  4n
- Decision to proliferate or not
- Detect DNA damage
- Centrosome duplication begins
- Major period of cell growth (organelle
replication)
- Genome completely replicated (only
once!)
- Any DNA damage gets repaired
G2 (Gap 2)
4-5 hours
4n
-Cell prepares for mitosis
-Centrosome replication finishes
-Monitor for DNA damage and make
sure DNA replication complete
M (mitosis)
1 hour
4n  2n
-creation of 2 new daughter cells by
mitosis and cytokinesis (see previous
noteset for more details)
It is also important to note that G0 is the quiescent phase that a cell enters if it chooses not to
proliferate it will enter G0 from G1.
Finally it is important to note that not all cells proliferate and that the cell cycle must have
orderly progression! Consequences of improper cell cycle control include the development of cancer.
The cell ensures orderly progression (and prevents regression) by cell cycle checkpoints that are
discussed below in LO3.
LO2: The CDK complex, including the components of the complex, the levels of activation and
inactivation, and the mechanism of CDK phosphorylation for cell cycle progression
The CDK complex is a collection of serine/threonine kinases which can be activated and inactived to
regulate the cell’s progression through the cell cycle checkpoints. CDK stands for cyclin dependent
kinase. The complex has 3 components:
1. CDK  catalytic kinase subunit (motor)
2. Cyclin  regulatory subunit (gas petal)
3. CDKI  inhibitor (brake)
CDKI’s inhibit the CDK complex. There are two types with different mechanisms of inhibition. You do not
need to know the superscripts of the members of the CDKI families so I have not included them .
1. CIP/KIP family: p21, p27, p57
a. Mechanism  binds the CDK-cyclin complex and inhibits the catalytic kinase activity
b. Very general, can bind any CDK complex and inhibit it
2. INK4 family: p16, p15, p18, p19
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Cell Cycle Regulation
a. Mechanism  bind CDK 4 or CDK 6 and displace it’s cyclin (D type) subunit so that the
catalytic activity cannot occur
b. Very specific, can only bind CDK4 or CDK6
Be sure to know which CDKIs are in which family, I like to remember that if p is less than 20 it’s in the
INK4 family and if it’s greater than 20 it’s in the CIP/KIP family.
Five factors regulate the kinase activity of the CDK complex:
1. Cyclin expression
a. Cyclin is so named because it’s expression oscillates
through the cell cycle, each checkpoint has a specific
cyclin associated with it
b. When cyclin expression is high there will be more CDK
activity
c. Cyclins are short lived (see degradation) and when
there is less Cyclin around there will be less CDK activity
2. Assembly
a. CDK complexes are only activity if they are bound to their cyclin
3. Phosphorylation
a. Once cyclin is bound, CDK is somewhat active, however to achieve maximal activity a
specific threonine residue gets phosphorylated
i. Cyclin binding CDK allows some activity and leads to a conformational change
that exposes the T-loop of the kinase that contains a specific threonine residue
ii. CDK activating kinase (CAK) phosphorylates that threonine allowing high
catalytic activity
4. CDKI
a. Inhibit the CDK complex as discussed above
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Cell Cycle Regulation
5. Degradation
a. Both cyclins and CDKIs are short-lived and get degraded by the ubiquitin mediated
protein degradation pathway
b. As they get degraded their ability to activate or inactivate CDK lessens
It is important to note that unlike cyclin and CDKIs, CDK expression is fairly consistent throughout the
cell cycle
The CDK, Cyclin and CDKI expressed are specific to each cell cycle checkpoint. You need to memorize
this specificity summarized in the table below
Checkpoint
CDK
Cyclin
CDKI
Mid –G1
4/6
D
CIP/KIP or INK4
G1/S
2
E
CIP/KIP
S
2
A
CIP/KIP
G2/M
1
B
CIP/KIP
Finally, Dr. Franklin conceptualized the CDK complexes with the example of the Retinoblastoma protein
(Rb). Rb functions to negatively regulate growth by binding to and inactivating transcription factors (E2F
specifically) that are needed to transcribe genes for S phase. The gene for Rb is the most commonly
inactivated gene in human cancers and is called the tumor suppressor gene. It’s regulation by the CDK
complex occurs as follows:
1. In G1 Rb is hypophosphorylated and active there for stopping cells from entering S-phase
a. Rb does this by binding and sequesting E2F transcription factors so S-phase gene
expression can’t occur
2. If conditions are met for the cell to enter the cell cycle then Cyclin D expression occurs activates
the early G1 CDKs (4/6) and then Cyclin D and CDK4 and CDK6 form complexes
3. The CDK complex now phosphorylates Rb and allows progression past the mid-G1 checkpoint to
the G1/S checkpoint; now the cells are committed to proliferation
4. If the conditions are met for entry to the S phase CDK2-cyclin E complex gets activates and
further phosphorylates Rb to a hyperphosphorylated and completely inactive state.
5. Inactivated Rb releases E2F transcription factors allowing entry into the S phase. E2F allows
expression of
a. S phase proteins
b. Cyclin E  accelerating the progression through the G1/S checkpoint
c. Cyclin A  allows activation of S phase function
6. As the cell completes mitosis Rb gets dephosphorylated and resets the cell cycle in G1
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Cell Cycle Regulation
LO3: The cell cycle checkpoints (G1, G1/S, S and G2/M) and the coordination of cell cycle events
There are four cell cycle checkpoints. A cell will not pass through a cell checkpoint unless its CDK
complex is formed and active (cyclin expressed and no CDKI.) The formation of the CDK complex won’t
occur unless the cell has met the requirements to proceed. The first two Dr. Franklin discussed primarily
in the context of Rb discussed above. The four checkpoints are as follows:
1. Mid-G1  determines whether the cell will proliferate or go to G0
a. Responsive to growth factors (stimulate proliferation)  leads to cyclin D expression
b. Main substrate of its CDK complex is Rb which gets phosphorylated and allows
progression through this checkpoint
c. When passed cells are committed to proliferate
2. G1/S  ensures G1 complete, cell big enough and no DNA damage
a. CDK2-cyclin E must be activated
b. CDK complex again phosphorylates Rb such that it is now completely inactive and E2F
can allow expression of S phase proteins, more cyclin E and cyclin A
c. Now origins of replications fire up and DNA replication can begin
3. S-phase  assesses origins of replication, that DNA replication complete (only 1 copy and no
damage unrepaired)
a. A CDKI (p27) helps maintain the CDK complex inactive (CDK2-cyclin E) but CDK2 and
cyclin E levels increase so you get some active CDK complex
b. The active CDK complex phosphorylates p27 causing it to be targeted for ubiquitin
mediated degradation leading to accelerated CDK complex activation and propulsion
into S phase
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Cell Cycle Regulation
c. p27 also inactives CDK2-CyclinA also allowing efficient firing of replication origins in S
phase
d. Eventually both of the CDK complexes get shutdown as cyclins become phosphorylated
(last substrate for phosphorylation) and subsequently degraded  this prevents the
reversal of the cell cycle!
4. G2/M  ensures centrosomes replicated and cell has everything it needs for mitosis
a. CDK1-Cyclin B complexes that were assembled in S phase have been maintained
inactive by Wee1 phosphorylation at an inhibitory site (Y15 in figure)
b. Once S phase is complete CDK1-cyclin B becomes activated by CAK phosphorylation at
an activating phosphorylation site (T161 in figure)
c. Now the phosphatase Cdc25 can remove the phosphate at the inhibitory site thereby
activating the CDK complex
d. The active CDK complex phosphorylates substrates to begin mitotic events
(chromosome condensation, nuclear break down, MT remodeling, kinetochore
assembly and activation of mitotic kinases)
Finally Dr. Franklin talked about some of the events that occur in Mitosis and factors that cause cell cycle
arrest. He skimmed over this material much quickly in lecture and doesn’t seem to be as emphasized in
the learning objectives.
In Anaphase, cohesion (a protein that holds the sister chromatids together at the kinetochore) is
degraded so that the sister chromatids can separate. Kleisin is a component of cohesion that gets
cleaved to the sister chromatids can separate



Separase = enzyme that cleave Kleisin
Securin = enzyme that keeps Separase inactive until anaphase
APC/C –cdc20 = complex that ubiquitinates securin for degradation
APC/C-cdc20  securin degradation  separase free to cleave Kleisin  sister chromatids separate
Mitotic Cyclin Destruction occurs at the end of mitosis in order to inactive the CDK complexes (CDK1cyclin B) so that the cell cycle returns to G1. This destruction occurs in late anaphase and is
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Cell Cycle Regulation
accomplished by APC/C-Cdh1 which will very specifically ubiquinate mitotic cyclins (B). This
ubiquitination only occurs at the end of mitosis because Cdh1 is maintained in an inactive
phosphorylated state by G1 CDK complexes until anaphase. Cdh1 is activated by dephosphorylation
carried out by the phosphatase Cdc14
Lastly, Cell Cycle arrest occurs in response to damage or improper DNA replication. ATM/ATR and
Chk1/2 are kinases that halt the cycle in 2 ways:
1. preventing Cdc25 from activating CDK complex (normally occurs in G1/S, S and G2/M
checkpoints)
2. induces CIP/KIP CDKIs by a p53 dependent mechanism
Cell cycle arrest also occurs if there is improper spindle attachment to the kinetochore by blocking
anaphase by inactivating APC/C mediated securin and cyclin B degradation
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