Chapter 30: The Cell Cycle and Checkpoint Controls

Question 1

Basics of Normal Cell Replication

Answer 1

• The replication of most normal cells is mitogen (growth factor) dependent
  
  • Human cells only proliferate when instructed to do so by mitogens. Ex: EGF, FGF, Epo, estrogen, testosterone
• Cell replication is anchorage-dependent
  
  • Cells in the body are attached to the extracellular matrix and proliferate only when attached.
• Cell replication is contact-inhibited
  
  • Normally, cells stop growing when the available space is filled. In a dish, this means a single cell layer.
• Cells are mortal
  
  • Cells normally have a limited number of cell divisions before they go into replicative senescence and die

Question 2

More Basics of Cell Replication

Answer 2

• Cell replication/division is one of the most fundamental processes of life, allowing cells to faithfully transmit their genetic information from cell to cell and from generation to generation.
• The decision on whether to divide or not is a critical and tightly controlled process regulated by both internal and external signals.
• In order to control this decision, a complex network known as the cell-cycle control system or cell cycle clock governs progression through the cell cycle.
• The term cell cycle refers to the ordered series of events that lead to cell division and the production of two daughter cells

Question 3

The Cell Cycle

Answer 3

• The two most basic functions of the cell cycle:
  
  • Chromosomal replication to faithfully produce two copies of the genetic information.
  • Chromosomal segregation to ensure each daughter cell receives a copy of the entire genome.

Question 4

Cell Replication Is Regulated by Both Positive and Negative Controls

Answer 4

• Positive controls (most typically growth factors) promote cell replication
• Negative controls, known as checkpoint controls operate to prevent cells from continuing division if the cell is not ready, most notably if the DNA is damaged or if mitotic spindle attachment is not complete.

Question 5

he Master Controllers of the Cell Cycle Are Heterodimeric Protein Kinases

Answer 5

The cyclin-cyclin dependent protein kinase complexes (cyclin-Cdks) regulate the activities of multiple proteins involved in cell replication such at those involved in entry into the cell cycle, DNA replication, and mitosis. The cyclin-Cdks do that by phosphorylating these proteins at specific regulatory serines or threonines.

Question 6

Cyclin-dependent kinases (Cdks)

Answer 6

• are serine/threonine protein kinases
• are inactive unless bound to cyclins
• The activated cyclin-Cdk complex phosphorylates proteins involved in the cell cycle
• Cdk specificity is determined by the cyclin bound to it

Question 7

Cyclins

Answer 7

• are specialized Cdk-activating proteins but have no enzyme activity
• help direct Cdks to the target proteins
• are only present during the cell cycle stage that they trigger, i.e. the amount of these proteins changes in a cyclical fashion.

Question 8

Cyclin D

Answer 8

• Cdk: Cdk4, Cdk6
• Cyclin-Cdk Complex: G₁ Cyclin-Cdk
• Function: Entry into the cell cycle

Question 9

Cyclin E

Answer 9

• Cdk: Cdk2
• Cyclin-Cdk Complex: G₁-S Cyclin-Cdk
• Function: Progression through restriction point

Question 10

Cyclin A

Answer 10

• Cdk: Cdk2
• Cyclin-Cdk Complex: S Cyclin-Cdk
• Function: Stimulates DNA synthesis

Question 11

Cyclin B

Answer 11

• Cdk: Cdk1
• Cyclin-Cdk Complex: M Cyclin-Cdk (Mitotic)
• Function: Stimulate entry into mitosis

Question 12

Cdk Activity Is Extensively Regulated

Answer 12

1. Regulated degradation of the cyclin subunits
2. The phosphorylation state of cyclin-Cdks regulates their activities
3. Cdk-inhibiting proteins (CKI’s, CIPs, INKs) interfere with kinase activity
4. The transcription of the cyclins and CKIs is regulated

Question 13

Cyclins “Rule the Cycle”

Answer 13

• Called cyclins because they undergo a cycle of synthesis and then regulated degradation by the proteasome.
• This ‘cycle’ ensures that:
  
  • Each checkpoint is ‘checked’ each round of cell division
  • Only 1 round of cell division occurs unless growth factors (mitogens) are still present
• During G1 and S phases, the SCF E3 ubiquitin ligase complex ubiquitinates and destroys the G1/S cyclins
  
  • Cyclin E and other targets get phosphorylated and thus are recognized as substrates for the SCF E3 ligase
• The anaphase-promoting E3 ligase complex (APC/C) ubiquitinates and destroys S- and M-cyclins
  
  • APC/C gets activated by binding of another protein (cdc20) at the metaphase-anaphase transition and targets the M cyclin and other proteins

Question 14

The SCF Ubiquitin Ligase Complex Targets the G1/S Cyclin and Other G1 Proteins for Proteasomal Degradation

Answer 14

SCF activity is dependent upon association with an F-box protein. There are many F-box proteins, and they are involved in target selection. The phosphorylation of the target protein (CKI here) allows it to be recognized by the F-box protein.

Question 15

The Cyclin-Cdk Complex Must Be in the Correct Phosphorylation State to be Active, Ex. M-Phase Cdk

Answer 15

• Both of the phosphorylated amino acids are in the ATP binding site of the Cdk, which likely interferes with ATP binding and thus kinase activity.
• These phosphorylation events are critical for activation of the M phase Cdk but also seem to be important in the control of G1/S and S phase Cdks.

Question 16

Wee1

Answer 16

Tyrosine kinase

Question 17

CAK

Answer 17

Cdk-activating kinase

Question 18

Cdc25

Answer 18

tyrosine protein phosphatase

Question 19

Regulation at the G1 Checkpoint

Answer 19

• G1 is a stable state of Cdk inactivity.
• G1 is the key point where cells decide whether or not to divide.
• That decision is based on whether or not:
  
  • External mitogenic signals indicate that additional cells are needed.
  • There is DNA damage.
  • The cell has grown sufficiently

Question 20

Regulation of the G1 Checkpoint Involves Four Major Mechanisms

Answer 20

1. Induction of the D cyclins (D1, D2, D3) by mitogens
2. Induction of the SCF E3 ubiquitin ligase and degradation of the CKIs (cyclin inhibitory proteins)
3. Inhibition of Rb, an inhibitor of E2F
4. Induction and activation of E2F, a transcriptional activator required to induce the synthesis of proteins needed in S phase

Question 21

All Cells Require 3 Classes of Cyclin-Cdks

Answer 21

1. G1/S Cyclin-Cdks (cyclin E-Cdks)
2. S-Phase Cyclin-Cdks (cyclin A-Cdk)
3. Mitotic Cyclin-Cdks (cyclin B-Cdk)

Question 22

G1/S Cyclin-Cdks (cyclin E-Cdks)

Answer 22

• control entry into S phase, (progression through the restriction point)
• phosphorylate transcription factors controlling genes whose proteins are needed for DNA replication
  
  • Examples: DNA polymerases, dNTP synthesis, S-phase Cdks

Question 23

S-Phase Cyclin-Cdks (cyclin A-Cdk)

Answer 23

• control DNA synthesis
• phosphorylate protein components of the prereplication complexes at origins of replication.

Question 24

Mitotic Cyclin-Cdks (cyclin B-Cdk):

Answer 24

• control mitosis
• Phosphorylate hundreds of proteins
  
  • Examples: chromatin condensation proteins, microtubule associated proteins, nuclear envelope and nuclear pore proteins, kinetochore proteins, all proteins required for mitosis.

Question 25

Most Cells Require a 4th Class of Cyclin-Cdks: G1-Cdks = Cyclin D-Cdks

Answer 25

• The G1 cyclin/Cdks:
• control the activities of the G1/S cyclins
• D cyclins are induced by mitogens•Mitogens are not required by embryonic stem (ES) cells to bypass G1
  
  • ES cells respond to an intrinsic timer or oscillator instead of mitogens, so they are autonomous.
  • ES cells are the only wild type cells that are tumorigenic

Question 26

Cells Enter G₀ Between Cell Divisions

Answer 26

• G₀ is imposed on all cells, at least temporarily, by the degradation of the M-phase cyclin after mitosis.
• G₀ can be very transient, where cells enter and leave rapidly (fibroblasts, intestinal cells)
• G₀ can be essentially irreversible, as with terminally differentiated cells (neurons, muscle cells). The cell cycle machinery is dismantled and even with mitogenic signals the cells won’t divide
• G₀ can be a quiescent state but one where the cell cycle machinery is intact (liver cells). These cells can divide with the appropriate external signals.

Question 27

Myc and Other Mitogen-Stimulated Transcription Factors Inhibit Rb

Answer 27

• A key function of cyclin D-Cdk is to activate the E2F transcription factors
• In the absence of mitogens, E2F is inhibited by members of the Rb (retinoblastoma) family
• When active cyclin D-Cdks (no CKIs!) accumulate after mitogen stimulation, they phosphorylate Rb, which inactivates it. This liberates E2F so it can activate the genes for cyclins E and A and other genes needed in S-phase
• Rb is a tumor suppressor
• The feedback loops depicted ensure that entry into the cell cycle is complete and irreversible.

Question 28

Negative Regulation of the G1 Checkpoint by TGFβ in Normal Cells

Answer 28

• TGFβ functions as a tumor suppressor and opposes cell proliferation by preempting the mitogenic pathway.
  1. TGFβ increases expression of CKIs
  2. TGFβ reduces the phosphorylation of Rb (p107 is an Rb family member), which increases its activity
  3. TGFβ represses expression of Myc

Question 29

Summary of Activation of the G1 Checkpoint

Answer 29

• Degradation of Cyclin B (M cyclin) initiates Go
• Mitogenic stimulation pushes cells into G1 by inducing transcription factors such as Myc, AP-1 (Fos & Jun), β-catenin, STATs, or by activating nuclear receptors
  
  • Induction of D cyclins
  • Induction of SCF and degradation of CKIs
  • Inactivation of Rb and activation of E2F
  • Synthesis of G1/S cyclins and other proteins needed for DNA synthesis

Question 30

Summary of Inhibitory Controls of the G1 Checkpoint

Answer 30

• TGFβ prevents progression into G1 by
  
  • Inhibiting the induction of Myc
  • Inducing the transcription of CKIs
  • Keeping Rb unphosphorylated
• Rb inhibits E2F so the G1-S cyclin is not synthesized
• Rb and TGFβ are thus major tumor suppressors of cell cycle progression through the G1 checkpoint

Question 31

Embryonic Stem Cells Are Not Subject to the G1 Checkpoint

Answer 31

• Rb is usually hyperphosphorylated all the time and is thus inactive, allowing E2F to be active
• Therefore, mitogens and MAP kinase signaling are not needed for progression through G1.
• There is no DNA damage checkpoint in G1
• Cyclin E expression is constant, not cyclical
• Net result: ESC pass through G1 rapidly, allowing rapid cell proliferation

Question 32

DNA Damage Is Detected by Kinases

Answer 32

• A cascade of DNA-dependent kinases detects damage to DNA, especially single- and double-strand breaks.
• DNA-dependent protein kinases must be bound to the DNA (directly or through bridging proteins) to be active
• These include the ATM and ATR kinases
  
  • ATM is mutated in ataxia telangiectasia
  • ATR = ATM-related
• ATM/ATR bind to and recognize the damaged DNA and send on the signal of DNA damage

Question 33

ATM/ATR Activation Blocks the Cell Cycle in Two Major Ways

Answer 33

• ATM and ATR activate the Chk1 and Chk2 kinases.
  
  • Chk1/Chk2 phosphorylate the Cdc25 tyrosine phosphatase, which leads to its degradation. (Cdc25 removes the inactivating phosphoryl from Cdks. Without cdc25, Cdks cannot be activated.)
• ​Both ATM/ATR and the Chk kinases increase the levels of the p53 transcription factor.
  
  • p53 regulates genes involved in cell cycle checkpoint controls, among other functions

Question 34

p53 Induces Target Genes Involved in Cell Cycle Control

Answer 34

In undamaged cells, p53 is highly unstable because it interacts with Mdm2, which is a ubiquitin ligase that targets p53 to the proteasome. Phosphorylation of p53 by Chk1/Chk2 reduces its binding to Mdm2, so p53 becomes stable.

Question 35

Summary of the DNA Damage Checkpoint

Answer 35

• DNA damage activates the ATM or ATR serine/threonine kinases
• The ATM/ATR kinases:
  
  • Phosphorylate and activate the Chk1 and Chk2 kinases
  • Phosphorylate and thus stabilize p53
• The Chk kinases phosphorylate the Cdc25 phosphatase, which targets it for proteosomal degradation
  
  • Cyclin-Cdk complexes do not reach the right phosphorylation state to be active
  • Can also phosphorylate and thus stabilize p53
• p53 induces target genes that inhibit cell division, such as the CKIs

Question 36

if DNA damage is too severe to be repaired, then normal cells go through…

Answer 36

Apoptosis

Question 37

Release at the Spindle-Assembly Checkpoint

Answer 37

• This checkpoint ensures that the cell does not enter anaphase until all the chromosomes are properly oriented on the mitotic spindle.
• Cells spend about half of mitosis in metaphase waiting for the APC/C signal that tells the chromatids to separate. This checkpoint relies on a sensor mechanism that monitors microtubule attachment at the kinetochore. Only when all microtubules are attached does APC/C get activated by Cdc20. Drugs that destabilize microtubules such as colchicine or vinblastin arrest the cells in this state for hours and days. Ultimately, prolonged activation of this checkpoint leads to cell death.