Learning Objectives

  • Explain the role of the pRB family of proteins in inhibiting the cell cycle.
  • Explain the role of P53 in inhibiting the cell cycle in response to DNA damage.
  • Differentiate between growth factor and mitogen.
  • Explain the role of cyclin D-CDK complex in triggering entry into the cell cycle.
  • Explain the difference between oncogene and tumor suppressor.

Keywords

  • Cyclin D
  • Cyclin E
  • E2F
  • pRB
  • P27/P21
  • P53
  • Mitogen
  • Growth factor
  • Start
  • Ink4

Pre-Lecture Reading

Overview

Cell division is tightly regulated in multicellular organisms to prevent the uncontrolled proliferation of cells. Most eukaryotic cells only divide in the presence of mitogens. Mitogens trigger cells to enter Start in G1 which is the point at which cells are committed to divide. Biochemically, Start is marked by the activation of G1/S cyclin-CDKs and S cyclin-CDKs that initiate DNA replication. The G1 cyclins (cyclin D) regulate entry into Start by turning on expression of G1/S and S cyclins and by removing and inhibitory protein from G1/S cyclin-CDK complexes. DNA damage also inhibits the cell cycle by inhibiting the activation of G1/S-CDK complexes.

Summary

  • Commitment to cell division occurs at Start when G1/S cyclin-CDK is activated.
  • Expression of G1/S cyclins is regulated by E2F proteins in conjunction with pRB proteins..
  • Mitogens stimulated cell division by increasing the amount of G1 cyclins.
  • G1 cyclin-CDK leads to active G1/S cyclin-CDK by increasing the transcription of G1/S cyclin and removing an inhibitor of G1/S cyclin-CDK.
  • DNA damage triggers activation of P53 that increases the amount of proteins that inhibit G1/S cyclin-CDK.

Introduction

Start represents the point in G1 after which cells are committed to divide. Consequently, cells intensely regulate progression past Start. In single cell organisms, such as yeast, entry into Start is usually tied to the availability of nutrients. In animals, where nutrients are usually abundant, Start is regulated by extracellular signals called mitogens and the absence of DNA damage. Start is marked by the activation of G1/S cyclin-CDKs. Expression of G1/S cyclins is increased prior to Start and proteins that inhibit the activation G1/S cyclin-CDKs are removed.

Transcriptional Regulation of G1/S Cyclins

The E2F family of proteins regulates the expression of G1/S cyclins. The family contains proteins that activate transcription of G1/S cyclins (E2F1, E2F2, E2F3) and proteins that repress transcription of G1/S cyclins (E2F4, E2F5). Whether the activators or inhibitors of G1/S cyclin are active depends upon the pRB family of proteins. pRB proteins bind to E2F proteins and control their activity. pRB binds and inhibits the activity of the transcription activators (E2F1, E2F2, E2F3), whereas other pRB family members, P107 and p130, function as corepressors with E2F4 and E2F5. Therefore, when the pRB proteins are expressed, in G0 for example, transcription of G1/S cyclins is repressed. Importantly, cells that lack the activity of the pRB proteins often fail to exit the cell cycle and proliferate uncontrollably. Many human cancers contain mutations in pRB genes.

How Mitogens Stimulate the Cell Cycle

In the absence of mitogens, most cells exist in G0 due to the presence of pRB proteins that prevent expression of the G1/S cyclins. Mitogens induce the phosphorylation of pRB proteins that dissociates them from E2F proteins. When E2F4 and E2F5 are not bound to p107 and p130, they are much less efficient inactivators of transcription. When E2F1-3 are not bound to pRB, they become proficient activators of G1/S cyclin transcription. The pRB proteins are phosphorylated by cyclinD-CDK4 and cyclinD-CDK6 complexes. Cyclin D is a G1 cyclin and its expression is increased in the presence of mitogens.

Mitogens and Expression of G1 Cyclins

Mitogens act through a classic tyrosine kinase pathway. Receptors for mitogens become active when bound to mitogens, and phosphorylate each other on the cytoplasmic domains. The phosphorylated domains are recognized by a guanine nucleotide exchange factor for Ras that increases the amount of Ras-GTP. Ras-GTP activates a MAP kinase pathway with the final kinase phosphorylating transcription factors that turn on expression of early response genes. The early response genes include FOS and MYC. Fos and Myc proteins form separate complexes that activate expression of G1 cyclins (cyclin D).

Regulation of Cyclin D-CDKs

The increased expression of cyclin D is not sufficient to generate active cyclin D-CDK. Ink4 proteins bind to cyclin D-CDK and inhibit its activity. Expression of Ink4 is increased in the presence of the transcription protein Miz1. The levels of Miz1 are elevated when cells bind anti-mitogenic factors, such as TGFβ-1. In the presence of mitogens, Myc inactivates Miz1, lowering the expression of Ink4 proteins. As Ink4 levels fall, the amount of active cyclin D-CDK increases. The importance of Ink4 in regulating he cell cycle is highlighted by the findings that many cancers contain mutations in INK4 genes, including 80% of pancreatic cancers.

A second mechanism that cells regulate cyclin D-CDK activity is through protein localization. Because most targets of cyclin D-CDK reside in the nucleus, cells can inhibit the effects of cyclin D-CDK by confining it to the cytoplasm. Glycogen synthase kinase phosphorylates cyclin D-CDK and prevents it from entering the nucleus. Mitogens, acting through a tyrosine kinase pathway (different from the MAP kinase pathway described above), inhibit the activity of glycogen synthase kinase, allowing cyclin D-CDK to enter the nucleus and phosphorylate its target proteins.

CDK Inhibitory Proteins

As mentioned, cyclin D-CDK increase the activity of G1/S cyclin-CDK by increasing the transcription of cyclin E, but cyclin D-CDK also activate cyclin E-CDK by another mechanism. Cyclin E-CDK are bound by a set of proteins, P27 and P21, that inhibit their activity. Cyclin D-CDK compete with cyclin E-CDK for P27 and P21. Interestingly, though P27 and P21 inhibit the activity of cyclin E-CDK, they increase the activity of cyclin D-CDK. As the concentration of cyclin D-CDK increases, more P27 and P21 are removed from cyclin E-CDK, leading to increased activity of cyclin E-CDK. Similar to INK4, a number of cancers contain mutations in the genes that encode P27 and P21. Eventually, the amount of active cyclin D-CDK rises to a level sufficient to produce enough active cyclin E-CDK that the cells are triggered to enter Start and begin the process of cell division.

DNA Damage Checkpoint

One of the most critical checkpoints in the cell cycle is DNA damage, as unfixed damage can introduce mutations during DNA replication, leading to potential changes in cell behavior. P53 is one of the central players in a cell’s response to DNA damage. P53 is activated by proteins that find and monitor DNA damage, and P53,as a transcription activator, turns on the expression of genes that inhibit the cell cycle (e.g. P27, P21). In some cases, P53 causes cells to undergo apoptosis.

In the absence of DNA damage, the levels of P53 are kept low by the protein Mdm2. Mdm2 catalyzes the ubiquitination of P53, marking it for degradation. Mdm2 also binds and inhibits the transcription activation domain in P53. DNA damage activates a set of kinases that phosphorylate Mdm2, causing it to dissociate from P53. Freed from Mdm2, P53 is no longer targeted for degradation and its concentration increases. In addition, its transcription activation domain is exposed.

Cells also regulate P53 activity through protein localization. In the absence of DNA damage P53 is a monomer. In its monomeric form, P53 exposes a nuclear export sequence. This causes any P53 that enters the nucleus to be rapidly exported. Because P53 functions as a transcription activator, it is impotent in the cytoplasm. DNA damage induces a conformation change in P53 that causes it to assemble into a tetramer. In its tetrameric form, the nuclear export sequence is hidden, so P53 that enters the nucleus is not exported.

In addition to activating P53, DNA damage also inhibits the cell cycle by inactivating Cdc25. Cdc25 is the phosphatase that removes the inhibitory phosphate group in ATP-binding pocket of CDKs. Activation of Cdc25 is thought to be the trigger that activates cyclin-CDK, starting the next phase of the cell cycle. Kinases activated by DNA damage phosphorylate Cdc25, targeting Cdc25 for ubiquitination and degradation.

Regulation of Cell Growth

The rate of cell growth of is largely determined by the rate of protein synthesis, as proteins make up the bulk of the cell. The rate of protein synthesis is controlled by the rate or ribosome synthesis and the rate of initiation of translation. TOR is considered the master regulator of growth rates in cells because it increases the rate of ribosome production by increasing expression of ribosome proteins and RNAs and it increases the rate of translation initiation by relieving the inhibition of initiation.

Growth factors (different than mitogens) stimulate the activity of TOR. Insulin-like growth factor (IGF) is one of the most well understood growth factor, and it activates TOR through a tyrosine kinase pathway. The receptor for IGF activates a phosphatidylinositol kinase that generates of patch of phosphatidylinositol 3-phosphate (PIP3) in the inner leaflet of the plasma membrane. PIP3 recruits and activates kinases that inhibit the GTPase activating protein (GAP) for Rheb. As Rheb-GTP levels increase, it activates TOR, leading to increased protein synthesis.

Coordination of Cell Growth and Division

In most single cell organisms, cell growth and cell division are coordinated as cells divide after reaching a certain size. The relationship between cell growth and division in mammalian cells is more complicated, as cell growth seems to regulate cell division in some cells (fibroblasts) but appears not to have an effect in other cells (muscle, neurons). Small fibroblasts remain longer in G1 than larger fibroblasts, suggesting that they must reach a minimal size before dividing. In contrast, muscle cells can grow to very large sizes without undergoing cell division.

Whether cell growth and cell division are coordinated seems to depend upon the wiring of the mitogen and growth factor signaling pathways. In some cells, mitogens or growth factors activate both cell growth and cell division, suggesting that there is crosstalk between the growth factor and mitogen signaling pathways. In other cells, the growth factor and mitogen signaling pathways seem to function independently as growth factors do not stimulate cell division and mitogens do not stimulate cell growth.

Questions

  1. Why are pRB and P53 called tumor suppressors?
  2. Answer: pRB and p53 both arrest or slow the rate cell division by either inhibiting the expression of G1 cyclins (pRB) or the activation of G1/S cyclin-CDKs (p53). Mutations in the genes that encode pRB and p53 that reduce the amount of pRB and p53 are associated with a variety of tumors, suggesting that both proteins have a role in preventing the development of tumors.
  3. Why is MYC considered an oncogene?
  4. Answer: Myc increases the rate of cell division by activating the expression of G1 cyclins. G1 cyclin-CDK increase the expression and activation of G1/S cyclin-CDKs that ultimately trigger entry into cell division. Increased levels of Myc in cells is associated with a variety of tumors, suggesting that Myc has a role in promoting the development of tumors.
  5. Why would the concentration of cyclin D-CDK be important for triggering cell division?
  6. Answer: Besides causing increased expression of cyclin E (G1/S cyclin), cyclin D-CDK4 also triggers the activation of cyclin E-CDK2 complexes. The cell cycle inhibitors p21 and p27 bind cyclin E-CDK2 and inhibit the activity of the complex. Cyclin D-CDK4 also bind p21 and p27, and when the concentration of cyclin D-CDK4 reaches a certain level, most of the p21 or p27 is bound to cyclin D-CDK4, creating enough active cyclin E-CDK2 to start the cell cycle.
  7. At what point in the cell cycle would cells no longer require mitogens for division?
  8. Answer: START is the point in the cell cycle where cell division becomes independent on mitogens. Mitogens indirectly activate cyclin E-CDK2 by increasing the expression of cyclin D which when bound to CDK4, increases the expression of cyclin E. At start, cyclin E-CDK2 is activated and through a positive feedback loop, cyclin E-CDK2 is able to maintain its own activity and mitogens are no longer needed to drive the cell cycle.