T

p21CiP

DNA-damage p53 checkpoint

ATM/R

Cdc25A

ATM/R

Cdc25A

DNA-damage checkpoint

Chk1/2

▲ FIGURE 21-32 Overview of checkpoint controls in the cell cycle. The unreplicated-DNA checkpoint (1) prevents activation of cyclin A-CDK1 and cyclin B-CDK1 (i.e., mitosis-promoting factor, MPF) by activation of an ATR-Chk1 protein kinase cascade that phosphorylates and inactivates Cdc25C, thereby inhibiting entry into mitosis. In the spindle-assembly checkpoint (2|), Mad2 and other proteins inhibit activation of the APC specificity factor (Cdc20) required for polyubiquitination of securin, thereby preventing entry into anaphase. The chromosome-segregation checkpoint ( 3) prevents release of the Cdc14 phosphatase from nucleoli, thereby blocking activation of the APC specificity factor (Cdh1) required for polyubiquitination of B-type cyclins as well as induction of Sic1. As a result, the decrease in MPF activity required for the events of telophase does not occur. In the initial phase of the DNA-damage checkpoint (4|), the ATM or ATR protein kinase (ATM/R) is activated. The active kinases then trigger two pathways: the Chk-Cdc25A pathway (4bl and 4cl), blocking entry into or through the S phase, and the p53-p21CIP pathway, leading to arrest in G1, S, and G2 (4a|-4b|). See text for further discussion. Red symbols indicate pathways that inhibit progression through the cell cycle.

The Presence of Unreplicated DNA Prevents Entry into Mitosis

Cells that fail to replicate all their chromosomes do not enter mitosis. Operation of the unreplicated-DNA checkpoint control involves the recognition of unreplicated DNA and inhibition of MPF activation (see Figure 21-32, 1). Recent genetic studies in S. pombe and biochemical studies with Xenopus egg extracts suggest that the ATR and Chk1 protein kinases, which also function in the DNA-damage checkpoint, inhibit entry into mitosis by cells that have not completed DNA synthesis.

The association of ATR with replication forks is thought to activate its protein kinase activity, leading to the phosphorylation and activation of the Chk1 kinase. Active Chk1 then phosphorylates and inactivates the Cdc25 phosphatase (Cdc25C in vertebrates), which normally removes the inhibitory phosphate from CDKs that function during mitosis. As a result, the cyclin A/B-CDK1 complexes remain inhibited and cannot phosphorylate targets required to initiate mitosis. ATR continues to initiate this protein kinase cascade until all replication forks complete DNA replication and disassemble.

Improper Assembly of the Mitotic Spindle Prevents the Initiation of Anaphase

The spindle-assembly checkpoint prevents entry into anaphase when just a single kinetochore of one chromatid fails to associate properly with spindle microtubules. Clues about how this checkpoint operates has come from isolation of yeast mutants in the presence of benomyl, a microtubule-depolymerizing drug. Low concentrations of benomyl increase the time required for yeast cells to assemble the mitotic spindle and attach kinetochores to microtubules. Wild-type cells exposed to benomyl do not begin anaphase until these processes are completed and then proceed on through mitosis, producing normal daughter cells. In contrast, mutants defective in the spindle-assembly checkpoint proceed through anaphase before assembly of the spindle and attachment of kinetochores is complete; consequently, they mis-segregate their chromosomes, producing abnormal daughter cells that die.

Analysis of these mutants identified a protein called Mad2 and other proteins that regulate Cdc20, the specificity factor required to target the APC to securin (see Figure 21-32, |2). Recall that APC-mediated polyubiquitination of securin and its subsequent degradation is required for entry into anaphase (see Figure 21-19). Mad2 has been shown to associate with kinetochores that are unattached to microtubules. Experiments with Mad2 fused to green fluorescent protein (GFP) indicate that kinetochore-bound Mad2 rapidly exchanges with a soluble form of Mad2. Current models propose that when Mad2 associates with a kinetochore complex that is not bound by a microtubule, it is converted to a short lived activated form that can interact with and inhibit Cdc20. Microtubule attachment prevents this activation of Mad2. Consequently, once all kinetochore complexes bind a microtubule, generation of the activated form of Mad2 ceases, the inhibition of Cdc20 is relieved, and Cdc20 is free to direct the APC to polyubiquitinate securin, thereby initiating the onset of anaphase.

Proper Segregation of Daughter Chromosomes Is Monitored by the Mitotic Exit Network

Once chromosomes have segregated properly, telophase commences. The various events of telophase and subsequent cytokinesis, collectively referred to as the exit from mitosis, require inactivation of MPF. As discussed earlier, dephos-phorylation of the APC specificity factor Cdh1 by the Cdc14 phosphatase leads to degradation of mitotic cyclins and loss of MPF activity late in anaphase (see Figure 21-10). During interphase and early mitosis, Cdc14 is sequestered in the nu-cleolus and inactivated. The chromosome-segregation checkpoint, which monitors the location of the segregating daughter chromosomes at the end of anaphase, determines whether active Cdc14 is available to promote exit from mitosis (see Figure 21-32, [3).

Operation of this checkpoint in ,S. cerevisiae depends on a set of proteins referred to as the mitotic exit network. A key component is a small (monomeric) GTPase, called Tem1. This member of the GTPase superfamily of switch proteins controls the activity of a protein kinase cascade similarly to the way Ras controls MAP kinase pathways (Chapter 14). During anaphase, Tem1 becomes associated with the spindle pole body (SPB) closest to the daughter cell bud. (The SPB, from which spindle microtubules originate, is equivalent to the cen-trosome in higher eukaryotes.) At the SPB, Tem1 is maintained in the inactive GDP-bound state by a specific GAP (GTPase-accelerating protein). The GEF (guanosine nucleotide-exchange factor) that activates Tem1 is localized to the cortex of the bud and is absent from the mother cell. When spindle microtubule elongation at the end of anaphase has correctly positioned segregating daughter chromosomes into the bud, Tem1 comes into contact with its GEF and is converted into the active GTP-bound state. The terminal kinase in the cascade triggered by Tem1 • GTP then phosphorylates the nu-cleolar anchor that binds and inhibits Cdc14, releasing it into the cytoplasm and nucleoplasm in both the bud and mother cell (Figure 21-33, 1). Once active Cdc14 is available, a cell can proceed through telophase and cytokinesis. If daughter chromosomes fail to segregate into the bud, Tem1 remains in its inactive state, Cdc14 is not released from the nucleolus, and mitotic exit is blocked (Figure 21-33, 2).

In the fission yeast S. pombe, formation of the septum that divides daughter cells is regulated by proteins homologous to those that constitute the mitotic exit network in sS. cerevisiae. Genes encoding similar proteins also have been found in higher organisms where the homologs probably

▲ FIGURE 21-33 Operation of the chromosome-segregation checkpoint. In S. cerevisiae, Cdc14 phosphatase activity is required for the exit from mitosis. (Top) During interphase and early mitosis, Cdc14 is sequestered and inactivated in the nucleolus. Inactive Tem1 ■ GDP (purple) associates with the spindle pole body (SPB) nearest to the bud early in anaphase with the aid of a linker protein (green) and is maintained in the inactive state by a specific GAP (GTPase-accelerating protein). If chromosome segregation occurs properly (1), extension of the spindle microtubules inserts the daughter SPB into the bud, causing Tem1 to come in contact with a specific GEF (guanine nucleotide-exchange factor) localized to the cortex of the bud (brown). This GEF converts inactive Tim1 ■ GDP to active Tem1 ■ GTP which triggers a protein kinase cascade leading to release of active Cdc14 and exit from mitosis. If the spindle apparatus fails to place the daughter SPB in the bud ( 2|), Tem1 remains in the inactive GDP-bound state and Cdc14 remains associated with nucleoli. Arrest in late mitosis results. [Adapted from G. Pereira and E. Schiebel, 2001, Curr. Opin. Cell Biol. 13:762.]

function in an analogous checkpoint that leads to arrest in late mitosis when daughter chromosomes do not segregate properly.

Cell-Cycle Arrest of Cells with Damaged DNA Depends on Tumor Suppressors

The DNA-damage checkpoint blocks progression through the cell cycle until the damage is repaired. Damage to DNA can result from chemical agents and from irradiation with ultraviolet (UV) light or y-rays.

Arrest in G1 and S prevents copying of damaged bases, which would fix mutations in the genome. Replication of damaged DNA also promotes chromosomal rearrangements that can contribute to the onset of cancer. Arrest in G2 allows DNA double-stranded breaks to be repaired before mitosis. If a double-stranded break is not repaired, the broken distal portion of the damaged chromosome is not properly segregated because it is not physically linked to a centromere, which is pulled toward a spindle pole during anaphase.

As we discuss in detail in Chapter 23, inactivation of tumor-suppressor genes contributes to the development of cancer. The proteins encoded by several tumor-suppressor genes, including ATM and Chk2, normally function in the DNA-damage checkpoint. Patients with mutations in both copies of ATM or Chk2 develop cancers far more frequently than normal. Both of these genes encode protein kinases.

DNA damage due to UV light somehow activates the ATM kinase, which phosphorylates Chk2, thereby activating its kinase activity. Activated Chk2 then phosphorylates the Cdc25A phosphatase, marking it for polyubiquitination by an as-yet undetermined ubiquitin ligase and subsequent pro-teasomal degradation. Recall that removal of the inhibitory phosphate from mammalian CDK2 by Cdc25A is required for onset of and passage through the S phase mediated by cy-clin E-CDK2 and cyclin A-CDK2. Degradation of Cdc25A resulting from activation of the ATM-Chk2 pathway in G1 or S-phase cells thus leads to G1 or S arrest (see Figure 21-32, l4bl and l4cl). A similar pathway consisting of the protein kinases ATR and Chkl leads to phosphorylation and polyubiquitination of Cdc25A in response to y-radiation. As discussed earlier for the unreplicated-DNA checkpoint, Chk1 also inactivates Cdc25C, preventing the activation of CDK1 and entry into mitosis.

Another tumor-suppressor protein, p53, contributes to arrest of cells with damaged DNA. Cells with functional p53 arrest in G1 and G2 when exposed to y-irradiation, whereas cells lacking functional p53 do not arrest in Gj. Although the p53 protein is a transcription factor, under normal conditions it is extremely unstable and generally does not accumulate to high enough levels to stimulate transcription. The instability of p53 results from its polyubiquitination by a ubiquitin ligase called Mdm2 and subsequent proteasomal degradation. The rapid degradation of p53 is inhibited by ATM and probably ATR, which phosphorylate p53 at a site that interferes with binding by Mdm2. This and other modifications of p53 in response to DNA damage greatly increase its ability to activate transcription of specific genes that help the cell cope with DNA damage. One of these genes encodes p21CIP, a generalized CIP that binds and inhibits all mammalian cyclin-CDK complexes. As a result, cells are arrested in G1 and G2 until the DNA damage is repaired and p53 and subsequently p21CIP levels fall (see Figure 21-32, I4al-I4di).

Under some circumstances, such as when DNA damage is extensive, p53 also activates expression of genes that lead to apoptosis, the process of programmed cell death that normally occurs in specific cells during the development of multicellular animals. In vertebrates, the p53 response evolved to induce apoptosis in the face of extensive DNA damage, presumably to prevent the accumulation of multiple mutations that might convert a normal cell into a cancer cell. The dual role of p53 in both cell-cycle arrest and the induction of apoptosis may account for the observation that nearly all cancer cells have mutations in both alleles of the p53 gene or in the pathways that stabilize p53 in response to DNA damage (Chapter 23). The consequences of mutations in p53, ATM, and Chk2 provide dramatic examples of the significance of cell-cycle checkpoints to the health of a multi-cellular organism. I

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