H

CAK. Cdc25A

Cyclin B Cdk1

CAK, weel

Cdc25B/C

Figure 16.1. The human cell cycle. The cell cycle is divided into four phases: Gj, S, G2, and M. Cyclin D-Cdk4/6 controls early Gj, cyclin E-Cdk2 controls late Gj, cyclin A-Cdk2/1 controls S and G2, and cyclin B-cdk1 controls G2 and entry into M phase. Activators and inhibitors of these cyclin-cdk complexes are shown. G0 is a state defined by quiescent, nondividing cells that have exited the cell cycle.

tion of the Cip/Kip inhibitors. The production of robust cyclin E/Cdk2 activity supplants cyclin D/Cdk4/6 activity as the driving force of progression from G¡ phase to S phase.4 At this point, the cell has passed the restriction point, as cyclin D/Cdk4/6 activity is no longer required. Cyclin E/Cdk2 serves to hyperphosphorylate pRb, completely removing its inhibition of E2F, allowing transcription of genes required for S phase, including cyclin A, the S-phase cyclin.

Cyclin A, similar to the D-type cyclins, can bind two distinct cdks, Cdk2 and Cdk1. Currently, any differences in the function between the two cyclin A-Cdk complexes are unknown. The active cyclin A-Cdk2/1 complex has a nuclear localization and is thought to control DNA replication, controlling initiation and restricting replication to once per cell cycle, by phosphorylation of substrates. RPA, Mcm4, and the DNA polymerase a-primase are known cyclin A substrates that are involved in DNA replication.5 In addition to a role in regulating DNA replication, cyclin A plays a key role in allowing cell-cycle progression through G2 phase into M.

During S phase, cyclin B levels begin to increase, and by G2 phase, two cdk complexes, cyclin A-Cdk 1 and cyclin B-Cdk1, are active. The accumulation of active cyclin B/Cdk1 is strictly dependent on cyclin A/Cdk1 activity. During the S and G2 phases, cyclin A/Cdk1 phosphorylates Cdh1, preventing it from targeting cyclin B to the anaphase-promoting complex (APC) for ubiquitination and degradation.5 Active cyclin B/Cdk1 first accumulates in the cytoplasm, where it is thought to prepare structural components of the cell for the upcoming cell division. Cyclin B/cdk1 activity reorganizes the cell's microtubules and microfilaments, and phosphorylates proteins in the nuclear lamina, resulting in this structure's breakdown.6 Just before the breakdown of the nuclear membrane, cyclin B/Cdk1 translocates to the nucleus to target further substrates, including those that control the shutdown of RNA polymerase III-mediated transcription.6 Finally, cyclin B and cyclin A are rapidly degraded by the APC before the end of mitosis. The APC also mediates the transition from metaphase to anaphase by degrading securin, the inhibitor of separase. Separase then proceeds to cleave Sccl, a protein found in the cohesin complex of proteins that holds the newly replicated chromosomes together, allowing anaphase to ensue.

In addition to being regulated by their cyclin partners, mammalian cdks are subject to regulation by a number of different inhibitors throughout the cell cycle. These inhibitors can arrest or slow the cell cycle in response to many signals including nutrient depletion, failures in DNA replication, and DNA damage. During the G1 to S transition, two distinct inhibitor classes function to inhibit Cdk4/6 and Cdk2 activity. The INK4 family of proteins (p16INK4A, p15INK4B, p18INK4C, and p19INK4D) are noncompetitive inhibitors of Cdk4 and Cdk6, binding to these kinases and causing allosteric changes that weaken binding to both cyclin D and adenosine triphosphate (ATP).4 With the exception of p19INK4D, the expression of the INK4 family does not appear to be cell-cycle dependent, and p16INK4A, which appears to be the most important of the INK4 proteins, has been shown to increase with age and population doubling. However, the exact mechanism of regulation of this family of proteins remains under investigation.

The Cip/Kip family of cdk inhibitors, which includes p21, p27, and p57, also function at the G1 to S transition. Cip/Kip inhibitors can inhibit multiple cyclin-cdk complexes with varying efficiencies, but, important for the G1 to S transition, as already mentioned, they inhibit cyclin E/Cdk2 strongly while inhibiting cyclin D/Cdk4/6 poorly.4 When bound to the cyclin E-Cdk2 complex, the Cip/Kip proteins bind the catalytic site of the cdk, blocking ATP from binding. In addition to being inactivated by titration away from cyclin E-Cdk2 complexes by cyclin D/Cdk4/6, p27 is subject to ubiquitin-mediated degradation during S phase. Although the regulation of all the Cip/Kip proteins is not completely understood, the gene for p21, CDKN1A, in particular, is subject to upregula-tion by the p53 tumor suppressor (see following) in response to DNA damage, allowing p53 to block cell-cycle progression.3,7-9 p21 is a particularly potent protein for inducing cell-cycle arrests, as it can also inhibit cyclin A/Cdk2 and, indirectly, cyclin A/cyclin B/Cdk1.6

During the S, G2, and M phases of the cell cycle, cyclin-cdk complexes containing Cdk2 and Cdk1 are also regulated by activating and inhibiting phosphorylations. All the cdks require an activating phosphorylation provided by a Cdk-activating kinase (CAK) composed of cyclin H, Cdk7,

Figure 16.2. Inactivation of pRb by cyclin-Cdk complexes in G1. Phosphorylation of pRb by cyclin D/Cdk4/6 in early G1 partially inactivates pRb, allowing some E2F-mediated transcription. Hyperphos-phorylation of pRb by cyclin E-sCdk2 in late G1 phase completely inactivates pRb, allowing uninhibited E2F-mediated transcription.

Figure 16.2. Inactivation of pRb by cyclin-Cdk complexes in G1. Phosphorylation of pRb by cyclin D/Cdk4/6 in early G1 partially inactivates pRb, allowing some E2F-mediated transcription. Hyperphos-phorylation of pRb by cyclin E-sCdk2 in late G1 phase completely inactivates pRb, allowing uninhibited E2F-mediated transcription.

and MAT1.5,6 Because the cyclin H-Cdk7 complex can be inhibited by the Cip/Kip inhibitor p21, p21 upregulation by DNA damage can induce a G1 arrest by direct inhibition of cyclin E/Cdk2 and a G2-M arrest by indirect inhibition of cyclin B/A/Cdkl via cyclin H/Cdk7 inactivation. However, the most potent control over Cdk2 and Cdkl activity comes from the inhibitory phosphorylation of two amino-terminal tyrosine residues by the Weel kinase.6 Phosphorylation of these residues blocks not only the ability of the kinase to transfer phosphate but also the ability to bind ATP. Removal of these inhibitory phosphorylations is accomplished by the actions of a Cdc25 phosphatase member. Cdc25A specifically dephosphorylates Cdk2 and Cdc25B and Cdc25C specifically dephosphorylates Cdkl.6,10 The effects of Cdc25B and Cdc25C on Cdk1 are additive, and after activation by Cdc25B, cyclin B/Cdk1 activates Cdc25C by phosphorylation in a positive feedback loop. By regulating the levels and activities of Wee1 kinase activity and Cdc25 phosphatase activity, the cell can stop or promote cell-cycle progression as required. For example, the detection of DNA damage during S phase results in the rapid degradation of Cdc25A and increased expression of Wee1, causing a cell-cycle arrest.10

Finally, cell-cycle progression can be blocked by other pathways not directly involving cdks and cdk inhibitors. The mitotic spindle checkpoint monitors the proper attachment of chromosomes to the mitotic spindle and the proper alignment of these chromosomes along the metaphase plate. Failures in the mitotic spindle activate a mitotic checkpoint complex (MCC) that includes Mad2, BubR1, and Bub3. Mad2 and BubR1 both can bind and inhibit Cdc20, the subunit of the APC that targets securin, as well as cyclin B, for degra-dation.11 Inhibition of APC-mediated degradation of securin prevents cleavage of the cohesin complex holding replicated sister chromosomes together, preventing mitosis.

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