▲ FIGURE 21-30 Regulation of Rb and E2F activities in mid-late G1. Stimulation of G0 cells with mitogens Induces expression of CDK4, CDK6, D-type cyclins, and the E2F transcription factors, all encoded by delayed-response genes. Rb protein initially inhibits E2F activity. When signaling from mitogens is sustained, the resulting cyclin D-CDK4/6 complexes begin phosphorylating Rb, releasing some E2F, which stimulates transcription of the genes encoding cyclin E, CDK2, and E2F (autostimulation). The cyclin E-CDK4 complexes further phosphorylate Rb, resulting in positive feedback loops (blue arrows) that lead to a rapid rise in the expression and activity of both E2F and cyclin E-CDK2 as the cell approaches the G-i ^ S transition.

Rb protein is one of the most significant substrates of mammalian G1 cyclin-CDK complexes. Phosphorylation of Rb protein at multiple sites prevents its association with E2Fs, thereby permitting E2Fs to activate transcription of genes required for entry into S phase. As shown in Figure 21-30, phosphorylation of Rb protein is initiated by cyclin D-CDK4 and cyclin D-CDK6 in mid G1. Once cyclin E and CDK2 are induced by phosphorylation of some Rb, the resulting cyclin E-CDK2 further phosphorylates Rb in late G1. When cyclin E-CDK2 accumulates to a critical threshold level, further phosphorylation of Rb by cyclin E-CDK2 continues even when cyclin D-CDK4/6 activity is removed. This is one of the principle biochemical events responsible for passage through the restriction point. At this point, further phosphorylation of Rb by cyclin E-CDK2 occurs even when mitogens are withdrawn and cyclin D and CDK4/6 levels fall. Since E2F stimulates its own expression and that of cyclin E and CDK2, positive cross-regulation of E2F and cyclin E-CDK2 produces a rapid rise of both activities in late G1.

As they accumulate, S-phase cyclin-CDK and mitotic cyclin-CDK complexes maintain Rb protein in the phospho-rylated state throughout the S, G2, and early M phases. After cells complete anaphase and enter early G1 or G0, the fall in cyclin-CDK levels leads to dephosphorylation of Rb by unopposed phosphatases. As a consequence, hypophosphory-lated Rb is available to inhibit E2F activity during early G1 of the next cycle and in G0-arrested cells.

Cyclin A Is Required for DNA Synthesis and CDK1 for Entry into Mitosis

High levels of E2Fs activate transcription of the cyclin A gene as mammalian cells approach the G1 ^ S transition. (Despite its name, cyclin A is a B-type cyclin, not a G1 cyclin;

see Table 21-1.) Disruption of cyclin A function inhibits DNA synthesis in mammalian cells, suggesting that cyclin A-CDK2 complexes may function like S. cerevisiae S-phase cyclin-CDK complexes to trigger initiation of DNA synthesis. There is also evidence that cyclin E-CDK2 may contribute to activation of pre-replication complexes.

Three related CDK inhibitory proteins, or CIPs (p27KIP1, p57KIP2, and p21CIP), appear to share the function of the sS. cerevisiae S-phase inhibitor Sic1 (see Figure 21-24). Phosphorylation of p27KIP1 by cyclin E-CDK2 targets it for poly-ubiquitination by the mammalian SCF complex (see Figure 21-2, step 5). The SCF subunit that targets p27KIP1 is synthesized as cells approach the G1 ^ S transition. The mechanism for degrading p21CIP and p57KIP2 is less well understood. The activity of mammalian cyclin-CDK2 complexes is also regulated by phosphorylation and dephosphorylation mechanisms similarly to those controlling the ,S. pombe mitosis-promoting factor, MPF (see Figure 21-14). The Cdc25A phosphatase, which removes the inhibitory phosphate from CDK2, is a mammalian equivalent of ,S. pombe Cdc25 except that it functions at the G1 ^ S transition rather than the G2 ^ M transition. The mammalian phosphatase normally is activated late in G1, but is degraded in the response of mammalian cells to DNA damage to prevent the cells from entering S phase (see Section 21.7).

Once cyclin A-CDK2 is activated by Cdc25A and the S-phase inhibitors have been degraded, DNA replication is initiated at pre-replication complexes. The general mechanism is thought to parallel that in ,S. cerevisiae (see Figure 21-26), although small differences are found in vertebrates. As in yeast, phosphorylation of certain initiation factors by cyclin A-CDK2 most likely promotes initiation of DNA replication and prevents reassembly of pre-replication complexes until the cell passes through mitosis, thereby assuring that replication from each origin occurs only once during each cell cycle. In metazoans, a second small protein, gemi-nin, contributes to the inhibition of re-initiation at origins until cells complete a full cell cycle.

The principle mammalian CDK in G2 and mitosis is CDK1 (see Figure 21-28). This CDK, which is highly homologous with sS. pombe Cdc2, associates with cyclins A and B. The mRNAs encoding either of these mammalian cyclins can promote meiotic maturation when injected into Xenopus oocytes arrested in G2 (see Figure 21-6), demonstrating that they function as mitotic cyclins. Thus mammalian cyclin A-CDK1 and cyclin B-CDK1 are functionally equivalent to the S. pombe MPF (mitotic cyclin-CDK). The kinase activity of these mammalian complexes also appears to be regulated by proteins analogous to those that control the activity of the S. pombe MPF (see Figure 21-14). The inhibitory phosphate on CDK1 is removed by Cdc25C phosphatase, which is analogous to ,S. pombe Cdc25 phosphatase.

In cycling mammalian cells, cyclin B is first synthesized late in the S phase and increases in concentration as cells proceed through G2, peaking during metaphase and dropping after late anaphase. This parallels the time course of cyclin B expression in Xenopus cycling egg extracts (see Figure 21-9).

In human cells, cyclin B first accumulates in the cytosol and then enters the nucleus just before the nuclear envelope breaks down early in mitosis. Thus MPF activity is controlled not only by phosphorylation and dephosphorylation but also by regulation of the nuclear transport of cyclin B. In fact, cyclin B shuttles between the nucleus and cytosol, and the change in its localization during the cell cycle results from a change in the relative rates of import and export. As in Xenopus eggs and ,S. cerevisiae, cyclins A and B are poly-ubiquitinated by the anaphase-promoting complex (APC) during late anaphase and then are degraded by proteasomes (see Figure 21-2, step 9).

Two Types of Cyclin-CDK Inhibitors Contribute to Cell-Cycle Control in Mammals

As noted above, three related CIPs—p21CIP, p27KIP2, and p57KIP2—inhibit cyclin A-CDK2 activity and must be degraded before DNA replication can begin. These same CDK inhibitory proteins also can bind to and inhibit the other mammalian cyclin-CDK complexes involved in cell-cycle control. As we discuss later, p21CIP plays a role in the response of mammalian cells to DNA damage. Experiments with knockout mice lacking p27KIP2 have shown that this CIP is particularly important in controlling generalized cell proliferation soon after birth. Although p27KIP2 knockouts are larger than normal, most develop normally otherwise. In contrast, p57KIP2 knockouts exhibit defects in cell differentiation and most die shortly after birth due to defective development of various organs.

A second class of cyclin-CDK inhibitors called INK4s (inhibitors of Ainase 4) includes several small, closely related proteins that interact only with CDK4 and CDK6 and thus function specifically in controlling the mid-G1 phase. Binding of INK4s to CDK4/6 blocks their interaction with cyclin D and hence their protein kinase activity. The resulting decreased phosphorylation of Rb protein prevents transcrip-tional activation by E2Fs and entry into the S phase. One INK4 called p16 is a tumor suppressor, like Rb protein discussed earlier. The presence of two mutant p16 alleles in a large fraction of human cancers is evidence for the important role of p16 in controlling the cell cycle (Chapter 23).

■ Mammalian cells use several CDKs and cyclins to regulate passage through the cell cycle. Cyclin D-CDK4/6 function in mid to late G1; cyclin E-CDK2 in late G1 and early S; cyclin A-CDK2 in S; and cyclin A/B-CDK1 in G2 and M through anaphase (see Figure 21-28).

■ Unphosphorylated Rb protein binds to E2Fs, converting them into transcriptional repressors. Phosphorylation of Rb by cyclin D-CDK4/6 in mid G1 liberates E2Fs to activate transcription of genes encoding cyclin E, CDK2, and other proteins required for the S phase. E2Fs also auto-stimulate transcription of their own genes.

■ Cyclin E-CDK2 further phosphorylates Rb, further activating E2Fs. Once a critical level of cyclin E-CDK2 has been expressed, a positive feedback loop with E2F results in a rapid rise of both activities that drives passage through the restriction print (see Figure 21-30).

■ The activity of cyclin A-CDK2, induced by high E2F activity, initially is held in check by CIPs, which function like an S-phase inhibitor, and by the presence of an inhibitory phosphate on CDK2. Proteasomal degradation of the inhibitors and activation of the Cdc25A phosphatase, as cells approach the G1 ^ S transition, generate active cyclin A-CDK2. This complex activates pre-replication complexes to initiate DNA synthesis by a mechanism similar to that in S. cerevisiae (see Figure 21-26).

■ Cyclin A/B-CDK1 induce the events of mitosis through early anaphase. Cyclins A and B are polyubiquitinated by the anaphase-promoting complex (APC) during late anaphase and then are degraded by proteasomes.

■ The activity of mammalian mitotic cyclin-CDK complexes also are regulated by phosphorylation and dephos-phorylation similar to the mechanism in ,S. pombe, with the Cdc25C phosphatase removing inhibitory phosphates (see Figure 21-14).

■ The activities of mammalian cyclin-CDK complexes also are regulated by CDK inhibitors (CIPs), which bind to and inhibit each of the mammalian cyclin-CDK complexes, and INK4 proteins, which block passage through G1 by specifically inhibiting CDK4 and CDK6.

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