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(quiescence)

CDKl/cyclinB/M earlyX CDK4,6/D-cyclins

CDKl/cyclin A \G2 M CDK2/cyclin E

CDK2/cyclin A

figure 9.1 General schematic of the cell cycle. The cell cycle can be broken down into four distinct phases: Gap 1 (G1), DNA synthesis (S), Gap 2 (G2), and mitosis (M). The phase-specific CDK/ cyclin complexes responsible for cell cycle regulation are indicated.

pointed to protein degradation (proteolysis) as a key regulator of their expression [2]. It was discovered that the cyclins become covalently bound to ubiquitin moieties which target the proteins for degradation via the proteasome, a large, compartmentalized protease complex that destroys ubiquitinated substrates (Fig. 9.2) (reviewed in [3]). In mammalian cells, the cyclins can be divided into the G1 cyclins (D-type cyclins 1-3, cyclin E), the S-phase cyclins (cyclins A, E) and the mitotic cyclins (cyclins A, B) (Fig. 9.1). Cyclin-Dependent Kinases (CDKs) require cyclin binding in order to form enzymatically active hetero-dimeric complexes competent for substrate phospho-rylation (addition of phosphate moieties). As cyclin levels are diminished, CDKs lose catalytic activity and are no longer capable of substrate phosphorylation. CDK substrates include the retinoblastoma protein (pRb), a key effector protein that mediates progression beyond the RP. Protein modification events such as phosphorylation (achieved by kinases) and de-phosphorylation (achieved by phosphatases) are critical regulatory mechanisms responsible for the activation or inactivation of proteins that control entry into and progression through the cell cycle, targeting proteins for degradation while activating others in order to maintain cell cycle integrity. As is the case with the cyclins, CDKs can be grouped according to their roles in the various phases of the cell cycle: the G1 CDKs (CDK4, CDK6, CDK2), the S-phase CDKs (CDK2) and the M-phase CDKs (CDK2, CDK1).

An additional level of cell cycle regulation is achieved by CDK Inhibitors (CDKIs). CDKIs are capable of abrogating catalytic kinase activity by binding to CDKs or CDK/cyclin complexes. There are two main families of CDKIs: the INhibitor of CDK4 (INK4) family of inhibitors and the CDK

1 j^j proteasome mediated degradation of substrate figure 9.2 The ubiquitin/proteasome-mediated degradation pathway. The ubiquitin conjugating enzymes E1, E2, and E3 sequentially transfer a ubiquitin moiety (Ub) (or polyubiquitin chain) from one enzyme to the next followed by transfer of the Ub chain from E2 to the substrate. The polyubiquitinated product is targeted for destruction by the large, self-compartmentalized protease known as the 26S proteasome. The 19S subunit of the proteasome (regulatory subunit) is responsible for substrate recognition, removal of Ub and substrate unfolding. Free Ub is recycled. The 20S subunit (protease subunit) is responsible for substrate degradation. See Plate 9.2 in Color Plate Section.

1 j^j proteasome mediated degradation of substrate figure 9.2 The ubiquitin/proteasome-mediated degradation pathway. The ubiquitin conjugating enzymes E1, E2, and E3 sequentially transfer a ubiquitin moiety (Ub) (or polyubiquitin chain) from one enzyme to the next followed by transfer of the Ub chain from E2 to the substrate. The polyubiquitinated product is targeted for destruction by the large, self-compartmentalized protease known as the 26S proteasome. The 19S subunit of the proteasome (regulatory subunit) is responsible for substrate recognition, removal of Ub and substrate unfolding. Free Ub is recycled. The 20S subunit (protease subunit) is responsible for substrate degradation. See Plate 9.2 in Color Plate Section.

Inhibitor Protein/Kinase Inhibitor Protein (CIP/KIP) family of inhibitors. INK4 inhibitors specifically inhibit CDK4 and CDK6 activity during G1 to elicit a G1/S arrest whereas the CIP/KIP proteins are capable of inhibiting all CDK/cyclin complexes [4]. INK4 and CIP/KIP proteins are mechanistically different in that INK4 CDKIs only bind to CDKs while CIP/KIP proteins bind to both CDK and cyclin molecules simultaneously, stabilizing the proteins in a ternary complex.

Recently, a complex critical to M-phase progression was discovered. The Anaphase Promoting Complex (APC) is an E3 ubiquitin ligase complex that triggers the timed, sequential ubiquitination and proteolysis of mitotic regulators such as cyclin A, securin, and cyclin B (reviewed in [5]). In much the same way that proteasomes mediate degradation of cyclins in a timely manner, the APC coordinates the destruction of proteins involved in both entry into and exit from mitosis. Activation of the APC in late S-phase initiates a signaling cascade that enables sister chromatid separation. Subsequent binding of the APC to another regulatory molecule directs exit from mitosis.

The molecular associations amongst regulators of the cell cycle and the critical protein modification events required for cycling will be discussed below. Genetic analyses of primary human brain tumors have revealed common mutations in genes encoding proteins critical for cell cycle regulation. Such observations indicate the importance of tight control of the cell cycle in maintaining normal cellular cycling and appropriate quiescence. Dysregulation of the cell cycle in cancer has researchers looking for new ways to inhibit cell cycle progression. A number of small molecular inhibitors that target CDK inactiva-tion have been investigated for use in clinical trials. Current research is also exploring the use of protea-some inhibitors to block the relentless cellular division associated with cancer cells. Some of the recent pharmacological interventions and their preclinical and clinical results are presented here.

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