Whereas activation of specific cyclin–Cdk complexes drives progression through the Start and G2/M transitions (see Figure 17–11), progression through the meta-phase-to-anaphase transition is triggered not by protein phosphorylation but by protein destruction, leading to the final stages of cell division.
The key regulator of the metaphase-to-anaphase transition is the anaphasepromoting complex, or cyclosome (APC/C), a member of the ubiquitin ligase family of enzymes. As discussed in Chapter 3, these enzymes are used in numerous cell processes to stimulate the proteolytic destruction of specific regulatory proteins. They polyubiquitylate specific target proteins, resulting in their destruction in proteasomes. Other ubiquitin ligases mark proteins for purposes other than destruction (discussed in Chapter 3).
Figure 17–12 The structural basis of Cdk activation. These drawings are based on three-dimensional structures of human Cdk2 and cyclin a, as determined by x-ray crystallography. The location of the bound aTp is indicated. The enzyme is shown in three states. (a) In the inactive state, without cyclin bound, the active site is blocked by a region of the protein called the T-loop (red). (B) The binding of cyclin causes the T-loop to move out of the active site, resulting in partial activation of the Cdk2. (C) phosphorylation of Cdk2 (by Cak) at a threonine residue in the T-loop further activates the enzyme by changing the shape of the T-loop, improving the ability of the enzyme to bind its protein substrates (Movie 17.1).
Figure 17–13 The regulation of Cdk activity by phosphorylation. The active cyclin–Cdk complex is turned off when the kinase Wee1 phosphorylates two closely spaced sites above the active site. Removal of these phosphates by the phosphatase Cdc25 activates the cyclin– Cdk complex. For simplicity, only one inhibitory phosphate is shown. Cak adds the activating phosphate, as shown in Figure 17–12.
Figure 17–14 The inhibition of a cyclin–Cdk complex by a CKI. This drawing is based on the three-dimensional structure of the human cyclin a–Cdk2 complex bound to the CkI p27, as determined by x-ray crystallography. The p27 binds to both the cyclin and Cdk in the complex, distorting the active site of the Cdk. It also inserts into the aTp-binding site, further inhibiting the enzyme activity.
The APC/C catalyzes the ubiquitylation and destruction of two major types of proteins. The first is securin, which protects the protein linkages that hold sister-chromatid pairs together in early mitosis. Destruction of securin in meta-phase activates a protease that separates the sisters and unleashes anaphase, as described later. The Sand M-cyclins are the second major targets of the APC/C. Destroying these cyclins inactivates most Cdks in the cell (see Figure 17–11). As a result, the many proteins phosphorylated by Cdks from S phase to early mitosis are dephosphorylated by various phosphatases in the anaphase cell. This dephosphorylation of Cdk targets is required for the completion of M phase, including the final steps in mitosis and then cytokinesis. Following its activation in mid-mitosis, the APC/C remains active in G1 to provide a stable period of Cdk inactivity. When G1/S-Cdk is activated in late G1, the APC/C is turned off, thereby allowing cyclin accumulation in the next cell cycle.
The cell-cycle control system also uses another ubiquitin ligase called SCF (see Figure 3–71). It has many functions in the cell, but its major role in the cell cycle is to ubiquitylate certain CKI proteins in late G1, thereby helping to control the activation of S-Cdks and DNA replication. SCF is also responsible for the destruction of G1/S-cyclins in early S phase.