As more mutant yeast were being screened for changes to their cell cycle, two other genes were found in which mutations gave rise to similar phenotypes. Nonfunctional cdc25 or overactive wee1 mutants generated the overly large cells with a single nucleus, and conversely, overactive cdc25 or inactive wee1 generated many severely undersized cells. Both cdc25 and wee1 gene products interact with cdk, and in fact, they are positive and negative regulators of cdk, respectively. Acting together with one more enzyme, CAK (cdk-activating kinase), they activate the cdk (fig. 3).
Figure 3. Activation of mitotic cyclin/cdk complex
Using the mitotic cyclin/cdk complex as an example, the cyclin (cdc13) and cdk (cdc2) come together to form an inactive complex. The cdk is then phosphorylated by wee1, a kinase. The phosphate it puts on tyrosine-15 is needed for the rest of the activation sequence, but it is inhibitory: it actually prevents final activation. But once Tyr-15 is phosphorylated, CAK can phosphorylate a neighboring threonine (Thr-161), which is required for activation. Finally, cdc25, a protein phosphatase, removes the phosphate on Tyr-15, allowing activation of the cdk by the phosphorylated Thr-161, and the MPF is finally on its way. There is self-amplification of the activation as well, because one of the targets of MPF is cdc25, so there is a positive feedback loop in which the activity of cdc25 is upregulated by phosphorylation.
As you will see in a later section of this chapter, MPF performs many functions, some of which prevent progress of mitosis past anaphase. Therefore, there must be a way to turn off MPF (and for that matter, any cyclin/cdk complex) quickly and completely when the cell reaches the appropriate stage of the cell cycle. This is borne out by time-course studies of MPF activity, which show a precipitous drop in activity in anaphase. This coincides with a depletion of the cyclin B (cdc13 in S. pombe) due to a combination of turning off transcription of the gene, and specific proteolytic degradation. The degradation pathway is now well understood, and is an interesting example of a sort of feedback regulation.
Figure 4. MPF activity and cyclin B protein expression rise as the cell enters mitosis but drop just before anaphase. However, cdk levels remain steady throughout the cell cycle.
Essentially, MPF ensures its own destruction: one of its phosphorylation targets is cdc20. Upon phosphorylation, cdc20 is activated and then activates anaphase promoting complex (APC). APC is a ubiquitin ligase (type E3) that polyubiquitinates the cyclin of the MPF complex, making it a target for proteolytic degradation by a proteosome. Note that only the cyclin is destroyed, while the kinase is left alone. Without the cyclin, the kinase is inactive and must wait for cyclin levels to rise again before it can be re- activated by a fresh round of phosphorylation and dephosphorylation.
Figure 5. MPF and other cyclin/cdk complexes are inactivated by destroying the cyclin.