oscillation: cyclin-B/CDK activates APC/C, which causes degradation of cyclin-B from metaphase onwards, restoring CDK1 to its inactive state. However, simple negative feedback loops lead to damped oscillations that eventually settle on a steady state. Kinetic models show that negative feedback loops coupled with bistable positive feedback motifs can lead to persistent, non-damped oscillations (see relaxation oscillator) of the kind required for long-term cell cycling. Positive feedback The positive feedback loop mentioned above, in which cyclin-B1/CDK1 promotes its own activation by inhibiting Wee1 and Myst1 and activating cdc25, does not inherently include a “trigger” mechanism to initiate the feedback loop. Recently, evidence has emerged suggesting a more important role for cyclin A2/CDK complexes in regulating the initiation of this switch. Cyclin A2/CDK2 activity begins in early S phase and increases during G2. Cdc25B has been shown to dephosphorylate Tyr15 on CDK2 in early-to-mid G2 in a manner similar to the aforementioned CDK1 mechanism. Downregulation of cyclin A2 in U2OS cells delays cyclin-B1/CDK1 activation by increasing Wee1 activity and lowering Plk1 and Cdc25C activity. However, cyclin A2/CDK complexes do not function strictly as activators of cyclin B1/CDK1 in G2, as CDK2 has been shown to be required for activation of the p53-independent G2 checkpoint activity, perhaps through a stabilizing phosphorylation on Cdc6. CDK2-/- cells also have aberrantly high levels of Cdc25A. Cyclin A2/CDK1 has also been shown to mediate proteasomal destruction of Cdc25B. These pathways are often deregulated in cancer. Spatial regulation In addition to the bistable and hysteretic aspects of cyclin B1-CDK1 activation, regulation of subcellular protein localization also contributes to the G2/M transition. Inactive cyclin B1-CDK1 accumulates in the cytoplasm, begins to be activated by cytoplasmic cdc25, and then is rapidly sequestered into the nucleus during prophase (as it is further activated). In mammals, cyclin B1/CDK1 translocation to the nucleus is activated by phosphorylation of five serine sites on cyclin B1's cytoplasmic retention site (CRS): S116, S26, S128, S133, and S147. In Xenopus laevis, cyclin B1 contains four analogous CRS serine phosphorylation sites (S94, S96, S101, and S113) indicating that this mechanism is highly conserved. Nuclear export is also inactivated by phosphorylation of cyclin B1's nuclear export signal (NES). The regulators of these phosphorylation sites are still largely unknown but several factors have been identified, including extracellular signal-regulated kinases (ERKs), PLK1, and CDK1 itself. Upon reaching some threshold level of phosphorylation, translocation of cyclin B1/CDK1 to the nucleus is extremely