The accurate regulation of phosphorylation at the kinetochore is essential for establishing chromosome bi-orientation. Phosphorylation of kinetochore proteins by the Aurora B kinase destabilizes improper kinetochore–microtubule attachments, whereas the phosphatase PP2A has a counteracting role. Imbalanced phosphoregulation leads to error-prone chromosome segregation and aneuploidy, a hallmark of cancer cells. However, little is known about the molecular events that control the balance of phosphorylation at the kinetochore. Here, we show that localization of SET/TAF1, an oncogene product, to centromeres maintains Aurora B kinase activity by inhibiting PP2A, thereby correcting erroneous kinetochore–microtubule attachment. SET localizes at the inner centromere by interacting directly with shugoshin 2, with SET levels declining at increased distances between kinetochore pairs, leading to establishment of chromosome bi-orientation. Moreover, SET overexpression induces chromosomal instability by disrupting kinetochore–microtubule attachment. Thus, our findings reveal the novel role of SET in fine-tuning the phosphorylation level at the kinetochore by balancing the activities of Aurora B and PP2A.

During mitosis, spatiotemporal regulation of phosphorylation at the kinetochore is essential for accurate chromosome alignment and proper chromosome segregation. Aurora B kinase phosphorylates kinetochore substrates to correct improper kinetochore-microtubule (KT-MT) attachments, whereas tension across the centromeres inactivates Aurora B kinase, and PP2A phosphatase dephosphorylates the kinetochore proteins to stabilize the attachments. However, the molecular entity of the tension sensing mechanism remains elusive. In a previous report, we showed that centromeric SET/TAF1 on Sgo2 up-regulates Aurora B kinase activity via PP2A inhibition in prometaphase. Here we show that Aurora B and Bub1 at the centromere/kinetochore regulate both kinase activities one another in an inter-kinetochore distance-dependent manner, indicating a positive feedback loop. We further show that the centromeric pool of SET on Sgo2 depends on Bub1 kinase activity, and the centromeric localization of SET decreases in a distance-dependent manner, thereby inactivating Aurora B in metaphase. Consistently, ectopic targeting of SET to the kinetochores during metaphase hyperactivates Aurora B via PP2A inhibition, and thereby rescues the feedback loop. Thus, we propose that SET, Aurora B and Bub1 form a distance-dependent positive feedback loop, which spatiotemporally may act as a tension sensor at centromeres.

The TWIK-related potassium channel-1 (TREK-1) is a member of two-pore domain potassium (K2P) channel family that plays an essential part in the regulation of resting membrane potential and cellular excitability. TREK-1 is involved in many physiological processes, such as chronic pain, epilepsy and overactive bladder. TREK-1 is activated downstream of mu receptor and involved in the antinociceptive activity of morphine without its adverse side effects. We discovered novel and potent TREK-1 activators, NCC-0768 and NCC-0850, which activated human TREK-1 channel and their EC 50 were 1.2 μM and 0.51 μM, respectively. EC 50 of BL-1249, a well-known TREK-1 activator, was 9.1 μM in our assay. TREK-1 is expressed in dorsal root ganglion (DRG) neurons that are associated pain perception. We investigated membrane potential changes in rat DRG neurons. NCC compounds showed hyperpolarization shift and decreased cell excitability. Oral administration of NCC-0768 and NCC-0850 dosedependently suppressed mouse phenyl benzoquinone-induced writhing behavior. Furthermore, NCC-0768 had antiallodynic effect in rat neuropathic pain induced by chronic constriction injury. The efficacy of NCC-0768 was equivalent to the pregabalin at 10 mg/kg. Our novel TREK-1 activator, NCC-0768, has a potent analgesic effect on neuropathic pain model, and our results suggest that TREK-1 activator may be an attractive anti-pain agent. In addition, TREK-1 activator can be expected to be applied to the other neuroexcitatory diseases.