Functional Delineation Of Kinetochore Motor Protein CENP-E With Its Binding Partners

Cell cycle is the universal process by which cells reproduce. It underlies the growth and development of all living organisms. The most important events of the cell cycle are those concerned with the duplicating and partitioning of the hereditary materials. In other words, that is replicating the genomic DNA during S phase and separating the replicated genomes into two daughter cells during mitosis. The onset of these events was regulated faithfully and delicately, and errors in their execution can and must be repaired to prevent catastrophic outcomes. The chromosome movements and separation during mitosis are mainly orchestrated by the interaction between the dynamic spindle microtubules and ldnetochore--a specialized chromosome domain located within the centromere.Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Our previous studies show that microtubule motor CENP-E represents a link between attachment of spindle microtubules and the mitotic checkpoint signaling cascade. However, the molecular function of CENP-E at the midbody had remained elusive. Here we show that CENP-E interacts with Skpi at the midbody and participates in cytokinesis. CENP-E interacts with Skpi in vitro and in vivo via its coiled-coil domain. Our yeast two-hybrid assays mapped the binding interfaces to the central stalk region of CENP-E (955-1571 aa) and the C-terminal 33 amino acids of Skpi, respectively. Our immunocytochemical studies revealed that CENP-E targets to the midbody prior to Skpi and the midbody localization of CENP-E becomes diminished as Skpi arrives at the midbody. Suppression of Skpi in mitotic HeLa cells by siRNA resulted in accumulation of telophase cells with elongated inter-cell bridges and with midbodies stretched 2-3 times longer than that of normal cells. These Skpi-eliminated or suppressed cells accumulate higher level of CENP-E, suggesting that spatiotemporal regulation of CENP-E degradation at the midbody is essential for cytokinesis. Over-expression of Skpi lacking the CENP-E-binding domain confirmed that Skp1-CENP-E interaction is essential for faithful cytokinesis. We hypothesize that CENP-E degradation is essential for faithful mitotic exit and the proteolysis of CENP-E is mediated by SCF via a direct Skpi link.CENP-E extends its motor domain on the spindle microtubule and anchors to kinetochore plate using its carboxyl terminus. To dissect the mechineary responsible for the CENP-E localization to the kinetochore, we conducted a search for proteins interacting with CENP-E C-terminus using a yeast two-hybrid assay, in which Nuf2, a core component of NDC80 comple, was identified by its ability to bind CENP-E. Further experiments such as Far-western and immunoprecipitation confirmed this interaction in vitro and in vivo. We also mapped the binding region to 341-424aa of Nuf2 by using GST pull down method. To explain the function of this novel interaction, we transfected siRNA of Nuf2, Hecl into Hela cells and found that CENP-E level was greatly reduced in Nuf2 depleted cells, while depletion of Hec1 can retain lower level CENP-E comparing with the control siRNA. To check the specificity of siRNA, we transfected GFP-tagged Nuf2 (281-462aa) into Nuf2 siRNA treated cells and found that CENP-E's fluorescence intensity on kinetochore was rescued in about 70% cells. To gain insight into the possible function of this interaction in stabilizing kinetochore-microtubule attachment, we performed more experiments and found that ACA distance which is a marker of kinetochore tension was shortened most in CENP-E and Nuf2 double depleted cells. This phenotype indicates that the interaction may helps CENP-E and Nuf2 collaborate to keep a stable kinetochore-microtubule attachment and also a tension between sister kinetochore which controls the spindle checkpoint.In summaiy, CENP-E functions with its accessory proteins to constitute the kinetochore-microtubule interface for orchestrating chromosome dynamics in mitosis. CENP-E is degraded by a two-step cascade during anaphase to cytokinesis.