E3 Ligase APC / C ^Cdh1 Mediated BRSK2 Through The Ubiquitin-proteasome Pathway For Periodic Degradation

Studies of brain-specific kinase2(BRSK2), an AMP-activated protein kinase (AMPK)-related kinase, and its homologs suggest that they are multifunctional regulators of cell-cycle progression. BRSK2, which contains a ubiquitin-associated (UBA) domain, is polyubiquitinated in cells. However, the regulatory mechanisms and exact biological function of BRSK2remain unclear. BRSK1, the homologue of BRSK2, regulates the activation of Cdkl by phosphorylating cyclin Cdc25B and Cdc25C, and thereby, ultimately inhibits cell cycle progression. Recent report showed that SADB (BRSK1) localizes to the centrosomes, and phosphorylates Ser131in y-tubulin, then controls the replication of centrosomes.Cells go smoothly through the process of the cell cycle requires a series of a complex signaling regulation pathway, including orderly completion of protelytic events mediated by phosphorylation and ubiquitination of regulators. Anaphase-promoting complex/cyclosome(APC/C) is required for the orderly progression of the cell cycle, two WD40-repeat proteins, Cdc20and Cdhl, function as substrate adaptors that activate APC/C and speciically recruit substrates having the D box or KEN box for ubiquitylation.Herein, we show that BRSK2co-localizes with the centrosomes during mitosis. We also demonstrate that BRSK2protein levels fluctuate during the cell cycle, peaking during mitosis and declining in G1phase. Furthermore, Cdhl, rather than Cdc20, promotes the degradation of BRSK2in vivo. Consistent with this finding, knock-down of endogenous Cdhl blocks BRSK2degradation during the G1phase. The conserved KEN box of BRSK2is required for anaphase-promoting complex/cyclosome-Cdhl (APC/CCdh1)-dependent degradation. Additionally, overexpression of either BRSK2(WT) or BRSK2(AKEN) increases the percentage of cells in G2/M. Thus, our results provide the first evidence that BRSK2regulates cell-cycle progression controlled by APC/CCdhl through the ubiquitin-proteasome pathway.In the second part of this paper, we study the biological function of a novel gene C19orf66. C19orf66belongs to UPF0515protein family, and the result of blasting shows that the C19orf66gene began to appear in the hemichordata phylum Saccoglossus kowalevskii. To date, research on the function of C19orf66is still very limited. Microarray data showed that C19orf66downregulates in the brain cancer, lung cancer and other tumors. Prompt the basis of the information, we began to carry out an initial functional studies on C19orf66. The C19orf66gene, which has2139base pairs in length and contains an open reading frame (ORF) encoding291amino acids, consists of7exons and6introns, and mapps to chromosome19p13.2. C19orf66was ubiquitously expressed in16human tissues by RT-PCR analysis. Subcellular localization of EGFP-C19orf66fusion protein revealed that C19orf66was distributed primarily in the cytoplasm of HeLa cells. Fingding after studying the cell signaling pathway by transfecting HEK293T cells with EGFP-C19orf66was that C19orf66significantly inhibited the activity of the Rb pathway. Further studies showed that either overexpression or interfering of C19orf66promoted apoptosis of HEK293T and HeLa cells.