Regulatory Role Of Aurora B Kinase In Cell Cycle Progression In Silkworm

Aurora B kinase,which belongs to the serine/threonine kinase family,is the catalytic subunit of the chromosomal passenger complex(CPC).Studies in mammal have shown that Aurora B kinase is an essential regulatory for chromosome alignment,chromosome condensation,sister chromosomes segregation,and cytokinesis during mitosis.To date,numerous studies have reported that Aurora B kinase regulates cell cycle progression in mammal.However,few study was focused on the model organism silkworm.In this study,we cloned the full-length cDNA sequence of silkworm Aurora B kinase(BmAurB)gene,analyzed the spatio-temporal expression profiling and verified BmAurB function in the silkworm cell cycle using RNAi,and identified the potential targets for BmAurB kinase using labeling quantitative phosphoproteomics.The main results were as follows: 1.Full-length cDNA cloning and spatio-temporal expression profiling of the silkworm BmAurB geneWe performed a homology search against silkworm genome database SilkDB by using the amino acid sequence of Drosophila Aurora B as a qurey.As a result,a predicted gene,namely BGIBMGA002350,shares a high identity to Drosophila Aurora B.We further applied 3' and 5' RACE method to clone the full-length cDNA of the BmAurB gene from silkworm ovary cDNA and acquired a GenBank accession number:KX420635.The full-length cDNA of the BmAurB gene was 1090 bp,and the opening read frame(ORF)in the region of 85-135 bp,coding 289 aino acides.By aligning the full-length cDNA sequence of the BmAurB gene on the assembly silkworm genome,we found that the BmAurB gene has six exons and five introns.We predicted that BmAurB gene includes a conserved S_TKc domain of Aurora B kinases using SMART online progarm.We further built a phylogenetic tree of Aurora kinases form silkworm and other species.As a result,the BmAurB gene and all other insect Aurora B kinase genes grouped together.All insect Aurora A kinase genes were also classified into a group.Although Aurora kinases from human and mouse grouped well together before grouping with insect Aurora kinases,the orthologs of Aurora A grouped together,and the same is as Aurora B or C.This indicates that the separation of Aurora kinases may have occurred after separation between mammalian and insect,and prior to radiation of mammalian or insects.For profiling the temporal expression pattern of the BmAurB gene,we investigated the BmAurB mRNA level in multiple tissues of silkworm larvae on day 3 of the fifth larval instar(L5D3)by using quantitative RT-PCR examination.The results demonstrated that the BmAurB gene was highly expressed in gonads(ovary and testis),and exhibited a low expression in head,hemocyte,and integument in L5D3.We further investigated the developmental expression of the BmAurB gene in two tissues of silkworm larvae,including brain with mitotic cycle and silk gland with endocycle.RT-PCR examination showed that the expression of the BmAurB gene could be detected in brain during feeding stage of the fourth larval instar(from L4D0 to L4D3)as well as day 1 and day 4 of the fifth larval instar(L5D1 and L5D4).However,the BmAurB gene was not expressed in silk gland at any time point during silkworm larval development.It suggests loss expression of BmAurB gene may be one of the influences on silk gland entrancing endocycle.2.The function of BmAurB kinase in silkworm cell cycleWe investigated the roles of the BmAurB kinase in BmN4-SID1 cells by using Aurora B RNAi and Aurora B kinase inhibitor.As a result,Aurora B RNAi and Aurora B kinase inhibitor treament not only caused an accumulation of polyploid cells having DNA content of >4N in BmN4-SID1 cells but also cell cycle arrest at G2/M phase.Further immunostaining analysis revealed that decrease of BmAurB activity led to a mitotic catastrophe in BmN4-SID1 cells,such as chromosome misalign-ment in metaphase,chromosome bridges in anaphase,and bi-nucleation induced by cytokinesis failure in interphase..Moreover,western blot analysis demonstrated that protein levels of both cyclin-dependent kinase 1(CDK1)as a key regulator on G2/M transition and the phosphorylated histone H3(pH3)as a mitotic marker were also reduced.The results showed that the reduction BmAurB kinase activity seriously impeded cell cycle procession in silkworm.We also overexpressed BmAurB gene in silkworm embryonate-derived BmE cells.Flow cytometry analysis demonstrated that BmAurB gene overexpression reduces the cell counts of G2 and M phase.Western blot analysis revealed that the protein levels of both pH3 and CDK1 was up-regulated by BmAurB gene overexpression,which further suggested that BmAurB played a regulated function of cell cycle progression in silkworm.3.Identification of the potential targets for Aurora B kinaseTo identify new potential targets for Aurora B kinase,we performed an isotope labelled quantitative phosphoproteomics analysis.In view of the silkworm cell cycle is difficult to synchronize,we employ the human embryo kidney-derived HEK293-FT cells for this study.First,human HEK293-FT cells were synchronized in late G2 using CDK1 inhibitor of RO3306 to ensure HEK293-FT cell cycle progression.Cells were released into mitosis after the synchronization and treated with AZD1152-HQPA inhibitor of Aurora B kinase.DMSO was used as a control.When the control cells entered into the mitotic metaphase 40 minutes later,collected the inhibitor and DMSO treated cells seperately.We extracted the total proteins from the collected cells and digested with trypin.Via TiO2 enrichment,phosphorylated peptides were enriched and analyzed by LC-MS/MS.In total,we identified 8903 phosphopeptides derived from 3364 proteins from the six samples of three biological replicates.Based on two criteria: fold change ?1.2 or ?0.83 and p value <0.05,the protemics data revealed that 25 phosphopeptides of 16 proteins were significantly up-regulated and 36 phosphopeptides of 32 proteins were significantly down-regulated.Through GO enrichment analysis,we found that most of the changed proteins were mainly distributed in the cell or cell membrane to perform catalytic activity and regulate cell cycle progression.In addition to the confirmed Aurora B kinase direct substrate H1 and H3,we identified that some other proteins may also the potential targets of Aurora B kinase,such as CBX5 and RP/EB.Further using RNAi to reduce CBX5 gene expression in BmN4-SID1 cells.Immunostaining analysis revealed that bi-nucleation induced by cytokinesis failure,consistent with Bm AurB gene interference results.Subsequently,we will thoroughly research the potential targets of BmAurB kinase by combining human and silkworm together.