RESUMO
The evolutionarily conserved Aurora family kinases, a family of mitotic serine/threonine kinases, has three members in humans (Aurora-A, -B and -C). Overexpression of Aurora family members, particularly Aurora-A, has been reported in many human cancers and cell lines. In this study, we present evidence based on comparative gene expression analysis via quantitative RT-PCR to delineate the relative contributions of these kinases in 60 cell lines and statistical analysis in five different human cancer microarray datasets. The analysis demonstrated the selective upregulation of these Aurora members in various cancers. In general, Aurora-A exhibited the highest expression levels, with substantially decreased quantities of the Aurora-C transcript detected relative to Aurora-A and -B. Moreover, to characterize the roles of each Aurora member, which share many similarities, we investigated the expression profiles of the family in normal tissues and a panel of different phases of the HeLa cell cycle. Finally, both Aurora-A and -B were overexpressed in a majority of esophageal tumor tissues in comparison to the normal variants. Taken together, the results show that each Aurora member exhibits distinct expression patterns, implying that they are engaged in different biological processes to accomplish more elaborate cell physiological functions in higher organisms.
Assuntos
Neoplasias Esofágicas/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Aurora Quinase C , Aurora Quinases , Neoplasias Esofágicas/genética , Células HeLa , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas/metabolismoRESUMO
The c-Fos has been implicated in the regulation of gene expression under a variety of stimuli. It is known that c-Fos undergoes protein phosphorylation, which may subsequently modulate diverse functions in cells. However, less is known about the role and phosphorylation status of c-Fos during mitosis. Here, we showed that c-Fos exhibited an electrophoretic mobility up-shift as detected by SDS-PAGE during mitosis, which is an indication of protein phosphorylation. Aurora-A, but not Aurora-B or -C, serves as one of the kinases catalyzing the mitotic phosphorylation of c-Fos. The mobility up-shift was partially abolished by introducing siRNA or a catalytically inactive form of Aurora-A. Moreover, ectopic expression of the wild type, but not the catalytically inactive form of Aurora-A resulted in the alteration of c-Fos complex formation, suggesting Aurora-A is engaged in the regulation of c-Fos protein-protein interaction. These findings imply that c-Fos may undergo cell cycle dependent phosphorylation, in which some kinases including Aurora-A play a role in catalyzing the post translational modification of c-Fos.
Assuntos
Mitose/fisiologia , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Aurora Quinase B , Aurora Quinases , Sequência de Bases , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Complexos Multiproteicos , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por SubstratoRESUMO
Human Aurora kinases have three gene family members: Aurora-A, Aurora-B, and Aurora-C. It is not yet established what the specificity of these kinases are and what signals relayed by their reactions. Therefore, we employed small pool expression screening to search for downstream substrates of Aurora-A. Interestingly, all of the identified Aurora-A substrates were resistant to serve as substrates for Aurora-B or Aurora-C, suggesting that these Aurora family members may have distinct substrate specificity for propagation of diverse signaling pathways, even though they share a conserved catalytic kinase domain. Of the candidate substrates, Aurora-A could increase the functional activity of RalA. Mutational analysis revealed that RalA-Ser194 was the phosphorylation site for Aurora-A. Ectopic expression of V23RalA-WT could enhance collagen I-induced cell migration and anchorage-independent growth in Madin-Darby canine kidney (MDCK) Aurora-A stable cell lines. In contrast, overexpression of V23RalA-S194A in MDCK Aurora-A-stable cell lines abolished the intrinsic migration and transformation abilities of Aurora-A. To our knowledge, this is the first systematic search for the downstream substrates of Aurora-A kinase. Moreover, these results support the notion that Aurora-A may act in concert with V23RalA through protein phosphorylation on Ser194 to promote collagen I-induced cell motility and anchorage-independent growth in MDCK epithelial cells.