Aurora-a Kinase Regulation Mechanism Of Ajuba Increase The Activation Mechanism,

Aurora kinases representing a novel family of serine/threonine kinases have been identified as key regulators of the mitotic and meiotic cell division process. The three members of this kinase family, identified so far, referred to as Aurora-A, Aurora-B and Aurora-C which are known to be involved in the regulation of centrosome function, bipolar spindle assembly and chromosome segregation processes. Significant interest in the subject was generated since all three Aurora kinases family members were reported to be overexpressed in many human cancers. Ectopic overexpression of one member of the family, Aurora-A, was shown to induce oncogenic transformation in cells. All three members of the mammalian kinase family have a catalytic domain that is highly conserved and an N-terminal domain of varying sizes.To investigate the role of the N-terminal of Aurora-A, we overexpressed the N-terminal(1-128aa), C-terminal(129-403aa) and full length of Aurora-A. We found that the C-terminal (129-403aa) has higher kinase activity than that of the full length by 4-fold. The N-terminal can inhibit the C-terminal kinase activity both in vitro and in vivo, and the function of its inhibition is dose-dependent. So we presume the N-terminal of Aurora-A can negatively regulate the Aurora-A kinase activity. Then, we use Co-ip, GST-pull down to identify the direct interaction motif between the two parts of the kinase. Using mutagenesis, we design a set of deletions to identify the interaction motif in the N-terminal and C-terminal. The most possible motif locates in N (64-128aa) and C (240-300aa). We Analyzed the crystallographic structure of the catalytic domain of Aurora-A and after screening several point mutants we found some mutants of Aurora-A catalytic domain affect the kinase activity. With further study, we found the (l-128aa) can inhibit the autophosphorylation of Thr288 in the active loop. So we suggest the active loop may play an important role in the regulation of Aurora-A.These years more and more proteins have been found to regulate the kinase activity of Aurora-A. The mechanisms how they regulate the Aurora-A are still unknown. TPX2 is the most understood of them. Conti and coworkers solved the crystallographic structure of the catalytic domain of Aurora-A in the presence or absence of the TPX2-derived activation domain (1-43aa). In the presence of the TPX2 peptide the active loop was constrained in a different conformation, so that the phosphorylated Thr288 was oriented inwardly, and in this buried position was protected from dephosphorylation by PP1. So TPX2 can lock Aurora-A in an active conformation. Ajuba is another important activator of Aurora-A. In this report we showed that Ajuba plays an important role in regulation of the kinase activity of Aurora-A. One possibility for the mechanism by which Ajuba mediates the activation of Aurora-A is that the Lim domain of Ajuba can bind the N-terminal of Aurora-A, and prevent this inhibitory interaction with the catalytic domain. The Prelim of Ajuba can be phosphorylated by catalytic domain of Aurora-A and induces an activating conformational change of Aurora-A.In the second section we report the cloning and characterization of a novel human LU domain-containing gene, LYPD7 (LY6/PLAUR domain containing 7), isolated from human testis cDNA library, and mapped to 2q22. 3-23.3 by searching the UCSC genomic database. The LYPD7 cDNA sequence consists of 1600 nucleotides and contains an open reading frame of 624bp, encoding a putative protein of 207 amino acid residues. RT-PCR analysis showed that LYPD7 was especially highly expressed in testis, lung, stomach and prostate. Subcellular localization of LYPD7 demonstrated that the protein was localized in the cytoplasm when overexpressed in Hela cells. Furthermore, the subsequent analysis based on reporter gene assays suggested that overexpression of LYPD7 in HEK 293T cells was able to activate the transcriptional activities of AP1 (PMA).In conclusion, we first proposed that N-terminal of Aurora-A kinase has autoinhibitory regulation to the kinase activity and presume a possible mechanism by which Ajuba activates Aurora-A. Our work shed light on the regulation of this critical mitotic kinase and helps us to well learn about the mechanism of oncogenesis and inhibitory reagent.