| Summary: | 博士 === 國立陽明大學 === 微生物暨免疫學研究所 === 88 === Protein phosphorylation is involved in many biological activities and plays important roles in cell cycle progression. In the present study, we have identified a serine/threonine kinase, hAIK, from human hepatic cells using degenerated polymerase chain reactions with a pair of primers derived from the highly conserved sequence in the catalytic domain of kinases. The full-length hAIK cDNA was then obtained, which contained 403 amino acids and was homologous to Drosophila Aurora2 and yeast Ipl1 proteins. Northern blotting analysis revealed that hAIK was highly expressed in the testis but not in other tissues. Expressions of hAIK drastically increased in cancer tissues/cell lines but not in fibroblasts or non-tumorigenic cell lines. The recombinant hAIK protein could phosphorylate itself and histone H1; this phosphorylation activity was totally abolished by a point mutation at the catalytic domain (hAIKm). In the interphase cell, hAIK was found mainly in the cytoplasm; during mitosis hAIK was noted to accumulate at the centrosomes. In addition, over-expression of hAIK in cancer cell lines (HEK293T and HeLa) appeared to inhibit cell cycle progression. Injection of the anti-hAIK antibodies into HeLa cells found that the cell cycle would be blocked at the early stage and delayed its progression. None of these phenomena was observed in hAIKm whose kinase activity was rendered inactive.
To further address the roles played by hAIK in modulation of cell division, we have performed several structural and functional assays. In the presence of hAIK, the diameters of microtubules nucleated from the centrosomes profoundly increased compared to the control samples. Microtubule co-sedimentation and polymerization assays also revealed that hAIK could not only bind to microtubules but also facilitate polymerization efficiency, especially at the tubulin concentrations below the critical concentration. Interestingly, the kinase-inactive hAIK mutants could neither bind to microtubules, nor interfere with the assembly kinetics of the microtubule cytoskeleton. In addition, we have found that one of the centrosomal proteins, the human ninein protein, was effectively phosphorylated by hAIK. Previous studies have suggested that ninein, as a microtubule-anchorage protein, might mediate the 烋ucleation and release of microtubules?from the centrosome, a microtubule-organization center. Taken together, I propose that hAIK may modulate the cell cycle progression by affecting the structures and functions of the mitotic machinery (i.e., the centrosomes and spindle microtubules) that segregate the chromosomes.
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