Molecular mechanism of G2/M Progression by protein kinases

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1. Aurora(1) We raised monoclonal antibodies against Aurora-A and determined the location of Aurora-A in human cells.(2) We showed that human Aurora-Adegradation is dependent on hCdh1 through the APC/C-ubiquitin-proteasome pathway.(3) We determined the substrate specificity of human Aurora-B. We found that this enzyme is an arginine-directed kinase that can phosphorylate histone H3 at serines 10 and 28 in vitro. We raised monoclonal antibodies against mitosis-specific autophosphorylation site in the T-loop of Aurora-A and determined the timing and location of Aurora-A activation in human cells.(4) We identified Aurora-B associated protein phosphatases as negative regulators of kinase activation.(5) We raised a monoclonal antibody which recognized all of Aurora family.(6) We developed an Aurora-A antisense oligonucleotide method and elucidated that depletion of Aurora-A by Aurora-A antisense oligonucleotide treatment causes a significant cell cycle arrest at prometaphase.(7) We found that MBD3, a component of the histone deacetylase NuRD complex, is phosphorylated in vivo in the late G2 and early M phases. Moreover, we found that Aurora-A phosphorylates MBD3 in vitro, physically associates with MBD3 in vivo, and co-localizes with MBD3 at the centrosomes in the early M phase.(8) We constructed a human stable cell-line in which Aurora-A, histone H3 and importina were differentially expressed as fusions to green, cyan, and red fluorescent proteins. Its molecular behavior in living mitotic cells was extensively analyzed by an advanced timelapse image analyzing system.2. SNK(1) We raised monoclonal antibodies against SNK and revealed that thses antibodies are useful diagnostic tools to find thyroid cancers at an early stage.