Application of ubiquitin ligase against tumor suppressor gene products for cancer therapy

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In this study, we analyzed the molecular mechanisms of proteolytic degradation of two major tumor suppressor gene products such as p27^<Kip1> and RB protein. Fast of all, we tried to identify the ubiquitin ligase for RB protein and analyze its function in malignant transformation process (2). The other project is identification of the down-regulation mechanisms of p27^<Kip1> (1).(1) Down-regulation mechanisms of p27^<Kip1>We as well as the other groups reported that p27^<Kip1> is efficiently degraded in human tumors with poor prognosis. Target disruption of Skp2, a ubiquitin ligase for p27^<Kip1>, resulted to decrease degradation of p27^<Kip1> in vivo. However, another mechanism of p27Kip1 degradation was stronglt suggested. We have reported that are down regulated by two mechanisms, one is ubiquitin dependent pathway and the other is site specific cleavage. We should identify the cleavage enzyme. In this study, we identified Ser10 as a major phosphorylation site for p27^<Kip1>. The phosphorylation at Ser10 stabilized p27^<Kip1> in cultured cells.(2) Identification of ubiquitin ligase for RB proteinWe established in vitro ubiquitination assay of RB protein and successfully obtain the candidate protein for ubiquitin ligase for RB protein using biochemical approach. In this study, it was confirmed by in vivo ubiquitination analysis and pulse chase experiment. It may be a novel mechanisms in malignant transformation via proteolytic degradation of RB protein.