臺灣大學: 生化科學研究所陳瑞華陳姿穎Chen, Tzu-YinTzu-YinChen2013-03-212018-07-062013-03-212018-07-062011http://ntur.lib.ntu.edu.tw//handle/246246/250975The expression of tumor suppressor promyelocytic leukemia (PML) is lost in a wide variety of solid tumors through a proteasome-dependent mechanism. Previous studies in our laboratory identified a KLHL20-based ubiquitin ligase complex that targets CDK1/2-phosphorylated PML for ubiquitination and degradation and in vitro analysis demonstrated PML S518 residue as the prime site for CDK1/2 phosphorylation. In this thesis, we first generated a PML S518-specific antibody and confirmed that both CDK1/2 were capable of phosphorylating PML at S518 in vivo. Next, we explored the effect of hypoxia on KLHL20-mediated PML degradation, because KLHL20 is known to be upregulated in hypoxic cells. We found that PML level was drastically decreased in hypoxia, correlating with a marked elevation of KLHL20 level. This opposite regulations of KLHL20 and PML were also detected by overexpression of a constitutively active HIF-1 in normoxia and were blocked by HIF-1 depletion in hypoxia. Importantly, we presented evidence indicating that CDK1/2 activities were responsible for PML S518 phosphorylation and PML degradation under hypoxia conditions. Thus, CDK1/2 activities are responsible for PML S518 phosphorylation in both hypoxic and normoxic cells and KLHL20 induction by HIF-1 likely contribute to enhanced PML degradation in hypoxia. Finally, we determined the residues flanking S518 that are important for KLHL20-mediated PML degradation. To this end, we generated a panel of PML point mutants. Mutations of S518 and P519 interfered with CDK1/2-induced PML S518 phosphorylation, whereas mutations of A516 and H521 blocked KLHL20-induced PML ubiquitination and degradation without affecting S518 phosphorylation. Taken together, our results suggest that the sequence of AxpSPxH between 516 and 521 of PML may act as a degron for KLHL20 recognition. This finding may aid in future identification of additional KLHL20 substrates.1269681 bytesapplication/pdfen-US磷酸化CDKPMLKLHL20HIF-1aDegronPhosphorylationthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/250975/1/ntu-100-R98b46006-1.pdf