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Title: | 探討抑癌基因PML中被KLHL20辨認之降解區塊 Characterization of the degron of KLHL20 on PML |
Authors: | Tzu-Yin Chen 陳姿穎 |
Advisor: | 陳瑞華(Ruey-Hwa Chen) |
Keyword: | 磷酸化,CDK,PML,KLHL20,HIF-1a,Degron, Phosphorylation,CDK,PML,KLHL20,HIF-1a,Degron, |
Publication Year : | 2011 |
Degree: | 碩士 |
Abstract: | The 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30352 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生化科學研究所 |
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