| Summary: | 碩士 === 國立清華大學 === 生物科技研究所 === 95 === Abstract
Skp2, an F-box protein of SCF E3 ubiquitin ligase, targets key cell cycle regulators for degradation including p21Cip1 and p27Kip1 to timely control the progression from late G1 until mitotic entry, while its protein level is keep low via APCCdh1 and 26S proteasome mediated destruction at G0-G1. Here we show a novel mechanism for the Skp2 destruction under arsenite stress. Arsenite decreased Skp2 protein and mRNA levels in a dose- and time-dependent manner. Under protein synthesis inhibition, arsenite markedly reduced Skp2 protein and enhanced its ubiquitination. Knockdown of Cdh1 lowered the arsenite-induced Skp2 ubiqutination and degradation; however, inhibition of 26S proteasome by MG132 or ALLN further enhanced the Skp2 degradation under arsenite. Intriguingly, inhibition of lysosomal proteases by leupeptin, E64, aprotinin, and pepstatin could effectively rescue the arsenite-induced Skp2 destruction. Depletion of p38 alpha/beta using siRNAs suppressed the Skp2 proteolysis under arsenite, whereas ectopic expression of p38MAPK enhanced the event. Surprisingly, the arsenite-induced Skp2 proteolysis still continued under blockage of p38MAPK kinase activity using SB202190 or via stably expressing a dominant-negative p38MAPK, or during over-expressing the MKK3/6-p38MAPK signaling. The Skp2 down-regulation was parallel to increases in p21Cip1 and p27Kip1 levels, causing marked delay of S entry in arsenite-treated G1 cells. Together, these results suggest that arsenite induces a novel mechanism for the Skp2 destruction by which lysosomal proteases override the action of 26S proteasome in spontaneous Skp2 turnover. Moreover, under arsenite the p38MAPK exhibits a kinase-independent function in triggering Skp2 destruction. This study also suggests that Skp2 down-regulation is a key contributor in the G1/S delay caused by arsenite.
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