PMC:5337621 / 14459-17670 JSONTXT

{"project":"2_test","denotations":[{"id":"28218735-18353650-79088313","span":{"begin":358,"end":360},"obj":"18353650"},{"id":"28218735-26782714-79088314","span":{"begin":2100,"end":2101},"obj":"26782714"}],"text":"AURKA modulates SCFFBXL7 to suppress Survivin degradation\nProtein proteasomal degradation precedes orchestrated events involving a series of enzymatic reactions comprising of E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. The E1 and E2 are common enzymes, while E3 ubiquitin ligases are highly substrate specific.27 To delineate the E3 ligase responsible for Survivin degradation controlled by AURKA in gastric cancer cells, we performed mass spectrometry analysis of Survivin immunoprecipitates after treatment with MG132 (data not shown) and identified a number of putative Survivin-interacting E3 ubiquitin ligases. To confirm further our mass spectrometry-based data, we selected seven Survivin-interacting proteins with potential E3 ligase activity, and had the genes cloned and transiently expressed in BGC823 cells (Figure 5a). Amongst these E3 ligases, only FBXL7 when overexpressed reduced Survivin expression at the protein level. We then examined Survivin and FBXL7 interaction by transiently co-expressing His-tagged Survivin and Flag-tagged FBXL7 in HEK293T cells followed by coimmunoprecipitation (Figure 5b). We found the Flag-tagged FBXL7 coprecipitated with His-tagged FBXL7 and vice versa, but not with the control IgG, suggesting that Survivin interacted with FBXL7 either directly or as part of a larger complex. Next, we transiently transfected AGS and BGC823 cells with increasing amounts of Flag-FBXL7 plasmid (Figure 5c) and found Survivin protein levels decreased in a dose-dependent manner in response to FBXL7 ectopic overexpression. To confirm the specificity of FBXL7 for Survivin, we purified all components of ubiquitylation reaction using the prokaryotic protein expression system and performed in vitro ubiquitylation analysis. As indicated in Figure 5d, SCFFBXL7 promoted the generation of polyubiquitylated Survivin species, thus confirming that Survivin was a specific substrate of the SCFFBXL7 E3 ligase complex.\nRecent evidence demonstrated that AURKA could regulate c-Myc expression through cooperating with hnRNP K,8 we hypothesized that AURKA regulated FBXL7 expression in order to control Survivin steady state. To test this hypothesis, we depleted AURKA in both AGS and BGC823 cells and analyzed FBXL7 expression. As shown in Figure 6a, AURKA depletion significantly increased FBXL7 protein levels. Consistent with the Western blot results, FBXL7 mRNA levels also increased in AURKA knockdown cells (Figure 6b), indicating AURKA regulates FBXL7 expression at the transcriptional level. It is noteworthy that endogenous FBXL7 mRNA and protein levels were low in AURKA wild-type gastric cancer cells; however, both FBXL7 mRNA and protein levels reached detectable levels only after AURKA depletion. To examine AURKA-mediated FBXL7 transcriptional activity, we cloned a 1.5 kb fragment of FBXL7 upstream of the transcriptional start site into the pGL3 basic vector and tested for its promoter activity. As shown in Figures 6c and d, both total AURKA depletion and kinase activity inhibition significantly increased FBXL7 promoter activity, suggesting that AURKA suppressed FBXL7 promoter activity in a kinase-dependent manner."}