Therefore, it is possible that this particular mutation may lead to an increased association with other E3 ubiquitin ligases targeting CHK1 leading to its reduced stability. Glucose\sensing pathways have also been shown to regulate the ability of E3 ubiquitin ligases to recognize and target proteins for ubiquitination and degradation. preventing tumorigenesis. Glucose deprivation is observed in many solid tumors due to high glycolytic rates of cancer cells and insufficient vascularization, yet cancer cells have devised mechanisms to survive in conditions of low glucose. Although CHK1 degradation through the ubiquitinCproteasome pathway following glucose deprivation has been previously reported, the detailed molecular mechanisms remain elusive. Here, we show that CHK1 is ubiquitinated and degraded upon glucose deprivation by the Skp1\Cullin\F\box (\TrCP) E3 ubiquitin ligase. Specifically, CHK1 contains a \TrCP recognizable degron domain, which is phosphorylated by AMPK in response to glucose deprivation, allowing for \TrCP to recognize CHK1 for subsequent ubiquitination and degradation. Our results provide a novel mechanism by which glucose metabolism regulates a DNA damage effector, and imply that glucose deprivation, which is often found in solid tumor microenvironments, may enhance mutagenesis, clonal expansion, and tumor progression by triggering CHK1 degradation. for 10?min at 4?C. Cleared lysates were then subjected to immunoprecipitation (IP) with bead\conjugated FLAG antibody (Sigma) with constant rotation at 4?C for 4?h. The immunoprecipitates were washed Tacalcitol monohydrate with lysis buffer six times for 5?min and assessed by immunoblotting (IB). Open in a separate window Figure 2 SCF \Tr CP targets CHK1 for degradation under glucose deprivation. (A) HEK293 cells were transfected with vectors encoding indicated FLAG\tagged Cullin proteins. Cells were lysed and immunoprecipitated with agarose\conjugated FLAG antibody. Immunoprecipitates and whole\cell extracts were separated on SDS/PAGE and immunoblotted for CHK1, FLAG, and ACTIN. (B) MDA\MB\231 and H1299 cells transfected with siCtrl or siCUL1 and grown in glucose\free media with 100?mgmL?1 cycloheximide (CHX) for the indicated time points. Cells were harvested and lysed and protein separated on SDS/PAGE and immunoblotted for CHK1, NEDD8, and ACTIN. (C) HEK293 cells transfected with empty vector, FLAG\\TrCP1, FLAG\FBXW7, or FLAG\SKP2 and grown in glucose\free media and treated with 10?m MG132 for 12?h. Cells were lysed and immunoprecipitated with agarose\conjugated FLAG antibody. Immunoprecipitates and whole\cell extracts were separated on SDS/PAGE and immunoblotted for CHK1, FLAG, and ACTIN. (D) MDA\MB\231, SK\BR3, and H1299 were treated with normal glucose or glucose\free media along with 20?m MG132 for 2?h before harvest. Cells were lysed and immunoprecipitated with either IgG or CHK1 antibody. Immunoprecipitates and whole\cell extracts were separated on SDS/PAGE and immunoblotted for \TrCP1, CHK1, and ACTIN. (E) MDA\MB\231, SK\BR3, and H1299 cells transfected with siCtrl or si\TrCP1?+?2 and grown in glucose\free media for the indicated time points. Cells were harvested and lysed and protein separated on SDS/PAGE and immunoblotted for CHK1, \TrCP1, and ACTIN. (F) MDA\MB\231, SK\BR3, and H1299 cells transfected Tacalcitol monohydrate with siCtrl or si\TrCP1?+?2 and grown in glucose\free media with 100?mgmL?1 CHX for the indicated time points. Cells were harvested and lysed and protein separated on SDS/PAGE and immunoblotted for CHK1, \TrCP1, and ACTIN. (G) HEK293 cells were transfected with HA\CHK1, FLAG\\TrCP1 WT, FLAG\\TrCP1 F, and His\Ub as indicated. Cells grown in glucose\free media and treated with 20?m MG132 for 5.5?h were lysed under denaturing conditions, and Ub\conjugated proteins were pulled down with Ni\NTA Rabbit polyclonal to ITPKB resin. Pull\downs and whole\cell extracts were separated on SDS/PAGE and immunoblotted for CHK1, FLAG, HA, and ACTIN. For experiment in Fig.?2D, cells were grown under normal glucose or glucose deprivation conditions together with MG132 (20?m) for 2?h. Following lysis, IPs were carried out by adding either 10?L mouse IgG (SC\2025 from Santa Cruz Biotechnology) or 20?L CHK1 Ab (SC\8408 from Santa Cruz) to each sample and incubating with constant rotation at 4?C for 16?h. Protein G beads (17\0618\01 from GE Healthcare, Chicago, IL, USA) were added to each IP and incubated for an additional 2?h at 4?C and subsequently assessed Tacalcitol monohydrate by IB. For direct IB analysis, cells were washed twice in PBS and lysed in RIPA buffer with phosphatase inhibitors and protease inhibitor cocktail. 2.5. ubiquitination assay HEK293 cells were transfected with indicated plasmids and grown in glucose\free media with 20?m MG132 for the indicated time points. Cells were then lysed in 6?M guanidine denaturing solution and incubated with Ni\NTA resin (Qiagen, Valencia, CA, USA) as described previously (Zhao kinase assay kinase assays were performed as previously described (Su.
Therefore, it is possible that this particular mutation may lead to an increased association with other E3 ubiquitin ligases targeting CHK1 leading to its reduced stability