| BackgroundAkt is a serine/threonine kinase that plays a key role in multiple cellular processes related to tumorigenesis such as apoptosis, cell proliferation, transcription, cell migration and cell cycle. It acts as a upstream signaling protein in the cellular pathways and a regulator of the other protein expression and function. CSN6is a oncoprotein that plays a pivotal role in the tumorigenesis, involving in the regulation of p53and MDM2. CSN6is overexpressed in many types of human cancers and is an attractive target for cancer therapies. Akt and CSN6both have important impact on the cell cycle and p53and MDM2, however whether Akt has interaction with CSN6is not known. We first scanned CSN6sequence using the NetPhos algorithm and identified a potential Akt phosphorylation site (55WIRMRS60), which is conserved among CSN6proteins from different species and shares the conserved core Akt phosphorylation site of Bad (134RGRSRS139) and p21(140RKRRQT146), so it was hypothesized that Akt can phosphrylate CSN6.ObjectiveTo investigate the activity of AKT on the CSN6and the mechanism of this action as well as its impact on the CSN6-mediated transformation of NSCLC.Materials and Method1. Cell lines, cell culture, plasmid and transfection.293T (human embryonic kidney cell line), A549(Human lung adenocarcinoma cell line), Ratl (Rat embryo fibroblast cell line) and MDA-MB453(Human breast cancer cell line) were enrolled. Ratl-akt cells were stably transfected Akt and MDA-MB453-DNAkt cells stably were transfected DN-Akt (Domain Negative Akt). The PCR-generated DN A fragments of human CSN6gene was cloned into pCMV5to generate a construct that encodes CSN6with a Flag tagged sequence or into pEGFP N1with a GFP tagged sequence or into pCDNA6with a myc-tagged sequence.2. Construction of mutants.CSN6(S60A) and CSN6(S60D) mutants were constructed using a site-directed mutagenesis technique. The primers were designed to change one or two bases to effect the change in the amino acid sequence and a PCR was performed with PFU Turbo polymerase to amplify plasmids in their entirety. After amplification, plasmids were treated with restriction enzyme DpnI to digest any remaining template.3. The effect of Akt kinase on the CSN6levelTo investigate the regulatory effect of Akt on the CSN6level,the plasmid encoding CSN6together with different dose of plasmid encoding Akt was cotransfected into293T cells. After48h, the cells were lysed in lysis buffer and the Western blotting assay was performed to examine the CSN6protein levels. To determine the influence of Akt on the endogenous CSN6level, the level of CSN6in the Akt stably transfected Ratl cells (Ratl/HA-Akt cells) and the dominant negative Akt (DNAkt) stably transfected MDA-MB453cells (MDA-MB453/HA-DNAkt cells) were examined using Western Blotting. Moreover, the level of CSN6in the293T cells transfected with CSN6and concurrently treated with PI3K inhibitor Ly294002were measured.To determine whether Akt signaling affects CSN6subcellular localization,the cells which were transfected with GFP-CSN6in the presence or absence of Akt and Ratl-Akt cells were probed with anti-CSN6. Then, Alexa Fluor488goat anti-rabbit antibody was added to detect CSN6. DAPI was applied to stain the nuclei. Immunofluorescence was observed by using a confocal microscope.4. The impact of Akt kinase on the CSN6ubiquitinationTo investigate the impact of Akt kinase on the polyubiquitination of the CSN6, the293T cells were cotransfected with HA-ubiquitin, HA-Akt and GFP-CSN6plasmids. At24h post-transfection, cells were treated with50μg/ml MG132for6h. Then the cells were harvested with lysis buffer.1mg protein was incubated with1μg/ml of anti-GFP antibodies at4℃overnight and subsequently incubated with50μl protein G conjugated to agarose beads at4℃for4h. The immunoprecipitates were washed with lysis buffer5times and bound proteins were eluted using2×SDS-loading buffer and then Western Blotting was performed using anti-ubiquitin antibody. 5. Turn over assayTo determine if the higher steady-state level of CSN6during Akt kinase overexpression was due to the slower degradation of CSN6, the turn over assay for the CSN6was carried out with protein synthesis inhibitor cycloheximide. The cells were transfected with indicated plasmids for24hours. Then Cycloheximide were added into the media at the final concentration of60μg/ml. The cell were harvested at the time points of0,1,2and4hours after treament. The protein levels were analysed by western blotting. Signals in the blots were quantitated by phosphorimaging and plotted to compare the degradation rates. Moreover, the MDA-MB453/DN Akt cells also were performed same experiment.6. Protein binding assayTo examine if CSN6has a binding relationship with Akt kinase, the experiment of co-immunoprecipitation (co-IP) of CSN6together with Akt was performed. To further investigate the potential site of CSN6binding with Akt,293T cells were cotransfected with the plasmids encoding Flag-CSN6(WT), Flag-CSN6(NH2-terminus, N-terminus) or Flag-CSN6(C-terminus) together with the plasmid encoding HA-Akt. The cell lysates were immunoprecipitated with anti-Flag antibody or mouse IgG as a control. And then immunoblotting was performed with anti-HA antibody to detect the binding association between both proteins.7. Akt Phosphorylates CSN6To verify CSN6to be a kinase substrate for Akt kinase. The in vitro kinase assays were performed. Recombinant CSN6substrates were produced by BL-21E. coli bacteria. Briefly, The bacterial cells were transformed with the pET-Flag-CSN6(wild type) and pET-Flag-CSN6(S60A) plasmid and grown in5mL LB media overnight and in500ml LB media for5h.1mM IPTG was added in culture media at3hours before harvested. Then the cells were lysed with NETN buffer and sonicated. The CSN6proteins were pulled down by M2beads and eluted by Flag peptide. Recombinant Akt kinase was incubated with immunopurified CSN6(wild type) and CSN6(S60A) substrates in1×kinase buffer with cold ATP and y32ATP at30℃for15min. Kinase reactions were analyzed by SDS/PAGE as described before. After the SDS/PAGE gel was dried, the radioactivity image was visualized using a phosphoimager cassette and a Typhoon Trio variable mode imager.8. Akt stabilizes CSN6via phosphorylating CSN6at Ser60To further verify Akt-mediated phosphorylation of CSN6on Ser60plays an important role in the stabilization of CSN6, we mutated CSN6Ser60into Ala and furthermore mutated CSN6Serine60into Aspartate (S60D) to mimic the phosphorylation state. After cotransfected the plasmid encoding CSN6(WT), CSN6(S60A) or CSN6(S60D) together with HA-Akt kinase in293T cells, the CSN6level, the turn over of CSN6and the ubiquitination of CSN6were determined.9. Quantitative real-time PCRTo investigate the influence of Akt on the CSN6mRNA transcription, we examined the CSN6mRNA level in293T cells transfected with different dose of plasmids encoding Akt. Total RNA was extracted from293T cells with TRIzol Reagents according to the manufacturer’s instructions. Reverse transcription (RT) was performed using the iScript cDNA synthesis kit. Primers of CSN6and GAPDH for real-time PCR were designed using software of Primer Bank and synthesized by Sigma company. Quantitative real-time PCR were performed with the iQ SYBR Green Supermix and iCycler thermocycler. The data were normalized by calculating ACT (CT of target-CT of the internal control (GAPDH)]. Use2-ΔΔCT method of relative quantification to analyze the relative changes in gene expression. Each experiment was conducted in triplicate.lO.The impact of Akt on the CSN6-mediated p53and MDM2stabilityTo verify whther CSN6played an important role in MDM2degration which is related with the p53stability, the impact of Akt on the CSN6-regulated p53and MDM2levels in A549cells were examined using Western Blot.11.Impacts of Akt-CSN6axis on ROS production and DNA damageTo determine the impact of the Akt-CSN6axis on DNA damage/chromosomal instability. The level of ROS and H2AX foci formation of HCT116cells were analyzed using immunofluorescence stainning.12. Akt kinases enhance the transforming activity of CSN6To investegate if Akt kinases can enhance the oncogenic transforming properties of CSN6by stabilizing CSN6, the MTS, foci formation assay and soft agar colony formation assay were performed, after cotransfected the plasmid encoding CSN6(WT), CSN6(S60A) or CSN6(S60D) together with HA-Akt kinase in A549cells.13.ImmunohistochemistryTwo paired tissue microarray slide with40case NSCLC were selected. DAKO LSAB kit was used to do immunohistochemistry stainning, performed according to protocol provided by the manufacturers. Results1. CSN6level is up-regulated by Akt kinase.After plasmid encoding CSN6together with different dose of plasmid encoding Akt was cotransfected in293T cells, the Western blotting assay showed that the protein levels of CSN6were enhanced in the presence of Akt kinase overexpression. Moreover, the elevation of CSN6level was increased with the increasing dose of Akt kinase. Compared with the control cells, the CSN6level was obviously increased in the Rat1/HA-Akt cells. While observing the CSN6level in the dominant negative Akt (DNAkt) stably transfected MDA-MB453cells (MDA-MB453/HA-DNAkt cells), we discovered the CSN6level was significantly reduced in MDA-MB453/HA-DNAkt cells compared with MDA-MB453cells. These results demonstrated that Akt kinase up-regulated the protein level of CSN6and took effect in the dose-dependent manner.GFP-CSN6seemed to be more abundant in the cytoplasm, while cotransfection with Akt promoted more nuclear localization of CSN6. Similar results have been observed in Rat1-Akt cells. While CSN6is located in the cytoplasm in Ratl cells, CSN6is relocated from the cytoplasm to the nucleus in Ratl-Akt cells.2. Akt kinase stabilizes the CSN6through repression of the ubiquitin-proteasome degradation of CSN6The turn over assay for the CSN6revealed that CSN6exhibited a slower degradation rate in the presence of Akt overexpression than empty vector, whereas, in the MDA-MB453/DNAkt cells, CSN6exhibited a faster degradation as compared to MDA-MB453cells. This illustrated that Akt kinase promoted the stabilization of CSN6protein. The ubiquitination assay showed that CSN6polyubiquitination was dramaticly reduced during Akt overexpression and the reduction was increased with the increasing dose of the Akt kianse. Concurrently, we found the Akt-mediated CSN6up-regulation was antagonized by the proteasome inhibitor MG132. These suggests Akt kinase stabilizes the CSN6by repressing the ubiquitin-proteasome degradation of CSN6.3. CSN6is bound with Akt kinaseThe experiment of co-immunoprecipitation (co-IP) of CSN6together with Akt showed that CSN6had a potent bond with Akt in293Tcells. Moreover, Akt bound both the N-terminus and the C-terminus of CSN6. However, the bond with N-terminus of CSN6was much more than the C-terminus at equal input amount of the proteins. These data demonstrated that Akt has a binding relationship with CSN6, especially, with N-terminus of CSN6.4. Akt Phosphorylates CSN6While in vitro kinase assay was performed to detect the phosphorylation of Akt on the CSN6,32P autoradiograph showed that both Flag-CSN6(WT) and Flag-CSN6(S60A) mutant were phosphorylated by Akt kinase, but the wild type CSN6phosphorylation was much higher than CSN6(S60A) mutant. These results suggested that Akt kinase have a potent effect on phosphorylating CSN6and the major site of phosphorylation is at serine60residues which is in the DNA binding domain.5. Akt stabilizes CSN6via phosphorylating CSN6at Ser60After cotransfected the plasmid encoding CSN6(WT) or CSN6(S60A) together with HA-Akt kinase in293T cells, we found the CSN6level in the cells transfected with CSN6S60A mutant was lower than that in the expressing WT CSN6cells in the presence of Akt. Consistently, the turn over of CSN6(S60A) mutant was faster than the CSN6(WT). Furthermore, the ubiquitination assay demonstrated that the ubiquitination of CSN6S60A mutant was increased as compared with the WT CSN6. These suggest that CSN6Ser60phosphorylation by Akt kinase contributes to the stabilization of CSN6. It was found that the CSN6level in the293T cells transfected with CSN6S60D mutant was increased as compared with WT CSN6and S60A CSN6. The turnover of CSN6S60D mutant was slower than the WT CSN6and S60A CSN6. The ubiquitination of CSN6S60D mutant was decreased, compared with the WT CSN6and S60A CSN6. Together, these results indicate that Akt-mediated phosphorylation on CSN6Ser60plays an important role in stabilizing CSN6.6. Akt affects CSN6gene transcriptionThe RT-PCR result showed the mRNA level of CSN6was elevated in the Akt-overexpressing cells compared with the control cells, and the elevation was enhanced with the increase of the transfected dose of Akt. It indicated Akt kinase has an activative effect on the CSN6transcription.7. The impact of Akt on the CSN6-mediated p53and MDM2stabilityThe results showed that expression of MDM2was higher in CSN6(WT)-overexpressing cells than the control and CSN6(S60A)-overexpressing cells, but lower than in CSN6(S60D)-overexpressing cells. Whreas, the expression of p53was lower in CSN6(WT)-overexpressing cells than the control and CSN6(S60A)-overexpressing cells, but higer than the CSN6(S60D)-overexpressing cells. These illustrated CSN6S60A mutants were resistant to Akt-mediated repression of p53, whereas S60D mutants remained this function. It suggests the phosphorylation of CSN6Ser60by Akt kinase enhanced the CSN6-mediated MDM2level, resulting in the repression of p53.8. Impacts of Akt-CSN6axis on ROS production and DNA damageCSN6-overexpressing HCT116cells had a higher level of ROS than HCT116parental cells. The production of ROS in CSN6-overexpressing HCT116cells was attenuated by PI3K inhibitor LY294002. There was a higher numbers of y-H2AX foci in CSN6-overexpressing than in HCT116cells. LY294002decreased numbers of y-H2AX foci caused by CSN6overexpression. 9. Akt kinases enhance the transforming activity of CSN6While cells were cotransfected with the plasmids encoding CSN6(WT) and Akt, the proliferation of the cells, the rate of plate foci formation and the number of the soft agar colony formation were increased than control without Akt transfection. When Ser60was mutated to alanine, the cell growth and foci formation were decreased than the WT CSN6when coexpressing Akt kinase, while the phosphomimic mutant of CSN6, S60D, exhibited more potency in transformation of A549cells as compared with WT CSN6and S60A CSN6mutant.Conclusion1. Akt kinase up-regulates the level of CSN6and acts as dose-dependent manner.2. Akt stabilizes the CSN6protein by phosphorylating CSN6, to attenuate the ubiquitination of CSN6and inhibit the degradation of CSN6.3. Akt kinase phosphorylates CSN6at Ser60site.4. Akt kinase functionally regulates the levels of MDM2and p53which are involved in cell cycle through up-regulation of CSN6.5. The up-regulation of CSN6by Akt kinase accelerates the CSN6-mediated transformation of NSCLC cell. |
PDF Full Text Request