SET domain-containing protein 2 (SETD2) is commonly mutated in renal cell carcinoma. SETD2 methylates histone H3 as well as a growing list of non-histone proteins. To explore SETD2-dependent regulation of the kidney cancer proteome, we performed a systems-wide analysis of protein lysine-methylation and expression in wild type (WT) and SETD2-knock out (KO) kidney cells. We observed decreased lysine methylation of the translation elongation factor eEF1A1. EEF1AKMT2 and EEF1AKMT3 are known to methylate eEF1A1, and we show here that their expression is dependent on SET-domain function of SETD2. Globally, we observe differential expression of hundreds of proteins in WT versus SETD2-KO cells, including increased expression of many involved in protein translation. Finally, we observe decreased progression free survival and loss of EEF1AKMT2 gene expression in SETD2-mutated tumors. Overall, these data suggest that SETD2-mutated ccRCC, via loss of enzymetic function of the SET domain, displays dysregulation of protein translation as a potentially important component of the transformed phenotype.
BACKGROUND: SET domain-containing protein 2 (SETD2) is commonly mutated in renal cell carcinoma. SETD2 methylates histone H3 as well as a growing list of non-histone proteins. OBJECTIVE: Initially, we sought to explore SETD2-dependent changes in lysine methylation of proteins in proximal renal tubule cells. Subsequently, we focused on changes in lysine methylation of the translation elongation factor eEF1A1. METHODS: To accomplish these objectives, we initially performed a systems-wide analysis of protein lysine-methylation and expression in wild type (WT) and SETD2-knock out (KO) kidney cells and later focused our studies on eEF1A1 as well as the expression of lysine methyltransferases that regulate its lysine methylation. RESULTS: We observed decreased lysine methylation of the translation elongation factor eEF1A1. EEF1AKMT2 and EEF1AKMT3 are known to methylate eEF1A1, and we show here that their expression is dependent on SET-domain function of SETD2. Globally, we observe differential expression of hundreds of proteins in WT versus SETD2-KO cells, including increased expression of many involved in protein translation. Finally, we observe decreased progression free survival and loss of EEF1AKMT2 gene expression in SETD2-mutated tumors predicted to have loss of function of the SET domain. CONCLUSION: Overall, these data suggest that SETD2-mutated ccRCC, via loss of enzymatic function of the SET domain, displays dysregulation of protein translation as a potentially important component of the transformed phenotype.
567 Background: SET domain-containing protein 2 ( SETD2) is among the most commonly mutated genes in clear cell renal cell carcinoma (ccRCC). Classically, SETD2 modulates chromatin structure via the methylation of lysine 36 on histone H3. However, histone-independent functions of SETD2 are emerging. We sought to discover novel SETD2-dependent changes to the cellular lysine methylation landscape during kidney cancer tumorigenesis. Methods: In this study, we used HKC human proximal tubule kidney cell lines, the likely nephrogenic region of origin for ccRCC. The proteomes of wild type (WT) and SETD2-knock out (KO) cells were labeled using stable isotope labeling by amino acids in cell culture (SILAC). Proteins were trypsinized and lysine-methylated peptides were immunoprecipitated. Relative abundance of lysine-methylated peptides and total peptides were calculated in the WT and SETD2-KO using nano-liquid chromatography/tandem mass spectrometry (LC/MS-MS). Results: > 30,000 peptides were quantified, including > 50 lysine methylated peptides. We observed decreased lysine methylation of eukaryotic elongation factor 1A1 (eEF1A1), including K165 trimethylation (K165me3) and K318 monomethylation (K318me1). eEF1A1 is classically known for delivery of aminoacyl-tRNA to the ribosome, and its function is modulated via the lysine methyltranferases eEF1AKMT1-4. eEF1AKMT3 is responsible for trimethylation of K165, and expression of this enzyme was decreased in SETD2-KO cells. In addition, we observed significant decrease in expression in proteins of the electron transport chain (ETC) complex 1 in SETD2-KO cells yet normal gene expression, suggesting a translational defect. Functionally, SETD2-KO cells are characterized by decreased cellular oxygen consumption rate and increased mitochondrial mass, suggestive of ETC dysfunction. Conclusions: We observe decreased expression of eEF1AKMT3 in SETD2-KO cells, resulting in decreased K165me3 of eEFA1A, decreased translation of ETC proteins, and mitochondrial dysfunction. As mitochondrial dysfunction is commonly observed in RCC, we expect this represents a novel mechanism of SETD2-mediated tumorigenesis.
https://content.iospress.com/articles/kidney-cancer/kca220009 On page 185, the order of the panels in Figure 3 in the published version did not correlate with the figure legend. An updated figure is included below so that it correlates accurately with the figure legend. In addition, an updated Results paragraph is included below that accurately correlates with this reorganized figure.None of the data changed. Rather, the order of the panels was adjusted. The error had no bearings on the interpretation of the results, nor did it influence the conclusions of this work.
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