Summary
The serine/threonine protein phosphatase-5 (PP5) regulates multiple cellular signaling networks. A number of cellular factors, including heat shock protein-90 (Hsp90) promote the activation of PP5. However, it is unclear whether post-translational modifications also influence PP5 phosphatase activity. Here, we show an “on/off switch” mechanism for PP5 regulation. The casein kinase-1 δ (CK1δ) phosphorylates T362 in the catalytic domain of PP5, which activates and enhances phosphatase activity independent of Hsp90. Overexpression of the phosphomimetic T362E-PP5 mutant hyperdephosphorylates the substrates such as the co-chaperone Cdc37 and the glucocorticoid receptor in cells. Our proteomic approach identified the tumor suppressor von Hippel-Lindau protein (VHL) to interact and ubiquitinate K185/K199-PP5 for proteasomal degradation in a hypoxia- and prolyl hydroxylation-independent manner. Finally, VHL-deficient clear cell renal cell carcinoma (ccRCC) cell lines and patient tumors exhibit elevated PP5 levels. Down-regulation of PP5 causes ccRCC cells to undergo apoptosis, suggesting a prosurvival role for PP5 in kidney cancer.
curve (AUC) was 0.85. Repeated analysis of a second measurement of urine samples provided comparable findings.CONCLUSIONS: This study provides a first evaluation of urinary volatilome in RCC. Urine VOCs profiling by e-nose seems a promising, non-invasive diagnostic tool with high accuracy in discriminating patients from controls. Ease of use, low costs and noninvasive nature makes this test a potential molecular biomarker in early RCC diagnostic settings.
INTRODUCTION AND OBJECTIVES: Xp11.2 translocation renal cell carcinoma (Xp11.2 tRCC) is a newly defined subset of RCC which is characterized by various translocations involving chromosome Xp11.2. All the Xp11.2 translocations produce chimeric TFE3 genes, which retain the coding sequences for the basic helix-loop-helix leucine zipper structure (bHLH-Zip) through which TFE3 binds to DNA. It is suggested that the TFE3 fusion proteins encoded by the chimeric TFE3 genes work as constitutively active transcription factors resulting in Xp11.2 tRCC development. We aim to clarify the cancer-causing molecular mechanism of Xp11.2 tRCC by analyzing the transcriptional function of the chimeric TFE3 proteins.METHODS: 1) We performed comprehensive gene expression analysis by RNA sequencing (RNA-seq) to identify the gene clusters whose expressions were changed predominantly by chimeric TFE3 proteins using cell lines that expressed chimeric TFE3 proteins (PRCC-TFE3, PSF-TFE3, NONO-TFE 3) in a doxycycline-dependent manner or wild-type TFE3. 2) We performed chromatin immunoprecipitation sequencing (Chip-seq) and mapped chimeric TFE3 and wild type TFE3 binding to the genome.RESULTS: WT-TFE3 and PRCC-TFE3 mapping on the genome revealed sharp peaks in the promoter regions, while PSF-TFE3 and NONO-TFE3 showed distinctly broader peaks. RNA-seq analysis demonstrated that PSF-TFE3 and NONO-TFE3 protein induction caused distinct gene expression changes compared to wild type TFE3 induction. In addition, gene expression changes caused by PRCC-TFE3 protein induction were also different compared to wild type TFE3 protein induction, despite the fact that mapping patterns in promoter regions were similar in both cases.CONCLUSIONS: Our key new finding is that PSF-TFE3 and NONO-TFE3 fusions show clearly different binding patterns to the genome, and these chimeric TFE3 proteins cause distinct gene expression changes compared to wild-type TFE3. These findings suggest that chimeric TFE3 proteins contribute to carcinogenesis of renal cell carcinoma as constitutively active transcription factors that induce altered binding patterns on the genome, which result in gene expression profiles that are distinct from wild-type TFE3.
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