pr3009429_si_009.xls (1.67 MB)
Quantitative Phosphoproteomic Analysis of Early Alterations in Protein Phosphorylation by 2,3,7,8-Tetrachlorodibenzo‑p‑dioxin
datasetposted on 2013-02-01, 00:00 authored by Melanie Schulz, Stefanie Brandner, Carola Eberhagen, Friederike Eckardt-Schupp, Martin R. Larsen, Ulrich Andrae
A comprehensive quantitative analysis of changes in protein phosphorylation preceding or accompanying transcriptional activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 5L rat hepatoma cells was performed using the SILAC approach. Following exposure of the cells to DMSO or 1 nM TCDD for 0.5 to 2 h, 5648 phosphorylated peptides corresponding to 2156 phosphoproteins were identified. Eight peptides exhibited a statistically significantly altered phosphorylation because of TCDD exposure and 22 showed a regulation factor of ≥1.5 in one of the experiments per time point. The vast majority of the TCCD-induced phosphorylation changes had not been reported before. The transcription factor ARNT, the obligate partner for gene activation by the TCDD-bound Ah receptor, exhibited an up-regulation of its Ser77 phosphorylation, a modification known to control the differential binding of ARNT homodimers and heterodimers to different enhancers suggesting that this phosphorylation represents a novel mechanism contributing to the alteration of gene expression by TCDD. Other proteins with altered phosphorylation included, among others, various transcriptional coregulators previously unknown to participate in TCDD-induced gene activation, regulators of small GTPases of the Ras superfamily, UBX domain-containing proteins and the oncogenic protein LYRIC. The results open up new directions for research on the molecular mechanisms of dioxin action and toxicity.
gene expressionProtein PhosphorylationARNT homodimersSILAC approachregulation factortranscriptional coregulatorsdioxin action2 hQuantitative Phosphoproteomic AnalysisSer 77 phosphorylationprotein phosphorylationUBXobligate partner5 L rat hepatoma cells5648 phosphorylated peptidesOther proteinstranscriptional activation1 nM TCDDtime point2156 phosphoproteinstranscription factor ARNTgene activationTCDD exposureoncogenic protein LYRICnovel mechanismRas superfamilyDMSO