Transcriptional and post-translational modifications of B-Raf in quinol-thioether induced tuberous sclerosis renal cell carcinoma

Jennifer D. Cohen, Matthew Labenski, Nicholas J. Mastrandrea, Ryan D. Canatsey, Terrence J. Monks, Serrine S. Lau

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

Increased activity of B-Raf has been identified in approximately 7% of human cancers. Treatment of Eker rats (Tsc-2EK/+), bearing a mutation in one allele of the tuberous sclerosis-2 (Tsc-2) gene, with the nephrocarcinogen 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of the wild-type allele of Tsc-2 in renal preneoplastic lesions and tumors. These tumors have increased protein expression of B-Raf, C-Raf (Raf-1), and increased expression and activity of ERK kinase. Similar changes are observed in Raf kinases following TGHQ-mediated transformation of primary renal epithelial cells derived from Tsc-2EK/+ rats (QTRRE cells), cells that are also null for tuberin. Herein, we utilized LC-MS/MS to identify constitutive phosphorylation of S345 and S483 in both 100- and 95-kDa forms of B-Raf in QTRRE cells. Using microRotofor liquid-phase isoelectric focusing, we identified four fractions of B-Raf that contain different post-translational modification profiles in QTRRE cells. Amplification of the kinase domain of B-Raf from QTRRE cells, outer-stripe of the outer medulla of 8-month TGHQ- or vehicle-treated Tsc-2+/+and Tsc-2EK/+ rats, as well as tumors excised from 8-month TGHQ-treated Tsc-2EK/+ rats revealed three splice variants of B-Raf within the kinase domain. These splice variants differed by approximately 340, 544, and 600 bp; confirmed by sequencing. No point mutations within the kinase domain of B-Raf were identified. In addition, B-Raf/Raf-1/14-3-3 complex formation in the QTRRE cells was decreased by sorafenib, with concomitant selective decreases in p-ERK levels. Transcriptional and post-translational characterization of critical kinases, such as B-Raf, may contribute to the progression of tuberous sclerosis RCC. (246/250)

Original languageEnglish (US)
Pages (from-to)1243-1250
Number of pages8
JournalMolecular Carcinogenesis
Volume55
Issue number8
DOIs
StatePublished - Aug 1 2016

Keywords

  • B-Raf
  • MAPK
  • Quinol-thioether
  • Raf-1
  • renal cell carcinoma

ASJC Scopus subject areas

  • Molecular Biology
  • Cancer Research

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