Transcriptional and post-translational modifications of B-Raf in quinol-thioether induced tuberous sclerosis renal cell carcinoma.
Increased activity of B-Raf has been identified in approximately 7% of human cancers. Treatment of Eker rats (Tsc-2(EK/+) ), 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-2(EK/+) 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-2(EK/+) rats, as well as tumors excised from 8-month TGHQ-treated Tsc-2(EK/+) 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) © 2015 Wiley Periodicals, Inc.
Southwest Environmental Health Sciences Center, College of Pharmacy, The University of Arizona, Tucson, Arizona.|Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona.
Jennifer D
JD
Cohen
Southwest Environmental Health Sciences Center, College of Pharmacy, The University of Arizona, Tucson, Arizona.|Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona.
Matthew
M
Labenski
Southwest Environmental Health Sciences Center, College of Pharmacy, The University of Arizona, Tucson, Arizona.|Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona.
Nicholas J
NJ
Mastrandrea
Southwest Environmental Health Sciences Center, College of Pharmacy, The University of Arizona, Tucson, Arizona.|Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona.
Ryan D
RD
Canatsey
Southwest Environmental Health Sciences Center, College of Pharmacy, The University of Arizona, Tucson, Arizona.|Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona.
Terrence J
TJ
Monks
Southwest Environmental Health Sciences Center, College of Pharmacy, The University of Arizona, Tucson, Arizona.|Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona.
Serrine S
SS
Lau
D000818:Animals; D002292:Carcinoma, Renal Cell / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives* / Q000633:toxicity; D006801:Humans; D006873:Hydroquinones / Q000633:toxicity*; D007680:Kidney Neoplasms / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism*; D008297:Male; D009374:Neoplasms, Experimental; D010766:Phosphorylation; D000072417:Protein Domains; D011499:Protein Processing, Post-Translational; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D012326:RNA Splicing / Q000187:drug effects; D051381:Rats; D014402:Tuberous Sclerosis / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism*; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000172:deficiency
D006873:Hydroquinones; C000624653:TSC2 protein, human; C000624655:Tsc2 protein, rat; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; C078765:2,3,5-(triglutathion-S-yl)hydroquinone; C482123:Braf protein, rat; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; C546782:Raf1 protein, rat; D005978:Glutathione
B-Raf; MAPK; Quinol-thioether; Raf-1; renal cell carcinoma