Functional analysis of mutations within the kinase activation segment of B-Raf in human colorectal tumors.
Mutations in the B-Raf gene have been reported in a number of human cancers, including colorectal carcinoma. More than 80% of the B-Raf mutations were V599E. Although other mutations have been reported, their functional consequences were unclear. Here, we examined the effect of colon tumor-associated B-Raf mutations within the kinase activation segment, including V599E, on extracellular signal-regulated kinase (Erk) and nuclear factor kappaB (NFkappaB) signaling, and on the transformation of NIH3T3 fibroblasts. Among the six mutations examined, only the B-Raf V599E and K600E mutations greatly increased Erk and NFkappaB signaling, and the transformation of NIH3T3 cells. The B-Raf F594L mutation moderately elevated Erk signaling and NIH3T3 transformation, but did not significantly increase NFkappaB signaling. Although the basal kinase activity of the B-Raf T598I mutant was comparable with that of wild-type, its oncogenic Ras-induced kinase activity was decreased to 60% of wild-type activity. The B-Raf D593V and G595R mutants showed severely reduced kinase activity and affected neither NFkappaB signaling nor NIH3T3 transforming activity. These results suggest that the B-Raf activation segment mutations other than V599E reported in colorectal tumors do not necessarily contribute to carcinogenesis by increasing kinase and transforming activities.
Ikenoue
Tsuneo
T
Division of Gastroenterology, The Institute for Adult Diseases, Asahi Life Foundation, Tokyo, Japan. t-ikenoue@asahi-life.or.jp
Hikiba
Yohko
Y
Kanai
Fumihiko
F
Tanaka
Yasuo
Y
Imamura
Jun
J
Imamura
Takaaki
T
Ohta
Miki
M
Ijichi
Hideaki
H
Tateishi
Keisuke
K
Kawakami
Takayuki
T
Aragaki
Jun
J
Matsumura
Masayuki
M
Kawabe
Takao
T
Omata
Masao
M
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D015179:Colorectal Neoplasms / Q000201:enzymology* / Q000235:genetics*; D004789:Enzyme Activation; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D048728:MAP Kinase Kinase Kinase 1; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000502:physiology; D041681:NIH 3T3 Cells; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014158:Transcription, Genetic; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D048948:14-3-3 Proteins; D018841:HSP90 Heat-Shock Proteins; D016328:NF-kappa B; D014446:Tyrosine 3-Monooxygenase; C482120:Braf protein, mouse; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D048728:MAP Kinase Kinase Kinase 1; D020930:MAP Kinase Kinase Kinases; C482576:MAP3K1 protein, human; C482578:Map3k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases