Functional consequences of mutations in a putative Akt phosphorylation motif of B-raf in human cancers.
Mutations in the B-raf gene have been reported in a number of human cancers, including melanoma and lung cancer. More than 80% of the reported B-raf mutations were V599E; however, non-V599E mutations have been frequently found in non-small cell lung cancers as compared with melanoma. Some non-V599E mutations have been found surrounding Thr439, which is thought likely to be one of the three Akt phosphorylation sites in the B-raf protein. However, as a previous report indicated that Thr439 was not phosphorylated by Akt, the functional consequences of these mutations have been unclear. Here, we examined the effects of cancer-related B-raf mutations surrounding Thr439 on the activation of the mitogen-activated protein/ extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (Erk) pathway and the transformation of NIH 3T3 fibroblasts. Among the three reported mutations (K438Q, K438T, and T439P) found in non-small cell lung carcinoma and melanoma, none elevated the activity of the MEK/Erk cascade as determined by in vitro kinase assays, immunoblots using antibody specific for phosphorylated Erk, or Elk1-dependent reporter assays. The inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling by LY294002 increased the Erk activation induced by the mutant B-raf proteins, as well as by wild-type B-raf. Furthermore, the B-raf mutants did not have increased NIH 3T3-transforming activities, as determined by colony-formation assays. These results suggest that the B-raf mutations surrounding Thr439 found in human cancers are unlikely to contribute to increased oncogenic properties of B-raf.
Ikenoue
Tsuneo
T
Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Japan.
Kanai
Fumihiko
F
Hikiba
Yohko
Y
Tanaka
Yasuo
Y
Imamura
Jun
J
Ohta
Miki
M
Jazag
Amarsanaa
A
Guleng
Bayasi
B
Asaoka
Yoshinari
Y
Tateishi
Keisuke
K
Kawakami
Takayuki
T
Matsumura
Masayuki
M
Kawabe
Takao
T
Omata
Masao
M
D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt
D011518:Proto-Oncogene Proteins; C494918:AKT1 protein, human; C482119:BRAF protein, human; D017346:Protein Serine-Threonine Kinases; D048493:Proto-Oncogene Proteins B-raf; D051057:Proto-Oncogene Proteins c-akt