Functional consequences of mutations in a putative Akt phosphorylation motif of B‐raf in human cancers

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Abstract
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.