DA-Raf, a dominant-negative antagonist of the Ras–ERK pathway, is a putative tumor suppressor

Highlights: • DA-Raf binds to oncogenic Ras and consequently suppresses the ERK pathway. • DA-Raf suppresses oncogenic Ras-induced transformation and tumorigenesis. • DA-Raf mutants cannot bind to oncogenic Ras or suppress the ERK pathway. • DA-Raf mutants cannot suppress oncogenic Ras-induced transformation and tumorigenesis. • DA-Raf is a tumor suppressor protein against Ras-induced tumorigenesis. Activating mutations of RAS genes, particularly KRAS, are detected with high frequency in human tumors. Mutated Ras proteins constitutively activate the ERK pathway (Raf–MEK–ERK phosphorylation cascade), leading to cellular transformation and tumorigenesis. DA-Raf1 (DA-Raf) is a splicing variant of A-Raf and contains the Ras-binding domain (RBD) but lacks the kinase domain. Accordingly, DA-Raf antagonizes the Ras–ERK pathway in a dominant-negative fashion and suppresses constitutively activated K-Ras-induced cellular transformation. Thus, we have addressed whether DA-Raf serves as a tumor suppressor of Ras-induced tumorigenesis. DA-Raf(R52Q), which is generated from a single nucleotide polymorphism (SNP) in the RBD, and DA-Raf(R52W), a mutant detected in a lung cancer, neither bound to active K-Ras nor interfered with the activation of the ERK pathway. They were incapable of suppressing activated K-Ras-induced cellular transformation and tumorigenesis in mice, in which K-Ras-transformed cells were transplanted. Furthermore, although DA-Raf was highly expressed in lung alveolar epithelial type 2 (AE2) cells, its expression was silenced in AE2-derived lung adenocarcinoma cell lines with oncogenic KRAS mutations. These results suggest that DA-Raf represents a tumor suppressor protein against Ras-induced tumorigenesis.