| Budget Amount *help |
¥13,200,000 (Direct Cost: ¥13,200,000)
Fiscal Year 2006: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Research Abstract |
Identification of the novel tumor suppressor gene DA-Raf and elucidation of its physiological functions
DA-Raf1 that we have identified is a novel splicing isoform of A-Raf and contains Ras-binding domain but lacks kinase domain. As inferred from its structure, DA-Raf1 bound to Ras and M-Ras and served as an intrinsic dominant-negative antagonist of the Ras-ERK pathway (B-Raf-MEK-ERK). By this mechanism, DA-Raf1 interfered with the transformation of NIH3T3 fibroblasts by v-K-Ras and tumor formation in nude mice transplanted the cells. Thus, DA-Raf1 might serve as a novel tumor suppressor gene.
The Ras-ERK pathway suppresses skeletal muscle cell differentiation by inhibiting the muscle-specific transcription factor myogenin. The expression of DA-Raf1 was prominently induced during differentiation of mouse C2C12 skeletal muscle cells. Differentiation phenotypes, i.e., growth arrest, expression of myogenin and late muscle-specific genes, and myotube formation by myoblast fusion were facilitated in C2C12 cells by the overexpression of DA-Rafl. In contrast, the knockdown of DA-Raf1 by RNAi markedly interfered with these differentiation phenotypes. Consequently, DA-Raf1 is essential for skeletal muscle cell differentiation by antagonizing the Ras-ERK pathway.
RSK activated by the Ras-ERK pathway inhibits apoptosis by phosphorylating Ser112 in Bad. DA-Raf1 overexpressed in COS-1 cells induced apoptosis. The phosphorylation of Bad Ser112 was suppressed and caspase-3 was activated in these cells. On the other hand, serum-starved NIH3T3 cells underwent apoptosis. DA-Raf1 expression was highly induced, the phosphorylation of Bad Ser112 was suppressed, and caspase-3 was activated in these cells. These results suggest that the elevation of DA-Raf1 expression under the serum starvation conditions induces apoptosis by suppressing Bad phosphorylation through inhibition of the Ras-ERK pathway.
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