Nuclear localized Raf1 isoform alters DNA‐dependent protein kinase activity and the DNA damage response

Abstract

Raf1/c-Raf is a well-characterized serine/threonine-protein kinase that links Ras family members with the MAPK/ERK signaling cascade. We have identified a novel splice isoform of human Raf1 that causes protein truncation and loss of the C-terminal kinase domain (Raf1-tr). We found that Raf1-tr has increased nuclear localization compared with full-length Raf1, and this finding was secondary to reduced binding of Raf1-tr to the cytoplasmic chaperone FK506 binding protein 5. We show that Raf1-tr has increased binding to DNA-dependent protein kinase (DNA-PK), which inhibits DNA-PK function and causes amplification of irradiation- and bleomycin-induced DNA damage. We found that the human colorectal cancer cell line, HCT-116, displayed reduced expression of Raf1-tr, and reintroduction of Raf1-tr sensitized the cells to bleomycin-induced apoptosis. Furthermore, we identified differential Raf1-tr expression in breast cancer cell lines and showed that breast cancer cells with increased Raf1-tr expression become sensitized to bleomycin-induced apoptosis. Collectively, these results demonstrate a novel Raf1 isoform in humans that has a unique noncanonical role in regulating the double-stranded DNA damage response pathway through modulation of DNA-PK function.-Nixon, B. R., Sebag, S. C., Glennon, M. S., Hall, E. J., Kounlavong, E. S., Freeman, M. L., Becker, J. R. Nuclear localized Raf1 isoform alters DNA-dependent protein kinase activity and the DNA damage response.

YWHAE-NUTM2 oncoprotein regulates proliferation and cyclin D1 via RAF/MAPK and Hippo pathways

“…These novel studies demonstrate that cyclin D1 overexpression in HG-ESS depends on YWHAE–NUTM2 activation of RAF/MAPK and Hippo pathways. The rationales for studying RAF/MAPK pathway roles were: (1) MAPK is a known positive regulator of cyclin D1 expression 18 ; (2) BRAF and RAF1 heterodimerization is generally required for MEK/MAPK phosphorylation and cell proliferation 19 , 20 ; (3) BRAF and RAF1 complex with YWHAE and other 14–3–3 proteins 19 , 21 , 22 ;, and (4) precedent for RAF1 translocation from cytoplasm to the nucleus 23 – 25 raised the possibility that RAF1 might interact with the predominantly nuclear YWHAE-NUTM2 oncoproteins. The rationales for studying Hippo pathway roles were: (1) upregulation of cyclin D1, along with CTGF and CYR61, is a well-known Hippo function, resulting from transcriptional upregulation by the Hippo effectors YAP and TAZ; (2) YAP and TAZ interact with YWHAE and other 14–3–3 proteins 14 – 17 ;, and (3) our recent studies show that Hippo YAP/TAZ mechanisms induce dramatic cyclin D1 overexpression and cell proliferation in another subtype of mesenchymal neoplasia, gastrointestinal stromal tumor 26 .…”

Targeted splicing therapy: new strategies for colorectal cancer

“…Alternative splicing results in the inclusion of a new exon 11 in the RAF1 mRNA, which causes a frameshift and introduces three premature stop codons, leading to the truncation of the RAF1 protein and the absence of its C-terminal kinase domain, The resulting splice isoform is named RAF1-tr ( 281 ). RAF1-tr can increase nuclear localization and inhibits the function of DNA damage–regulating protein.…”

“…RAF1-tr can increase nuclear localization and inhibits the function of DNA damage–regulating protein. This leads to an increase in the levels of DNA damage after the exposure to bleomycin and radiation, and enhances the apoptotic response of CRC cells to double-stranded DNA damage ( 281 ).…”