RAF kinases regulate cell proliferation and survival and can be dysregulated in tumors1,2. The role of RAF in cell proliferation has been linked to its ability to activate mitogen-activated protein kinase kinase 1 (MEK) and mitogen-activated protein kinase 1 (ERK). Here we identify a MEK-independent role for RAF in tumor growth. Specifically, in mitotic cells, CRAF becomes phosphorylated on Ser338 and localizes to the mitotic spindle of proliferating tumor cells in vitro as well as in murine tumor models and in biopsies from individuals with cancer. Treatment of tumors with allosteric inhibitors, but not ATP-competitive RAF inhibitors, prevents CRAF phosphorylation on Ser338 and localization to the mitotic spindle and causes cell-cycle arrest at prometaphase. Furthermore, we identify phospho-Ser338 CRAF as a potential biomarker for tumor progression and a surrogate marker for allosteric RAF blockade. Mechanistically, CRAF, but not BRAF, associates with Aurora kinase A (Aurora-A) and Polo-like kinase 1 (Plk1) at the centrosomes and spindle poles during G2/M. Indeed, allosteric or genetic inhibition of phospho-Ser338 CRAF impairs Plk1 activation and accumulation at the kinetochores, causing prometaphase arrest, whereas a phospho-mimetic Ser338D CRAF mutant potentiates Plk1 activation, mitosis and tumor progression in mice. These findings show a previously undefined role for RAF in tumor progression beyond the RAF-MEK-ERK paradigm, opening new avenues for targeting RAF in cancer.
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We thank K. Elliott and J. Lesperance for their assistance with the mouse experiments and immunohistochemistry, and M. Schmid, M. Kaulich and J. Desgrosellier for discussions. We thank K. Lee (US National Institutes of Health National Cancer Institute) and R. Erikson (Harvard University) for providing Plk1 constructs. D.A.C. was supported by grants CA78045, CA119335, CA95262 and CA104898 from the US National Institutes of Health.
The authors declare no competing financial interests.
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Nuclear localized Raf1 isoform alters DNA-dependent protein kinase activity and the DNA damage response
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