Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1–deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-α to the plasma membrane. Raf-1 physically associates with Rok-α in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

The mechanism by which Raf-1 antagonizes Rok-α to promote migration and tumorigenesis is revealed.

Activation of the Raf/MEK/ERK pathway, often by gainof-function mutations of RAS or RAF, is observed in many human cancers. The extracellular signal-regulated kinase (ERK) pathway is required for the proliferation of cancer cell lines harboring activating BRAF or, to a lesser extent, activating RAS mutations. It is still unclear, however, whether the pathway is required in vivo for tumor development, particularly in tumors in which B-Raf is not mutationally activated. During embryonic development, B-Raf is essential for angiogenesis in the placenta. To address the question of whether B-Raf contributed to tumor angiogenesis in vivo we conditionally ablated B-Raf in a model of pancreatic islet carcinoma driven by the functional inactivation of tumor suppressors (RIP1Tag2), which critically depends on angiogenesis for growth. We find that B-Raf is dispensable for the proliferation of tumor cells in culture, but necessary for ERK activation and for the expression of angiogenic factors by tumor cells in vivo and in vitro. In vivo, these defects result in the formation of hollow tumors with decreased vessel density and strongly reduced proliferation. The progression from adenoma to carcinoma is also significantly impaired. Thus, endogenous B-Raf contributes to the development of RIP1Tag2 tumors by supporting the stromal response and tumor progression.