Raf family kinases: old dogs have learned new tricks

Abstract

First identified in the early 1980s as retroviral oncogenes, the Raf proteins have been the objects of intense research. The discoveries 10 years later that the Raf family members (Raf-1, B-Raf, and A-Raf) are bona fide Ras effectors and upstream activators of the ubiquitous ERK pathway increased the interest in these proteins primarily because of the central role that this cascade plays in cancer development. The important role of Raf in cancer was corroborated in 2002 with the discovery of B-Raf genetic mutations in a large number of tumors. This led to intensified drug development efforts to target Raf signaling in cancer. This work yielded not only recent clinical successes but also surprising insights into the regulation of Raf proteins by homodimerization and heterodimerization. Surprising insights also came from the hunt for new Raf targets. Although MEK remains the only widely accepted Raf substrate, new kinase-independent roles for Raf proteins have emerged. These include the regulation of apoptosis by suppressing the activity of the proapoptotic kinases, ASK1 and MST2, and the regulation of cell motility and differentiation by controlling the activity of Rok-α. In this review, we discuss the regulation of Raf proteins and their role in cancer, with special focus on the interacting proteins that modulate Raf signaling. We also describe the new pathways controlled by Raf proteins and summarize the successes and failures in the development of efficient anticancer therapies targeting Raf. Finally, we also argue for the necessity of more systemic approaches to obtain a better understanding of how the Ras-Raf signaling network generates biological specificity.

Keywords: Raf kinases; apoptosis; cancer; kinase inhibitors; signal transduction.

Figure 1.

The prototypical Ras-Raf-MEK-ERK pathway. Activated receptor tyrosine kinases (RTKs) recruit the guanine nucleotide exchange factor SOS, which activates Ras proteins by exchanging GDP for GTP. Activated GTP-loaded Ras binds to Raf, initiating Raf activation. Active Raf phosphorylates and activates MEK, which in turn phosphorylates and activates ERK. While the phosphorylation cascade comprising Raf, MEK, and ERK is linear, ERK features more than 150 substrates both in the cytosol and nucleus. Protein interactions and phosphorylation reactions are modulated by a number of scaffolding proteins (see Fig. 5).

Figure 2.

Structure and regulatory phosphorylation sites of Raf proteins. (A) Common structure of the Raf proteins. Color-coded regions are described in the text. (B) Comparison of the structure and phosphorylation residues of the 3 Raf isoforms. Red residues indicate activating phosphorylation sites, black are inhibitory sites, and blue are sites that have been described as both activating and inhibitory. The major in vitro autophosphorylation sites in Raf-1 and B-Raf are in green.

Figure 3.

The Raf-1 activation/deactivation cycle. This scheme shows the salient steps in Raf-1 activation/deactivation. Activating events are coded red, inactivating processes are in black, and activation states are in blue. In quiescent cells, Raf-1 is phosphorylated on both 14-3-3 binding sites pS259 and pS621, and 14-3-3 maintains the closed inactive conformation. Upon membrane recruitment by activated Ras, pS259 is dephosphorylated by the corecruited phosphatases PP1 or PP2A. Subsequently, phosphorylation of the N-region and activation loop and homodimerization or heterodimerization (with B-Raf) cause full activation of Raf-1. Deactivation is initiated by pS338 inducing PP5 binding and dephosphorylation of pS338. In addition, ERK-mediated feedback phosphorylation suppresses Raf-1 catalytic activity. Eventually, PP2A (and maybe other unknown phosphatases) dephosphorylates the remainder of activating sites and the ERK feedback sites. Rephosphorylation of S259 allows intramolecular bidentate 14-3-3 rebinding and return to the inactive state.

Figure 4.

New Raf-1 signaling pathways that depend on protein interactions but not Raf-1 kinase activity or MEK. Raf-1 can suppress apoptosis in a MEK-independent fashion in several ways: 1) by binding to and inhibiting ASK1; 2) by suppressing cytochrome C release through voltage-dependent anion channels (VDACs) at the mitochondria; 3) by acting as a scaffold to recruit PKCθ to phosphorylate and inactivate BAD; 4) by inhibiting the mammalian MST2 pathway; and 5) by inhibiting Rok-α–induced Fas maintenance and clustering at the cell membrane. In addition, the inhibition of Rok-α by Raf-1 is also required for motility by regulating the actin cytoskeleton and for skin tumorigenesis by preventing keratinocyte differentiation and sustaining Myc expression.

Figure 5.

Scaffolding proteins in Raf-MEK-ERK signaling. Scaffolding proteins form Raf-MEK-ERK signaling platforms at different subcellular localizations. See text for details.

Figure 6.

Schematic diagram of feedback mechanisms in the Ras-ERK pathway. Short- and long-term negative feedbacks are colored light and dark blue, respectively. Short- and long-term positive feedbacks are colored orange and red, respectively.