The serine/threonine kinase activity of the Raf-1 proto-oncogene product is stimulated by the activation of many yrosine kinases, including growth factor receptors and pp6OV'. Recent studies of growth factor signal transduction pathways demonstrate that Raf-1 functions downstream of activated tyrosine kinases and p2l' and upstream of mitogen-activated protein kinase. However, coexpression of both activated tyrosine kinases and p2l1 is required for maximal activation of Raf-1 in the baculovirus-Sf9 expression system. In this study, we investigated the role of tyrosine kinases and tyrosine phosphorylation in the regulation of Raf-1 activity. Using the baculovirus-Sf9 expression system, we identified Tyr-340 and Tyr-341 as the major tyrosine phosphorylation sites of Raf-1 when coexpressed with activated tyrosine kinases. Introduction of a negatively charged residue that may mimic the effect of phosphorylation at these sites activated the catalytic activity of Raf-1 and generated proteins that could transform BALB/3T3 cells and induce the meiotic maturation of Xenopus oocytes. In contrast, substitution of noncharged residues that were unable to be phosphorylated produced a protein that could not be enzymatically activated by tyrosine kinases and that could block the meiotic maturation of oocytes induced by components of the receptor tyrosine kinase pathway. These findings demonstrate that mutation of the tyrosine phosphorylation sites can dramatically alter the function of Raf-1.In addition, this is the first report that a transforming Raf-1 protein can be generated by a single amino acid substitution.One of the key pathways involved in the transmission of proliferative, developmental, and oncogenic signals from receptor tyrosine kinases to the nucleus involves the activation of p2lmS and the serine/threonine kinases Raf-1 and mitogen-activated protein kinase (MAPK, also known as ERK [reviewed in references 28 and 34]). Biochemical and genetic studies have revealed that this pathway functions in many cell types in organisms as diverse as Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, and mammals (8-10, 20, 42). In mammalian cells, activation of p2lras and Raf-1 by receptors that stimulate cellular tyrosine phosphorylation results in the sequential activation of the serine/threonine kinase activities of MEK (5) (also known as MKK1 [50]), MAPK, and RSK (28,34). The activation of Raf-1 in many cases is dependent upon the activity of p21ras and can be induced by expression of oncogenic p2lvras, indicating that Raf-1 may function downstream of p2lras (27,41,49). In addition, p2lras enhances the kinase activity of Raf-1 when these proteins are coexpressed in the baculovirus-Sf9 system (48). However, for maximal activation of Raf-1 in this system, the presence of activated tyrosine kinases is also required. Because oncogenic forms of Raf-1 can phosphorylate and activate MEK, thereby activating MAPK, and RSK, these kinases are thought to function downstream of 18). In addition, immunoprecipitates of mitoge...

Recent reports have demonstrated the in vivo association of Raf-1 with members of the 14-3-3 protein family. To address the significance of the Raf-1-14-3-3 interaction, we investigated the enzymatic activity and biological function of Raf-1 in the presence and absence of associated 14-3-3. The interaction between these two molecules was disrupted in vivo and in vitro with a combination of molecular and biochemical techniques. Biochemical studies demonstrated that the enzymatic activities of Raf-1 were equivalent in the presence and absence of 14-3-3. Furthermore, mixing of purified Raf-1 and 14-3-3 in vitro was not sufficient to activate Raf-1. With a molecular approach, Cys-165 and Cys-168 as well as Ser-259 were identified as residues of Raf-1 required for the interaction with 14-3-3. Cys-165 and Cys-168 are located within the conserved cysteine-rich region of the CR1 domain, and Ser-259 is a conserved site of serine phosphorylation found within the CR2 domain. Mutation of either Cys-165 and Cys-168 or Ser-259 prevented the stable interaction of Raf-1 with 14-3-3 in vivo. Consistent with the model in which a site of serine phosphorylation is involved in the Raf-1-14-3-3 interaction, dephosphorylated Raf-1 was unable to associate with 14-3-3 in vitro. Phosphorylation may represent a general mechanism mediating 14-3-3 binding, because dephosphorylation of the Bcr kinase (known to interact with 14-3-3) also eliminated its association with 14-3-3. Finally, mutant Raf-1 proteins unable to stably interact with 14-3-3 exhibited enhanced enzymatic activity in human 293 cells and Xenopus oocytes and were biologically activated, as demonstrated by their ability to induce meiotic maturation of Xenopus oocytes. However, in contrast to wild-type Raf-1, activation of these mutants was independent of Ras. Our results therefore indicate that interaction with 14-3-3 is not essential for Raf-1 function.

Ras and Raf-1 are key proteins involved in the transmission of developmental and proliferative signals generated by receptor and nonreceptor tyrosine kinases. Genetic and biochemical studies demonstrate that Raf-1 functions downstrem of Ras in many sialig pathways. Although directly associates with GTP-bound Ras, an effect of this interaction on Raf-1 activity in vivo has not been established. To examine the biological consequence of the Ras/Raf-1 interaction in vivo, we set out to identify key residues of Raf-1 required for Ras binding. In this report, we show that a single amino acid mutation in Raf-1 (Arg8 to Leu) disrupted the interaction with Ras in vitro and in the yeast two-hybrid system. This mutation prevented Ras-mediated but not tyrosine kinase-mediated enzymatic activation of Raf-1 in the baculovirus/5f9 expression system. Furthermore, kinase-defective Raf-1 proteins containing the Arg'" -) Leu mutation were no longer dominantinhibitory or capable of blocking Ras-mediated signal transduction in Xenopus laevis oocytes. These results demonstrate that the association of Raf-1 and Ras modulates both the kinase activity and the biological function of Raf-1 and identify Arg8' as a critical residue involved in this interaction. In addition, the fng that tyrosine kinases can stimulate the enzymatic activity of Raf-1 proteins containing a mutation at the Rasinteraction site suggests that Raf-i can be activated by Rasindependent pathways. The Raf-1 and Ras protooncogene products serve as central intermediates in many signaling pathways by connecting upstream tyrosine kinases with downstream serine/threonine kinases, such as mitogen-activated protein kinase (MAPK) and MAPK kinase (MKK, also known as MEK) (1, 2). Ras is a membrane-localized guanine nucleotide-binding protein that is biologically active in the GTP-bound state (3, 4). Raf-1 is a protein-serine/threonine kinase located primarily in the cytosol (5, 6). Growth factors that stimulate cellular proteintyrosine kinase activity enhance both the kinase activity of Raf-1 and the proportion ofRas bound to GTP (reviewed in ref. 7). The activation of Raf-1 in many cases is dependent on the activity ofRas, suggesting that Raf-1 functions downstream of . Further evidence positioning Raf-1 downstream of Ras comes from studies using deregulated and dominantinhibitory mutants ofRas and Raf-1 in mammalian cells (9-15), as well as from studies examining developmental pathways in Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans (16)(17)(18)(19). Recently, Raf-1 has been shown to interact directly with GTP-bound forms of Ras in vitro and in yeast two-hybrid expression systems (20-25). Ras has also been reported to coimmunoprecipitate with Raf-1 from stimulated, but not unstimulated, mammalian cells (26,27). On the basis of these experiments and genetic and biochemical studies positioning Raf-1 downstream ofRas, Raf-1 has been proposed to be a direct effector ofRas. However, whether Raf-1 activity is modulated by the association with Ras ha...