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...

The serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase kinase, but the mechanisms of Raf-1 activation are incompletely understood. To dissect these mechanisms, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras-and v-Src-transformed cells (transformed membranes). Wild-type (His) 6 -and FLAG-Raf-1 were activated in a Ras-and ATP-dependent manner by transformed membranes; however, Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS) were not. Raf-1 proteins lacking putative regulatory sites for an unidentified kinase (S259A) or protein kinase C (S499A) were activated but with apparently reduced efficiency. The kinase(s) responsible for activation by Ras or Src may reside in the plasma membrane, since GTP loading of plasma membranes from quiescent NIH 3T3 cells (parental membranes) induced de novo capacity to activate Raf-1. Wild-type Raf-1, possessing only basal activity, was not activated by parental membranes in the absence of GTP loading. In contrast, Raf-1 Y340D, possessing significant activity, was, surprisingly, stimulated by parental membranes in a Ras-independent manner. The results suggest that activation of Raf-1 by phosphorylation may be permissive for further modulation by another membrane factor, such as a lipid. A factor(s) extracted with methanol-chloroform from transformed membranes or membranes from Sf9 cells coexpressing Ras and Src Y527F significantly enhanced the activity of Raf-1 Y340D or active Raf-1 but not that of inactive Raf-1. Our findings suggest a model for activation of Raf-1, wherein (i) Raf-1 associates with Ras-GTP, (ii) Raf-1 is activated by tyrosine and/or serine phosphorylation, and (iii) Raf-1 activity is further increased by a membrane cofactor.The proto-oncogene product Raf-1 is a member of a family of serine/threonine protein kinases (Raf-1, B-Raf, and A-Raf) that function in protein kinase cascades important for mitogenic signaling (31). Raf-1 phosphorylates and activates mitogen-activated protein (MAP) kinase kinase (MKK, also known as MEK), the specific activator of MAP kinase (23). MAP kinase in turn phosphorylates several regulatory proteins (4, 21) in the cytoplasm (e.g., PHAS1, p90 rsk , and cPLA 2 ) and nucleus (e.g., p62TCF and c-Myc) to alter the program of transcription and translation required for mitogenesis. Raf-1 exhibits restricted substrate specificity in vitro (12). Although other in vivo substrates for Raf-1 than MKK may exist, none have been definitively established.The mechanism of Raf-1 activation is incompletely understood. Activation of Raf-1 in vivo occurs at the plasma membrane (20, 28) and is dependent upon association with Ras-GTP (24). Association of purified Raf-1 and Ras proteins in the presence of ATP in vitro is not sufficient to cause activat...

Abstract. The role of Raf and MAPK (mitogenactivated protein kinase) during the maturation of Xenopus oocytes was investigated. Treatment of oocytes with progesterone resulted in a shift in the electrophoretic mobility of Raf at the onset of germinal vesicle breakdown (GVBD), which was coincident with the activation of MAPK. Expression of a kinasedefective mutant of the human Raf-1 protein (KD-RAF) inhibited progesterone-mediated MAPK activation. MAPK activation was also inhibited by KD-Raf in oocytes expressing signal transducers of the receptor tyrosine kinase (RTK) pathway, including an activated tyrosine kinase (Tpr-Met), a receptor tyrosine kinase (EGFr), and Ha-Ras w2. KD-RAF completely inhibited GVBD induced by the RTK pathway. In contrast, KD-RAF did not inhibit GVBD and the progression to Meiosis II in progesterone-treated oocytes. Injection of Mos-specific antisense oligodeoxyribonucleotides inhibited MAPK activation in response to progesterone and Tpr-Met, but failed to inhibit these events in oocytes expressing an oncogenic deletion mutant of Raf-1 (AN'Raf). Injection of antisense oligodeoxyribonucleotides to Mos also reduced the progesterone-and Tpr-Met-induced electrophoretic mobility shift of Xenopus Raf. These results demonstrate that RTKs and progesterone participate in distinct yet overlapping signaling pathways resulting in the activation of maturation or M-phase promoting factor (MPF). Maturation induced by the RTK pathway requires activation of Raf and MAPK, while progesterone-induced maturation does not. Furthermore, the activation of MAPK in oocytes appears to require the expression of Mos.T I-IE c-raf-1 gene is the normal cellular counterpart of the v-raf transforming gene of the murine sarcoma virus 3611 (59). The proto-oncogene product encoded by the c-raf-1 gene, Raf-1, is a 70-74-kD phosphoprotein with intrinsic kinase activity towards serine and threonine residues (for review see Morrison [45]). The Raf-1 protein consists of a carboxyl-terminal kinase domain and an aminoterminal regulatory region that is deleted in v-raf, generating a constitutively active kinase (29,68,69). Raf-1 is ubiquitously expressed and is hyperphosphorylated, primarily on serine residues, in many cell lines in response to mitogen treatment (30, 46). A close correlation has been established between Raf-1 hyperphosphorylation and activation of its kinase activity in cells stimulated by growth factors (30,40). Moreover, Raf-1 activity is required for growth factorinduced proliferation of NIH/3T3 cells and certain erythroid cells (11,34).Recently, genetic and biochemical evidence has placed Raf-1 in a signal transduction cascade downstream of both receptor tyrosine kinases (RTKs) 1 and ras, and upstream of 1. Abbreviations used in this paper: EGFr, EGF receptor; GVBD, germinal vesicle breakdown; IGF, insulin-like growth factor; KD-Raf, kinasemitogen-activated protein kinase (MAPK) (for review see Roberts [60]). Furthermore, recent reports suggest that the protein which activates MAPK (also known as MAPK kinase [M...

B-Raf, a member of the Raf family of serine/threonine kinases, is expressed primarily in the brain and in the nervous system. In this study, the biochemical properties of the B-Raf protein were investigated in nerve growth factor (NGF)-responsive cell lines and in brain tissues. B-Raf was identified by using phosphopeptide mapping analysis and cDNA analysis as a 95-kDa protein which is primarily localized in the cytosol. NGF rapidly stimulated both serine and threonine phosphorylation in vivo and autophosphorylation activity