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• W2034681985 abstract "Random mutagenesis and genetic screens for impaired Raf function in Caenorhabditis elegans were used to identify six loss-of-function alleles of lin-45 raf that result in a substitution of a single amino acid. The mutations were classified as weak, intermediate, and strong based on phenotypic severity. We engineered these mutations into the homologous residues of vertebrate Raf-1 and analyzed the mutant proteins for their underlying biochemical defects. Surprisingly, phenotype strength did not correlate with the catalytic activity of the mutant proteins. Amino acid substitutions Val-589 and Ser-619 severely compromised Raf kinase activity, yet these mutants displayed weak phenotypes in the genetic screen. Interestingly, this is because these mutant Raf proteins efficiently activate the MAPK (mitogen-activated protein kinase) cascade in living cells, a result that may inform the analysis of knockout mice. Equally intriguing was the observation that mutant proteins with non-functional Ras-binding domains, and thereby deficient in Ras-mediated membrane recruitment, displayed only intermediate strength phenotypes. This confirms that secondary mechanisms exist to couple Ras to Raf in vivo. The strongest phenotype in the genetic screens was displayed by a S508N mutation that again did not correlate with a significant loss of kinase activity or membrane recruitment by oncogenic Ras in biochemical assays. Ser-508 lies within the Raf-1 activation loop, and mutation of this residue in Raf-1 and the equivalent Ser-615 in B-Raf revealed that this residue regulates Raf binding to MEK. Further characterization revealed that in response to activation by epidermal growth factor, the Raf-S508N mutant protein displayed both reduced catalytic activity and aberrant activation kinetics: characteristics that may explain the C. elegans phenotype. Random mutagenesis and genetic screens for impaired Raf function in Caenorhabditis elegans were used to identify six loss-of-function alleles of lin-45 raf that result in a substitution of a single amino acid. The mutations were classified as weak, intermediate, and strong based on phenotypic severity. We engineered these mutations into the homologous residues of vertebrate Raf-1 and analyzed the mutant proteins for their underlying biochemical defects. Surprisingly, phenotype strength did not correlate with the catalytic activity of the mutant proteins. Amino acid substitutions Val-589 and Ser-619 severely compromised Raf kinase activity, yet these mutants displayed weak phenotypes in the genetic screen. Interestingly, this is because these mutant Raf proteins efficiently activate the MAPK (mitogen-activated protein kinase) cascade in living cells, a result that may inform the analysis of knockout mice. Equally intriguing was the observation that mutant proteins with non-functional Ras-binding domains, and thereby deficient in Ras-mediated membrane recruitment, displayed only intermediate strength phenotypes. This confirms that secondary mechanisms exist to couple Ras to Raf in vivo. The strongest phenotype in the genetic screens was displayed by a S508N mutation that again did not correlate with a significant loss of kinase activity or membrane recruitment by oncogenic Ras in biochemical assays. Ser-508 lies within the Raf-1 activation loop, and mutation of this residue in Raf-1 and the equivalent Ser-615 in B-Raf revealed that this residue regulates Raf binding to MEK. Further characterization revealed that in response to activation by epidermal growth factor, the Raf-S508N mutant protein displayed both reduced catalytic activity and aberrant activation kinetics: characteristics that may explain the C. elegans phenotype. The importance of the Ras signaling pathway in the regulation of cellular proliferation, differentiation, and survival has been well established using a variety of biochemical and genetic systems. One of the main Ras effectors is Raf, a serine/threonine kinase that is evolutionarily conserved in higher eukaryotes. Raf is a critical part of a signaling cascade that connects cell-surface receptors with regulatory events within the cell (1Dickson B. Sprenger F. Morrison D. Hafen E. Nature. 1992; 360: 600-603Crossref PubMed Scopus (244) Google Scholar, 2Han M. Golden A. Han Y. Sternberg P.W. Nature. 1993; 363: 133-140Crossref PubMed Scopus (192) Google Scholar, 3Troppmair J. Bruder J.T. App H. Cai H. Liptak L. Szeberenyi J. Cooper G.M. Rapp U.R. Oncogene. 1992; 7: 1867-1873PubMed Google Scholar, 4Wood K.W. Sarnecki C. Roberts T.M. Blenis J. Cell. 1992; 68: 1041-1050Abstract Full Text PDF PubMed Scopus (662) Google Scholar). Activated Raf proteins phosphorylate MEKs 1The abbreviations used are: MEK, MAPK/extracellular signal-regulated kinase kinase; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; RBD, Ras-binding domain; CRD, cysteine-rich domain; GFP, green fluorescent protein; BHK, baby hamster kidney cells. on two serine residues, which in turn phosphorylate and activate ERKs (5Alessi D.R. Saito Y. Campbell D.G. Cohen P. Sithanandam G. Rapp U. Ashworth A. Marshall C.J. Cowley S. EMBO J. 1994; 13: 1610-1619Crossref PubMed Scopus (468) Google Scholar, 6Gardner A.M. Vaillancourt R.R. Lange-Carter C.A. Johnson G.L. Mol. Biol. Cell. 1994; 5: 193-201Crossref PubMed Scopus (80) Google Scholar, 7Zheng C.F. Guan K.L. EMBO J. 1994; 13: 1123-1131Crossref PubMed Scopus (300) Google Scholar). Raf is localized to the cytoplasm as an inactive multi-protein complex. The activation of eukaryotic endogenous Raf molecules is dependent on relieving the interaction between the N-terminal regulatory and C-terminal kinase domain of the Raf molecule (8Heidecker G. Huleihel M. Cleveland J.L. Kolch W. Beck T.W. Lloyd P. Pawson T. Rapp U.R. Mol. Cell. Biol. 1990; 10: 2503-2512Crossref PubMed Scopus (206) Google Scholar, 9Stanton Jr., V.P. Nichols D.W. Laudano A.P. Cooper G.M. Mol. Cell. Biol. 1989; 9: 639-647Crossref PubMed Scopus (189) Google Scholar). The initial event in Raf activation is the recruitment of Raf from the cytosol to the plasma membrane through a high affinity interaction between the switch 1 region of activated Ras-GTP and the N-terminal minimal Ras-binding domain of Raf (Raf RBD) (10Finney R. Herrera D. Methods Enzymol. 1995; 255: 310-323Crossref PubMed Scopus (14) Google Scholar, 11Marais R. Light Y. Paterson H.F. Marshall C.J. EMBO J. 1995; 14: 3136-3145Crossref PubMed Scopus (527) Google Scholar, 12Stokoe D. Macdonald S.G. Cadwallader K. Symons M. Hancock J.F. Science. 1994; 264: 1463-1467Crossref PubMed Scopus (845) Google Scholar, 13Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar, 14Zhang X. Settleman J. Kyriakis J.M. Takeuchi-Suzuki E. Elledge S.J. Marshall M.S. Bruder J.T. Rapp U.R. Avruch J. Nature. 1993; 364: 308-365Crossref PubMed Scopus (687) Google Scholar). This step is critical for Raf activation as point mutations within Ras or Raf that disrupt this interaction block Raf activation (15Block C. Janknecht R. Herrmann C. Nassar N. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 244-251Crossref PubMed Scopus (124) Google Scholar, 16Fabian J.R. Vojtek A.B. Cooper J.A. Morrison D.K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5982-5986Crossref PubMed Scopus (157) Google Scholar). However, the interaction between Ras and Raf alone is not sufficient for full Raf activation, because Ras-GTP cannot activate Raf unless Ras-GTP is membrane bound (12Stokoe D. Macdonald S.G. Cadwallader K. Symons M. Hancock J.F. Science. 1994; 264: 1463-1467Crossref PubMed Scopus (845) Google Scholar). Full Raf activation involves interaction of the Raf cysteine-rich domain (Raf CRD) with Ras and membrane phospholipids, dephosphorylation of specific Raf serine residues, and a complex series of phosphorylation events at serine, tyrosine, and threonine residues. Several proteins bind to Raf and regulate its activity. The chaperone proteins Hsp90 and Cdc37 bind to and stabilize mammalian Raf proteins, holding them in a conformation permissive for recruitment by activated Ras (17Grammatikakis N. Lin J.H. Grammatikakis A. Tsichlis P.N. Cochran B.H. Mol. Cell. Biol. 1999; 19: 1661-1672Crossref PubMed Scopus (227) Google Scholar, 18Silverstein A.M. Grammatikakis N. Cochran B.H. Chinkers M. Pratt W.B. J. Biol. Chem. 1998; 273: 20090-20095Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Disruption of the Raf·chaperone complex in vivo reduces the half-life of Raf and abrogates Ras-dependent membrane recruitment (19Cissel D.S. Beaven M.A. J. Biol. Chem. 2000; 275: 7066-7070Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 20Schulte T.W. Blagosklonny M.V. Ingui C. Neckers L. J. Biol. Chem. 1995; 270: 24585-24588Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). Raf also binds to dimerized 14–3–3 at two phosphorylated serine residues, Ser-259 and Ser-621 (21Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1188) Google Scholar). 14–3–3 also binds to the Raf CRD (22Clark G.J. Drugan J.K. Rossman K.L. Carpenter J.W. Rogers-Graham K. Fu H. Der C.J. Campbell S.L. J. Biol. Chem. 1997; 272: 20990-20993Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), an interaction that serves to stabilize the Raf·14–3–3 complex (23McPherson R.A. Harding A. Roy S. Lane A. Hancock J.F. Oncogene. 1999; 18: 3862-3869Crossref PubMed Scopus (57) Google Scholar). 14–3–3 binding to cytosolic Raf maintains Raf in an inactive conformation permissive for Ras recruitment. Recruitment of Raf to the plasma membrane destabilizes the interaction of 14–3–3 with the N terminus, which allows phosphatases PP1 and PP2A to dephosphorylate Ser-259 thus removing 14–3–3 and allowing full Raf activation (24Abraham D. Podar K. Pacher M. Kubicek M. Welzel N. Hemmings B.A. Dilworth S.M. Mischak H. Kolch W. Baccarini M. J. Biol. Chem. 2000; 275: 22300-22304Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 25Jaumot M. Hancock J.F. Oncogene. 2001; 20: 3949-3958Crossref PubMed Scopus (154) Google Scholar, 26Mitsuhashi S. Shima H. Tanuma N. Matsuura N. Takekawa M. Urano T. Kataoka T. Ubukata M. Kikuchi K. J. Biol. Chem. 2003; 278: 82-88Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 27Roy S. McPherson R.A. Apolloni A. Yan J. Lane A. Clyde-Smith J. Hancock J.F. Mol. Cell. Biol. 1998; 18: 3947-3955Crossref PubMed Scopus (116) Google Scholar). In mammals there are three Raf isoforms: A-Raf, B-Raf, and Raf-1 (C-Raf). Transgenic experiments indicate that the different Raf isoforms are non-redundant (28Huser M. Luckett J. Chiloeches A. Mercer K. Iwobi M. Giblett S. Sun X.M. Brown J. Marais R. Pritchard C. EMBO J. 2001; 20: 1940-1951Crossref PubMed Scopus (277) Google Scholar, 29Mikula M. Schreiber M. Husak Z. Kucerova L. Ruth J. Wieser R. Zatloukal K. Beug H. Wagner E.F. Baccarini M. EMBO J. 2001; 20: 1952-1962Crossref PubMed Scopus (265) Google Scholar, 30Pritchard C.A. Bolin L. Slattery R. Murray R. McMahon M. Curr. Biol. 1996; 6: 614-617Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 31Wojnowski L. Zimmer A.M. Beck T.W. Hahn H. Bernal R. Rapp U.R. Zimmer A. Nat. Genet. 1997; 16: 293-297Crossref PubMed Scopus (251) Google Scholar, 32Wojnowski L. Stancato L.F. Zimmer A.M. Hahn H. Beck T.W. Larner A.C. Rapp U.R. Zimmer A. Mech. Dev. 1998; 76: 141-149Crossref PubMed Scopus (98) Google Scholar, 33Wojnowski L. Stancato L.F. Larner A.C. Rapp U.R. Zimmer A. Mech. Dev. 2000; 91: 97-104Crossref PubMed Scopus (97) Google Scholar). A-Raf knockout mice are viable but suffer intestinal and/or neurological defects depending on genetic background (30Pritchard C.A. Bolin L. Slattery R. Murray R. McMahon M. Curr. Biol. 1996; 6: 614-617Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). B-Raf knockout mice have defects in neuroepithelial differentiation and the maintenance of endothelial cell viability and die in utero at 10–13 days post coitum due to vascular hemorrhage (31Wojnowski L. Zimmer A.M. Beck T.W. Hahn H. Bernal R. Rapp U.R. Zimmer A. Nat. Genet. 1997; 16: 293-297Crossref PubMed Scopus (251) Google Scholar). Raf-1 knockout mice are anemic and die in utero or shortly after birth, with vascular defects in the yolk sac and placenta as well as an increase in the number of apoptotic cells throughout the embryo (28Huser M. Luckett J. Chiloeches A. Mercer K. Iwobi M. Giblett S. Sun X.M. Brown J. Marais R. Pritchard C. EMBO J. 2001; 20: 1940-1951Crossref PubMed Scopus (277) Google Scholar, 29Mikula M. Schreiber M. Husak Z. Kucerova L. Ruth J. Wieser R. Zatloukal K. Beug H. Wagner E.F. Baccarini M. EMBO J. 2001; 20: 1952-1962Crossref PubMed Scopus (265) Google Scholar, 32Wojnowski L. Stancato L.F. Zimmer A.M. Hahn H. Beck T.W. Larner A.C. Rapp U.R. Zimmer A. Mech. Dev. 1998; 76: 141-149Crossref PubMed Scopus (98) Google Scholar). Intriguingly, Raf-1 Y340F/Y341F “knockin” mice, which express the kinase-inactive RafFF mutant in place of wild-type Raf-1, survive to adulthood with no detectable phenotype (28Huser M. Luckett J. Chiloeches A. Mercer K. Iwobi M. Giblett S. Sun X.M. Brown J. Marais R. Pritchard C. EMBO J. 2001; 20: 1940-1951Crossref PubMed Scopus (277) Google Scholar). One conclusion from this study is that Raf-1 kinase function may not be required for normal mouse development. Instead, Raf-1 could play an anti-apoptotic role during development via a mechanism that is independent of its kinase function, perhaps by an interaction with the pro-apoptotic, stress activated protein kinase apoptosis signal-regulating kinase 1 (34Chen J. Fujii K. Zhang L. Roberts T. Fu H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7783-7788Crossref PubMed Scopus (270) Google Scholar). Investigation as to whether Raf-1 kinase activity is critical for Raf-1 biological function is hampered in mammalian systems due to the overlapping activities of different Raf isoforms (33Wojnowski L. Stancato L.F. Larner A.C. Rapp U.R. Zimmer A. Mech. Dev. 2000; 91: 97-104Crossref PubMed Scopus (97) Google Scholar). Invertebrates have only a single Raf isoform (D-Raf in Drosophila and LIN-45 in C. elegans). In C. elegans, signaling pathways that involve Raf are used multiple times during development to control a variety of cell fate decisions (2Han M. Golden A. Han Y. Sternberg P.W. Nature. 1993; 363: 133-140Crossref PubMed Scopus (192) Google Scholar). These signaling pathways have been characterized most extensively during the formation of the hermaphrodite vulva, a specialized epithelial structure used for egg laying (35Horvitz H.R. Sternberg P.W. Nature. 1991; 351: 535-541Crossref PubMed Scopus (193) Google Scholar). A mutation that reduces the activity of a gene in the Raf signaling pathway generates a vulvaless (Vul) phenotype. By contrast, a mutation that results in constitutive activity of one of these genes results in a multivulva (Muv) phenotype. Thus, vulva formation serves as an easily visualized readout of the activity of the Ras/Raf/MEK/ERK-signaling pathway. This signaling pathway regulates additional cell fate decisions including differentiation of the excretory cell, which is necessary for larval viability, and progression of germ cells through pachytene stage, which is necessary for fertility (36Yochem J. Sundaram M. Han M. Mol. Cell. Biol. 1997; 17: 2716-2722Crossref PubMed Scopus (62) Google Scholar, 37Church D.L. Guan K.L. Lambie E.J. Development. 1995; 121: 2525-2535PubMed Google Scholar). As there is only a single Raf isoform, genetic analysis in C. elegans is ideally suited to examine the functional significance of Raf within a physiological context. To characterize the function of the Raf gene, we used random chemical mutagenesis and genetic screens for worms with developmental defects to identify a collection of lin-45 raf alleles (38Hsu V. Zobel C.L. Lambie E.J. Schedl T. Kornfeld K. Genetics. 2002; 160: 481-492PubMed Google Scholar). The molecular lesions in these alleles were identified, and the mutations were classified based on phenotypic severity; they ranged from weak loss-of-function mutations that cause mild defects to strong or complete loss-of-function mutations that cause severe phenotypes. Six of the alleles contain missense mutations that result in substitution of a single amino acid. To understand the biochemical defects caused by these mutations, we engineered them into homologous residues of vertebrate Raf-1 and characterized the biochemical properties of the mutant proteins in vertebrate cells. These assays included measurements of basal kinase activity, Ras-stimulated kinase activity, membrane recruitment, and binding to 14–3–3, Hsp90, Cdc37, and MEK. Our analysis identifies functionally significant residues in the Ras-binding domain, protein kinase domain as well as MEK and 14–3–3-binding domains and demonstrates the necessity of these residues and domains for Raf activity in an animal. An interesting observation that mirrors the Raf-1 knockout mice studies recently published is that in vitro Raf kinase activity does not necessarily correlate to the in vivo function of the Raf protein. The significance of this apparently paradoxical observation is discussed. Plasmids and Mutagenesis—Raf mutant constructs were generated using the QuikChange site-directed mutagenesis kit (Stratagene) using a human Raf-1 or B-Raf clone tagged with a FLAG or myc epitope. All constructs were sequenced prior to use. EXV-K-RasG12V, EXV-FLAG-Raf, EXV-FLAG-RafDD, and EXV-FLAG-RafCAAX have all been previously described (39Roy S. Lane A. Yan J. McPherson R. Hancock J.F. J. Biol. Chem. 1997; 272: 20139-20145Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Raf-GFP was constructed by subcloning the Raf-1 cDNA into pEGFP-N3 (Clontech). Cell Culture and Antibodies—COS cells and baby hamster kidney cells (BHK) were grown and maintained in HEPES-buffered Dulbecco's modified Eagle's medium containing 10% donor calf serum as described previously (39Roy S. Lane A. Yan J. McPherson R. Hancock J.F. J. Biol. Chem. 1997; 272: 20139-20145Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Mouse monoclonal antibodies, anti-Raf-1, Hsp90, and Cdc37, were obtained from Transduction Laboratories, and anti-FLAG from Eastman Kodak Co. Polyclonal anti-14–3–3 antibody was obtained from Santa Cruz Biotechnology, Inc. Anti-Ras rat monoclonals (Y13–259 and Y13–238) were made from hybridomas acquired from the American Type Culture Collection. Polyclonal GFP antibody was obtained from I.A. Prior (University of Queensland) and monoclonal GFP antibody from Roche Applied Science. Phospho-MEK polyclonal and phospho-ERK monoclonal antibodies were purchased from New England Biolabs. Polyclonal MEK-1/2 (New England Biolabs) and ERK-1 polyclonal (Santa Cruz) were used as input control antibodies where indicated. Cell Transfection and Immunofluorescence—COS cells were electroporated as described previously (40Huang D.C.S. Marshall C.J. Hancock J.F. Mol. Cell. Biol. 1993; 13: 2420-2431Crossref PubMed Scopus (59) Google Scholar). After 54 h cells were switched to serum-free medium and incubated for a further 18 h before harvesting. BHK cells were seeded onto coverslips for immunofluorescence or 10-cm dishes for biochemical assays, and transfected using LipofectAMINE (Invitrogen) according to the manufacturer's instructions. Cells on 10-cm dishes were maintained in serum-free Dulbecco's modified Eagle's medium for 16 h after lipofection before being harvested. Cells were washed and scraped on ice into 0.5 ml of buffer A (10 mm Tris-HCl, pH7.5, 25 mm NaF, 5 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, 100 μm NaVO4). After 10 min on ice, cells were passed 25× through a 23-gauge needle and the nuclei removed by low speed centrifugation. Post-nuclear supernatants were spun at 100,000 × g. The supernatant (S100) was removed, and the sedimented fraction (P100) was rinsed and sonicated for 5 min in 100 μl of ice-cold buffer A. The S100 fraction and resuspended P100 fractions were snap-frozen and stored at –70 °C in aliquots after measuring protein content by the Bradford reaction. Cells on coverslips were fixed in 4% paraformaldehyde 24 h after lipofection. The coverslips were washed for 10 min in phosphate-buffered saline, permeabilized with 0.2% Triton X-100 in phosphate-buffered saline, and blocked in 3% bovine serum albumin in phosphate-buffered saline. The primary antibody Y13–238 (Ras) was diluted in blocking buffer at a 1:2 to 1:30 dilution and the secondary antibody, anti-rat CY-3, used at 1:300 dilution. Raf-GFP was visualized by direct fluorescence. Western Blotting—Expression and subcellular localization of ectopically expressed proteins were determined by immunoblotting. Cellular fractions, normalized for protein content, were resolved on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes using semi-dry transfer. The membranes were probed with anti-Raf-1, Y13–259 for Ras, or anti-14–3–3, anti-Hsp90, anti-CDC37, or anti-GFP monoclonal and polyclonal antibodies as appropriate, then developed using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. Where indicated immunoblots were quantified by phosphorimaging (Bio-Rad). Raf Kinase Assays—P100 aliquots of transfected cells were normalized for protein content and assayed for Raf activity using a two-stage coupled MEK/ERK assay with phosphorylation of myelin basic protein as readout (39Roy S. Lane A. Yan J. McPherson R. Hancock J.F. J. Biol. Chem. 1997; 272: 20139-20145Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). For assays of cytosolic Raf proteins, S100 fractions were normalized for Raf content and Raf proteins immunoprecipitated with M2 anti-FLAG monoclonal or anti-GFP polyclonal antibodies as previously described (23McPherson R.A. Harding A. Roy S. Lane A. Hancock J.F. Oncogene. 1999; 18: 3862-3869Crossref PubMed Scopus (57) Google Scholar). Immunoprecipitates were then assayed for Raf activity as above. After the kinase assays, immunoprecipitates were taken up in SDS-PAGE sample buffer, resolved by SDS-PAGE, and immunoblotted. Identification and Molecular Characterization of lin-45 raf Alleles—Alleles of lin-45 raf were identified by conducting genetic screens for C. elegans hermaphrodites with defective vulval development or sterility. To investigate how these mutations affect the activity of lin-45 and the role of lin-45 during development, the phenotypes of these mutants were characterized extensively (38Hsu V. Zobel C.L. Lambie E.J. Schedl T. Kornfeld K. Genetics. 2002; 160: 481-492PubMed Google Scholar). Based on these findings, the alleles were arranged in a series of increasing severity that is likely to correspond to an increasing loss of lin-45 activity; the series is the same whether larval lethality, vulval formation, or sterility are considered (Ref. 38Hsu V. Zobel C.L. Lambie E.J. Schedl T. Kornfeld K. Genetics. 2002; 160: 481-492PubMed Google Scholar and Table I).Table Ilin-45 mutations cause larval lethality, sterility, and abnormal vulva formationGenotypeLIN-45 mutationRaf-1 mutationLarval lethalaThe percentage of hatched eggs judged to be homozygous mutants that generated dead larvae; most dead larvae displayed rigid, rod-like morphology.Abnormal vulvabThe percentage of all adult hermaphrodites judged to be homozygous mutants that displayed a severe egg-laying defect, no discernable vulva, or a protruding vulva (abnormal vulva) or that generated no larval progeny (sterile). A more detailed genetic analysis of the mutants is displayed in Hsu et al. (38).SterilebThe percentage of all adult hermaphrodites judged to be homozygous mutants that displayed a severe egg-laying defect, no discernable vulva, or a protruding vulva (abnormal vulva) or that generated no larval progeny (sterile). A more detailed genetic analysis of the mutants is displayed in Hsu et al. (38).ClassificationcW, weak; I, intermediate; and S, strong.%%%Wild-type000n1925R108WH79W110Wn1924I726FV589F501Wn2520S754FS619F000Wn2018P92SP63S76241In2506R118WR89W86933Ioz201S645NS508N55100100Sa The percentage of hatched eggs judged to be homozygous mutants that generated dead larvae; most dead larvae displayed rigid, rod-like morphology.b The percentage of all adult hermaphrodites judged to be homozygous mutants that displayed a severe egg-laying defect, no discernable vulva, or a protruding vulva (abnormal vulva) or that generated no larval progeny (sterile). A more detailed genetic analysis of the mutants is displayed in Hsu et al. (38Hsu V. Zobel C.L. Lambie E.J. Schedl T. Kornfeld K. Genetics. 2002; 160: 481-492PubMed Google Scholar).c W, weak; I, intermediate; and S, strong. Open table in a new tab The Effect of lin-45 Loss-of-function Mutations on Ras Mediated Raf-1 Activation—To elucidate the biochemical basis of the inactivating mutations in lin-45, we initially generated the loss-of-function point mutations in lin-45. However, when expressed in mammalian cell lines the LIN-45 protein was catalytically inactive. We therefore turned to the well characterized model system of Ras-dependent Raf-1 activation in COS cells. K-Ras was used for these experiments because this mammalian Ras isoform most closely resembles C. elegans let-60 ras. Amino acid substitutions were introduced into FLAG-Raf (Raf-1 with an N-terminal FLAG epitope tag) at residues corresponding to the lin-45 loss-of-function point mutations identified in the genetic screens (Table I and Fig. 1). Briefly, the lin-45 mutations P92S, R108W, R118W, S645N, I726F, and S754F were introduced at the homologous Raf-1 residues P63S, H79W, R89W, S508N, V589F, and S619F, respectively. Wild-type and mutated FLAG-Raf-1 constructs were co-expressed with constitutively active RasG12V in COS cells. Membrane fractions of these cells were normalized for Raf-1 content, and Raf-1 kinase activity was measured in a coupled MEK/ERK assay. Fig. 2 shows that all of the lin-45 point mutations, except H79W, profoundly abrogated K-Ras-dependent Raf-1 activation. Raf-1 mutants resistant to RasG12V activation may be deficient in membrane recruitment or refractory to later membrane activation events. To examine Ras-dependent plasma membrane recruitment, the lin-45 point mutations were introduced into Raf-GFP (Raf-1 with a C-terminal GFP epitope tag). BHK cells were then co-transfected with the Raf-GFP constructs and RasG12V. Plasma membrane recruitment was assessed using confocal microscopy. Fig. 3 shows that Raf-GFP plasma membrane recruitment was abrogated by the two RBD point mutations P63S and R89W but was unaffected by the H79W substitution. None of the mutations outside of the Raf RBD compromised Raf-GFP membrane recruitment.Fig. 2Ras-dependent activation of lin-45 loss-of-function mutations. COS cells transfected with activated RasG12V and Raf-1 constructs indicated were fractionated and crude membrane (P100) fractions were immunoblotted for Ras and Raf-1 (lower panel). P100 fractions from each transfection were normalized for Raf-1 content and assayed for Raf-1 kinase activity using a coupled MEK/ERK assay (upper panel). The results show mean Raf specific activity from a single transfection assayed in duplicate. Similar results were obtained in three independent COS cell transfections.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Ras-dependent plasma membrane recruitment of lin-45 loss-of-function mutations. BHK cells were co-transfected with activated RasG12V and Raf-GFP containing lin-45 mutations. Raf-GFP proteins were detected by direct fluorescence. Representative cells are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Effect of lin-45 Loss-of-function Mutations on Raf-1 Basal Kinase Activity and Associated Proteins—We next examined the effect of the lin-45 point mutations on Raf-1 basal kinase activity and interaction with known associated proteins. FLAG-Raf and Raf-GFP constructs containing lin-45 point mutations were expressed equivalently in COS cells and anti-FLAG or anti-GFP immunoprecipitates prepared from the cytosolic S100 fraction. Basal Raf kinase activity in immunoprecipitates normalized for Raf-1 protein was measured in a coupled MEK/ERK assay. The results in Fig. 4 show that point mutations within the Raf RBD, P63S, H79W, and R89W, had no effect on Raf-1 basal kinase activity. In contrast, point mutations within the Raf kinase domain, S508N and V589F, and a point mutation directly adjacent to the COOH-terminal 14–3–3 binding motif (S619F) all markedly reduced basal kinase activity. Immunoprecipitates were then blotted for 14–3–3, Cdc37, and Hsp90, which are important co-factors for Raf-1 activation. (FLAG immunoprecipitates could not be blotted for Cdc37, or GFP immunoprecipitates for 14–3–3, due to secondary antibody species cross-reactivity.) The mutations P63S, V589F, and S619F severely reduced the association of 14–3–3 with Raf-1, but no mutation had any significant effect on Hsp90 or Cdc37 interactions. Thus reduced 14–3–3 association has a minimal effect on Cdc37 or Hsp90 interactions with Raf-1. Basal Raf-1 kinase activity could be reduced because of increased interaction between the N-terminal regulatory and catalytic domains, or structural disruption of the Raf-1 kinase domain. Replacement of tyrosines 340 and 341 with aspartic acid partially relieves the neg" @default.
• W2034681985 created "2016-06-24" @default.
• W2034681985 creator A5001938439 @default.
• W2034681985 creator A5037758372 @default.
• W2034681985 creator A5053309905 @default.
• W2034681985 creator A5077272790 @default.
• W2034681985 date "2003-11-01" @default.
• W2034681985 modified "2023-09-30" @default.
• W2034681985 title "Identification of Residues and Domains of Raf Important for Function in Vivo and in Vitro" @default.
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