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• W2037449911 abstract "Identifying 14-3-3 isoform-specific substrates and functions may be of broad relevance to cell signaling research because of the key role played by this family of proteins in many vital processes. A multitude of ligands have been identified, but the extent to which they are isoform-specific is a matter of debate. Herein we demonstrate, both in vitro and in vivo, a specific, functionally relevant interaction of human 14-3-3γ with the molecular scaffold KSR1, which is mediated by the C-terminal stretch of 14-3-3γ. Specific binding to 14-3-3γ protected KSR1 from epidermal growth factor-induced dephosphorylation and impaired its ability to activate ERK2 and facilitate Ras signaling in Xenopus oocytes. Furthermore, RNA interference-mediated inhibition of 14-3-3γ resulted in the accumulation of KSR1 in the plasma membrane, all in accordance with 14-3-3γ being the cytosolic anchor that keeps KSR1 inactive. We also provide evidence that KSR1-bound 14-3-3γ heterodimerized preferentially with selected isoforms and that KSR1 bound monomeric 14-3-3γ. In sum, we have demonstrated ligand discrimination among 14-3-3 isoforms and shed light on molecular mechanisms of 14-3-3 functional specificity and KSR1 regulation. Identifying 14-3-3 isoform-specific substrates and functions may be of broad relevance to cell signaling research because of the key role played by this family of proteins in many vital processes. A multitude of ligands have been identified, but the extent to which they are isoform-specific is a matter of debate. Herein we demonstrate, both in vitro and in vivo, a specific, functionally relevant interaction of human 14-3-3γ with the molecular scaffold KSR1, which is mediated by the C-terminal stretch of 14-3-3γ. Specific binding to 14-3-3γ protected KSR1 from epidermal growth factor-induced dephosphorylation and impaired its ability to activate ERK2 and facilitate Ras signaling in Xenopus oocytes. Furthermore, RNA interference-mediated inhibition of 14-3-3γ resulted in the accumulation of KSR1 in the plasma membrane, all in accordance with 14-3-3γ being the cytosolic anchor that keeps KSR1 inactive. We also provide evidence that KSR1-bound 14-3-3γ heterodimerized preferentially with selected isoforms and that KSR1 bound monomeric 14-3-3γ. In sum, we have demonstrated ligand discrimination among 14-3-3 isoforms and shed light on molecular mechanisms of 14-3-3 functional specificity and KSR1 regulation. The 14-3-3 proteins comprise a large family of highly conserved, acidic polypeptides of 28–33 kDa that are expressed ubiquitously in all eukaryotic species (1Aitken A. Semin. Cancer Biol. 2006; 16: 162-172Crossref PubMed Scopus (646) Google Scholar). Seven isoforms, each encoded by a distinct gene, have been described in mammals: β, γ, ϵ, η, σ, τ/θ, and ζ. In plants, up to 15 isoforms have been identified, and in yeast, Drosophila melanogaster and Caenorhabditis elegans, only two isoforms have been reported (1Aitken A. Semin. Cancer Biol. 2006; 16: 162-172Crossref PubMed Scopus (646) Google Scholar). Initially described in 1967 (2Moore B.W. Perez V.J. Carlson F Physiological and Biochemical Aspects of Nervous Integration. Prentice-Hall, Englewood Cliffs, NJ1967: 343-359Google Scholar), these proteins were characterized a decade ago as the first distinct phosphoserine (pSer) 6The abbreviations used are: pSer, phosphoserine; KSR, kinase suppressor of Ras; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; PP2A, protein phosphatase type 2A; CTS, C-terminal stretch; CRD, cysteine-rich domain; CAT, chloramphenicol acetyltransferase; HA, hemagglutinin; GVBD, germinal vesicle breakdown; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase; PBS, phosphate-buffered saline; EYFP, enhanced yellow fluorescent protein; WB, Western blot; IP, immunoprecipitation; HA, hemagglutinin; GVBD, germinal vesicle breakdown; siRNA, small interfering RNA; EGF, epidermal growth factor. -binding proteins (3Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1195) Google Scholar). Since then, a varied multitude of interacting partners have been identified, participating in cellular processes as diverse as signal transduction, cell-cycle control, apoptosis, regulation of metabolism, protein trafficking, cell morphology, transcription, stress response, and oncogenic transformation (1Aitken A. Semin. Cancer Biol. 2006; 16: 162-172Crossref PubMed Scopus (646) Google Scholar, 4Dougherty M.K. Morrison D.K. J. Cell Sci. 2004; 117: 1875-1884Crossref PubMed Scopus (397) Google Scholar), thereby highlighting 14-3-3 proteins as key mediators of intracellular signaling. Large scale analyses aimed at identifying potential 14-3-3 ligands have consistently resulted in long lists of proteins. Two laboratories independently have identified more than 200 interacting partners using and in vitro affinity chromatography protocol (5Meek S.E. Lane W.S. Piwnica-Worms H. J. Biol. Chem. 2004; 279: 32046-32054Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 6Pozuelo Rubio M. Geraghty K.M. Wong B.H. Wood N.T. Campbell D.G. Morrice N. Mackintosh C. Biochem. J. 2004; 379: 395-408Crossref PubMed Scopus (385) Google Scholar), and a recent direct proteomic analysis has identified as many as 170 specific 14-3-3-interacting proteins (7Jin J. Smith F.D. Stark C. Wells C.D. Fawcett J.P. Kulkarni S. Metalnikov P. O'Donnell P. Taylor P. Taylor L. Zougman A. Woodgett J.R. Langeberg L.K. Scott J.D. Pawson T. Curr. Biol. 2004; 14: 1436-1450Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Further, transgenic mouse proteomics allowed the identification of 147 brain proteins interacting with 14-3-3ζ (8Angrand P.O. Segura I. Volkel P. Ghidelli S. Terry R. Brajenovic M. Vintersten K. Klein R. Superti-Furga G. Drewes G. Kuster B. Bouwmeester T. Acker-Palmer A. Mol. Cell. Proteomics. 2006; 5: 2211-2227Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). With the seven mammalian isoforms sharing a 70% identity, the question arises as to how they achieve specificity in regulating hundreds of different proteins. On a structural level, 14-3-3 proteins form U-shaped dimers, each monomer containing nine anti-parallel α-helices, named A to I (9Gardino A.K. Smerdon S.J. Yaffe M.B. Semin. Cancer Biol. 2006; 16: 173-182Crossref PubMed Scopus (222) Google Scholar). Helices A to D are involved in dimer formation, and helices C, E, G, and I form a large amphipathic groove critically involved in binding to pSer/Thr-containing proteins (9Gardino A.K. Smerdon S.J. Yaffe M.B. Semin. Cancer Biol. 2006; 16: 173-182Crossref PubMed Scopus (222) Google Scholar). Screening of phosphopeptide libraries and structural analysis of 14-3-3/phosphopeptide complexes have identified two high-affinity binding motifs: RSXpSXP (mode 1) and RXXXpSXP (mode 2) (10Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar). In addition, exoenzyme S is able to interact with 14-3-3 in a nonphosphorylated form (11Ottmann C. Yasmin L. Weyand M. Veesenmeyer J.L. Diaz M.H. Palmer R.H. Francis M.S. Hauser A.R. Wittinghofer A. Hallberg B. EMBO J. 2007; 26: 902-913Crossref PubMed Scopus (119) Google Scholar), and recently a C-terminal mode 3-binding motif has been described in some 14-3-3 ligands having a general consensus of p(S/T)X1–2-COOH (12Coblitz B. Wu M. Shikano S. Li M. FEBS Lett. 2006; 580: 1531-1535Crossref PubMed Scopus (130) Google Scholar). Despite these exceptions, the majority of ligands bind to 14-3-3 proteins through the unique mode 1 or mode 2 sequence. This, in addition to extensive sequence conservation among 14-3-3 proteins, makes it difficult to uncover any specific role for each isoform and to understand the molecular determinants contributing to substrate specificity. Published data regarding substrate discrimination by 14-3-3 isoforms are scarce, with experiments having been performed mainly in vitro and often including only a few isotypes. Thus far, a detailed molecular analysis of 14-3-3 specificity both in vitro and in vivo is lacking (1Aitken A. Semin. Cancer Biol. 2006; 16: 162-172Crossref PubMed Scopus (646) Google Scholar). An important 14-3-3-interacting protein is the molecular scaffold kinase suppressor of Ras 1 (KSR1) (13Therrien M. Chang H.C. Solomon N.M. Karim F.D. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 879-888Abstract Full Text PDF PubMed Scopus (340) Google Scholar). KSR1 facilitates transduction of Ras-dependent signals by bringing into contact the individual components of the Raf/MEK/ERK cascade (14Claperon A. Therrien M. Oncogene. 2007; 26: 3143-3158Crossref PubMed Scopus (109) Google Scholar, 15Lozano J. Xing R. Cai Z. Jensen H.L. Trempus C. Mark W. Cannon R. Kolesnick R. Cancer Res. 2003; 63: 4232-4238PubMed Google Scholar). Consistent with such a role, KSR1 can interact transiently with Raf and ERK1/2 (16Xing H. Kornfeld K. Muslin A.J. Curr. Biol. 1997; 7: 294-300Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 17Cacace A.M. Michaud N.R. Therrien M. Mathes K. Copeland T. Rubin G.M. Morrison D.K. Mol. Cell. Biol. 1999; 19: 229-240Crossref PubMed Scopus (172) Google Scholar) and can be found in multiprotein signaling complexes, which include MEK1/2 and 14-3-3 (18Stewart S. Sundaram M. Zhang Y. Lee J. Han M. Guan K.L. Mol. Cell. Biol. 1999; 19: 5523-5534Crossref PubMed Scopus (181) Google Scholar). KSR1 also interacts with regulatory enzymes such as the Ser/Thr phosphatase PP2A (19Ory S. Zhou M. Conrads T.P. Veenstra T.D. Morrison D.K. Curr. Biol. 2003; 13: 1356-1364Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar) and the Ser/Thr kinases C-TAK1 (Cdc25C-associated kinase) and CK2 (casein kinase 2) (20Ritt D.A. Zhou M. Conrads T.P. Veenstra T.D. Copeland T.D. Morrison D.K. Curr. Biol. 2007; 17: 179-184Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 21Muller J. Ory S. Copeland T. Piwnica-Worms H. Morrison D.K. Mol. Cell. 2001; 8: 983-993Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Binding of KSR1 to 14-3-3 is regulated by phosphorylation on Ser-392 by C-TAK1 and on Ser-297 by an unknown kinase (17Cacace A.M. Michaud N.R. Therrien M. Mathes K. Copeland T. Rubin G.M. Morrison D.K. Mol. Cell. Biol. 1999; 19: 229-240Crossref PubMed Scopus (172) Google Scholar). The KSR1-bound PP2A activity dephosphorylates pSer-392 in response to Ras activation, resulting in the dissociation of the KSR1/14-3-3 complex and translocation of KSR1 to the plasma membrane, likely mediated by exposure of its cysteine-rich domain (CRD) (19Ory S. Zhou M. Conrads T.P. Veenstra T.D. Morrison D.K. Curr. Biol. 2003; 13: 1356-1364Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). In the membrane, KSR1 facilitates ERK activation by a dual mechanism involving the assembling of a signaling complex (scaffold function) and activation of Raf kinases through its associated CK2 activity (20Ritt D.A. Zhou M. Conrads T.P. Veenstra T.D. Copeland T.D. Morrison D.K. Curr. Biol. 2007; 17: 179-184Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Thus, 14-3-3 proteins critically regulate KSR1 function by sequestering it in the cytosol until Ras activation. It is not known whether KSR1 interacts promiscuously with 14-3-3 proteins or, on the contrary, the interaction is isoform-specific. Therefore, we chose KSR1 as a functionally relevant ligand to study 14-3-3 isoform specificity and to gain insight into its own regulation. Here, we demonstrate the existence of functional specificity among 14-3-3 isoforms by showing that 14-3-3γ interacts specifically with KSR1, regulating its ability to translocate to the plasma membrane and facilitate Ras-induced ERK2 activation. We show that the flexible C-terminal tail of 14-3-3γ is required for a full and specific interaction with KSR1 and that 14-3-3γ heterodimerizes preferentially with selected isoforms when bound to KSR1. Further, we provide data from molecular modeling that rationalizes the reported lower affinity binding of some mode 1-deviating binding sites (such as pSer-297 in KSR1) and also show that KSR1 can bind monomeric 14-3-3γ. Antibodies—Antibodies against KSR1 and 14-3-3ϵ were from BD Biosciences. Antibodies specific for the 14-3-3-β, -γ, -η, -τ and -ζ isoforms, GST, Myc, and an anti-14-3-3 broad antibody were obtained from Santa Cruz Biotechnology. An antibody specific for 14-3-3σ was from Lab Vision. The anti-FLAG antibody was from Sigma-Aldrich. Antibodies against Xenopus XMpk1 mitogen-activated protein kinase (MAPK) and Cdc2 have been described (22Perdiguero E. Nebreda A.R. Methods Mol. Biol. 2004; 250: 299-314PubMed Google Scholar). An antibody specific for KSR1 phosphorylated on residue Ser-392 was produced by immunizing rabbits with the KLH-conjugated phosphopeptide KSR-pSer-392 (H-LRRTEpSVPSDINC-OH). Phosphospecific antibodies were purified from serum by two-step affinity purification using HiTrap protein A (GE Healthcare) and phosphopeptide-coupled SulfoLink columns (Pierce). Cell Culture—293 and COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone) and antibiotics. U2OS cells, kindly provided by Dr. M. A. Medina (Universidad de Málaga, Spain), were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum and antibiotics. Transfections were performed with the FuGENE 6 reagent (Roche Applied Science). Xenopus laevis oocytes were maintained in modified Barth saline (mBarth) medium as described (22Perdiguero E. Nebreda A.R. Methods Mol. Biol. 2004; 250: 299-314PubMed Google Scholar). Cell Lysates—293 or COS-7 cells were washed once with cold PBS and lysed in NP-40 buffer (20 mm Tris-HCl, pH 7.4, 137 mm NaCl, 10% glycerol, 1% Nonidet P-40, 1 mm EDTA, 1 mm dithiothreitol, plus protease and phosphatase inhibitors) (15Lozano J. Xing R. Cai Z. Jensen H.L. Trempus C. Mark W. Cannon R. Kolesnick R. Cancer Res. 2003; 63: 4232-4238PubMed Google Scholar). Oocyte lysates were prepared by resuspending 3–5 frozen oocytes in 10 μl/oocyte of H1K buffer (80 mm β-glycerophosphate, pH 7.5; 20 mm EGTA, 15 mm MgCl2, 2.5 mm benzamidine, and protease inhibitors) and passing them several times through a micropipette tip. For immunoprecipitation, 250 μg of lysates expressing FLAG-tagged proteins were incubated with 20 μl (50% vol/vol) of FLAG affinity resin (Sigma-Aldrich) for 1 h at 4 °C and washed three times with NP-40 buffer. Myc-tagged KSR1 was immunoprecipitated with 2 μg of a polyclonal c-Myc antibody for 2 h at 4 °C followed by a 1-h incubation with 30 μl of 50% protein A/G-agarose beads (Santa Cruz Biotechnology). Plasmids—pCMV-FLAG-KSR1, encoding mouse FLAG-tagged KSR1, was kindly provided by Dr. Richard N. Kolesnick (Memorial Sloan-Kettering Cancer Center, New York). This plasmid was used as a template to obtain the pCMV-FLAG-CA3mut, pCMV-FLAG-KSR1-S297A, pCMV-FLAG-KSR1-S392A, and pCMV-FLAG-KAA plasmids by site-directed mutagenesis with the QuikChange kit (Stratagene). The pCMV-FLAG-KRK/KSR1 plasmid was constructed exactly as described (23Zhou M. Horita D.A. Waugh D.S. Byrd R.A. Morrison D.K. J. Mol. Biol. 2002; 315: 435-446Crossref PubMed Scopus (70) Google Scholar). The seven isoforms of 14-3-3 proteins (β, γ, ϵ, η, σ, τ, and ζ), each in a pGEX-6P1 vector, were provided by Dr. Cheryl L. Walker (M. D. Anderson Cancer Center, Houston, TX). These constructs were used to generate their Myc-tagged counterparts for expression in mammalian cells as follows. The β, η, σ, and τ isoforms were subcloned into the BamHI and EcoRI sites of pCMV-Tag3B (Stratagene), and the ϵ and γ isoforms were subcloned into the BamHI and XhoI sites. The 14-3-3ζ cDNA was amplified by PCR using the pGEX-6P1-14-3-3ζ plasmid as template and primers zBamHI and zEcoRIr (the sequences of all primers used in this study can be found in supplemental Table 1). The PCR product was digested with BamHI/EcoRI and ligated into pCMV-Tag3B. The plasmid pCMV-FLAG-14-3-3γ was obtained by digestion of pCMV-myc-14-3-3γ with BamHI/XhoI and ligation of the resulting fragment into pCMV-Tag2B (Stratagene). To obtain the plasmids for the mammalian two-hybrid experiments, mouse KSR1 was amplified by PCR using pCMV-FLAG-KSR1 as template and primers KSR6b and KSR17. The PCR product was digested with EcoRI/XbaI and subcloned into the pM vector (Stratagene). Primers MycBglII and pCMV3-XbaI were used to amplify the cDNAs coding for the seven 14-3-3 isoforms, using the Myc-tagged constructs as templates. The PCR products were digested with BglII/XbaI (β, γ, ϵ, η, τ, and ζ isoforms) or with BglII/HindIII (σ isoform) and ligated into the pVP16 vector (Stratagene). The 14-3-3 C-terminal deletion mutants (14-3-3-ΔC mutants) were generated by PCR using the Myc-tagged constructs as templates, a common T3 forward primer, and the following isoform-specific reverse primers: ϵ3′-ΔC, γ3′-ΔC, η3′-ΔC, σ3′-ΔC, and τ3′-ΔC. The 14-3-3β-ΔC and 14-3-3ζ-ΔC inserts were amplified using the T3 forward primer and a common β/ζ3′-ΔC reverse primer. PCR products were digested with NotI/XhoI except those corresponding to the βΔC and ζΔC inserts that were digested with NotI/EcoRI. After gel purification they were ligated into pCMV-Tag3B. Plasmids encoding the 14-3-3γ/ϵ, 14-3-3γ/η, 14-3-3γ/ζ, 14-3-3ϵ/γ, and 14-3-3η/γ chimeric molecules were constructed in two steps as follows. A common T3 forward primer was used in combination with reverse primers γ3′-EcoRV, ϵ3′-EcoRV, and η3′-EcoRV and templates pCMV-myc-14-3-3γ, pCMV-myc-14-3-3ϵ, and pCMV-myc-14-3-3η, respectively, to amplify nucleotides coding for residues 1–235 of 14-3-3γ (lacking the last 12 residues), 1–233 of 14-3-3ϵ (lacking the last 22 residues), and 1–236 of 14-3-3η (lacking the last 10 residues). The PCR fragments were digested with NotI/EcoRV and ligated into pCMV-Tag3B to generate plasmids pCMV-myc-14-3-3γC′, pCMV-myc14-3-3ϵC′, and pCMV-myc-14-3-3ηC′. Then, fragments containing the C-terminal residues of the γ, ϵ, η, and ζ isoforms plus vector sequence downstream of the multicloning site were amplified with forward primers γ5′-EcoRV, ϵ5′-EcoRV, η5′-SmaI, ζ5′-SmaI, and the common reverse primer pCMV3′-DraIII. Finally, the PCR products were digested with EcoRV/DraIII or SmaI/DraIII and ligated into pCMV-myc-14-3-3γC′, pCMV-myc14-3-3ϵC′, or pCMV-myc-14-3-3ηC′ to generate the appropriate chimeric constructs. Plasmids for in vitro mRNA synthesis were generated as follows. A mouse KSR1 fragment was PCR-amplified from pCMV-FLAG-KSR1 with primers KEcoRIf and MKSRSalI. The PCR product was digested with BglII/SalI and ligated into pFTX5 precut with BamHI/XhoI. Constructs FTX4-14-3-3β, FTX4-14-3-3η, FTX4-14-3-3σ, and FTX4-14-3-3τ were subcloned from their pGEX6P1-derived counterparts by ligation of a BamHI/EcoRI fragment into pFTX4. Constructs FTX4-14-3-3γ and FTX4-14-3-3ϵ were generated after ligation of BamHI/XhoI fragments from their pGEX6P1 counterparts into pFTX4. The plasmid FTX4-14-3-3ζ was generated by PCR amplification from pGEX6P1-14-3-3ζ using primers zBamHI and zEcoRIr. The amplified cDNA was digested with BamHI/EcoRI and ligated into pFTX4. Vectors pFTX4 and pFTX5 have been described (22Perdiguero E. Nebreda A.R. Methods Mol. Biol. 2004; 250: 299-314PubMed Google Scholar, 24Howell M. Hill C.S. EMBO J. 1997; 16: 7411-7421Crossref PubMed Scopus (57) Google Scholar). The pCMV-myc14-3-3γ-K50E mutant was obtained with mutagenic primers 1433γK50Ef and 1433γK50Er. The 1433γS59Df and 1433γS59Dr primers were used to clone the mutant pCMV-myc-14-3-3γ-S59D and pCMV-FLAG-14-3-3γ-S59D constructs. The EYFP-difopein and EYFP-R18(Lys) plasmids were gifts from Dr. Haian Fu (Emory University, Atlanta, GA). GST Pulldown Assays—GST-14-3-3 proteins were expressed in Escherichia coli DH5α cells essentially as described (25Liu M.Y. Cai S. Espejo A. Bedford M.T. Walker C.L. Cancer Res. 2002; 62: 6475-6480PubMed Google Scholar). Lysates from 293 cells expressing FLAG-KSR1 (250 μg) were incubated with recombinant GST-14-3-3 for 30 min (4 °C). Complexes were recovered by adding 30 μl of 50% (vol/vol) glutathione-Sepharose resin (GE Healthcare) for an additional 30 min (4 °C). After three washes in NP-40 buffer, the amount of FLAG-KSR1 bound to GST-14-3-3 isoforms was determined by Western blot (WB) with a FLAG antibody and quantitated as the percent of input material by loading 12.5 μg of the lysate in each gel. For the competition assays, 150 μm of the phospho- or non-phosphopeptides were incubated with 1 μg of GST-14-3-3 for 30 min at 4 °C before the addition of 150 μg of FLAG-KSR1 lysates. Endogenous B-KSR1 was pulled down by incubating 10 μg of each GST-14-3-3 isoform with 1.5 mg of mouse brain lysates prepared as described (20Ritt D.A. Zhou M. Conrads T.P. Veenstra T.D. Copeland T.D. Morrison D.K. Curr. Biol. 2007; 17: 179-184Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Pulldown/IP Depletion Assay—A FLAG-KSR1-expressing cell lysate (1.5 mg) was subjected to pulldown with 180 μl of 50% (vol/vol) GST-14-3-3γ-prebound beads (10 μg protein/30 μl resin). One-third of the supernatant (∼500 μg), containing unbound proteins, was immunoprecipitated with 30 μl of an anti-FLAG resin, and the remaining (∼1000 μg) was again pulled down with 120 μl of GST-14-3-3γ beads. After that, half of the supernatant (∼500 μg) was subjected to a second round of immunoprecipitation with 30 μl of FLAG resin. The activation state of FLAG-KSR1 was determined with an anti-pKSR1 antibody. Endogenous 14-3-3 still associated with FLAG-KSR1 after each pulldown was detected in the immunoprecipitates with a pan-14-3-3 antibody. ELISA Format Binding Assay—We followed a protocol similar to a published assay (26Paschal B.M. Delphin C. Gerace L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7679-7683Crossref PubMed Scopus (79) Google Scholar). Briefly, different amounts of GST fusion proteins were diluted in coating buffer (PBS, 1 mm phenylmethylsulfonyl fluoride, and 4 mm dithiothreitol) and allowed to bind to the wells of a microtiter plate by overnight incubation at 4 °C. Nonoccupied sites were blocked with blocking buffer (PBS, 0.3% bovine serum albumin, 0.1% Tween 20) for 1 h at 4 °C. Lysates from 293 FLAG-KSR1-expresing cells were diluted in blocking buffer at a final concentration of 0.5 μg/μl and added to the wells (50 μg/well) loaded with the GST-14-3-3 proteins. After 1 h (4 °C), the wells were washed three times with PBS. A horseradish peroxidase-conjugated FLAG antibody diluted 1:3000 in PBST (PBS plus 0.2% Tween 20) was then added to the wells and incubated for 30 min at room temperature. Wells were washed twice with PBS and incubated with 200 μl of solution D (0.04% o-phenylenediamine, 8 mm citric acid, 17 mm Na2HPO4, 0.012% H2O2) at room temperature until color developed (usually 15 min). The reaction was stopped by adding 50 μl/well 1 n H2SO4 and the amount of FLAG-KSR1 recovered on each well was determined by measuring absorbance at 492 nm in a microplate reader. Data were normalized to values obtained from wells containing GST alone. Mammalian Two-hybrid Assay—293 cells were seeded in 60-mm plates (500,000 cells/plate) and transfected with a combination of the following plasmids: pM-CAT reporter (0.2 μg), pM-KSR1 (1.5 μg), and pVP16-myc-14-3-3 specific for each isoform (0.5 μg for γ, 1.0 μg for η, σ, and τ, and 2.0 μg for β, ϵ, and ζ). Forty-eight hours later, CAT expression, indicative of the in vivo KSR1/14-3-3 interaction, was measured by a colorimetric enzyme immunoassay using the CAT ELISA kit (Roche Applied Science). Oocyte Meiotic Maturation Assay—X. laevis oocytes were prepared essentially as described (22Perdiguero E. Nebreda A.R. Methods Mol. Biol. 2004; 250: 299-314PubMed Google Scholar). Plasmids pFTX5-KSR1 and pFTX4-14-3-3 (2–3 μg) were linearized and used as templates for capped mRNA synthesis with the mMESSAGE mMACHINE in vitro transcription kit (Ambion Inc.). To avoid inhibition of Ras-induced maturation due to KSR1 scaffolding activity (17Cacace A.M. Michaud N.R. Therrien M. Mathes K. Copeland T. Rubin G.M. Morrison D.K. Mol. Cell. Biol. 1999; 19: 229-240Crossref PubMed Scopus (172) Google Scholar), the amount of injected KSR1 mRNA was titrated previously. Oocytes were microinjected with 50 nl of the in vitro transcribed mRNAs (2.5 ng for Myc-KSR1 and 35 ng for HA-14-3-3) and maintained at 18 °C in mBarth medium. Five hours later the oocytes were reinjected with 50 ng of a mRNA encoding Myc-H-RasG12K and scored for germinal vesicle breakdown (GVBD) as a measure of meiotic maturation. GVBD was scored when 5–10% of the oocytes injected with Ras alone had undergone GVBD (usually 6–8 h after injection) (17Cacace A.M. Michaud N.R. Therrien M. Mathes K. Copeland T. Rubin G.M. Morrison D.K. Mol. Cell. Biol. 1999; 19: 229-240Crossref PubMed Scopus (172) Google Scholar). RNA Interference—Synthetic siRNAs specific for human 14-3-3γ (NM_012479) or 14-3-3τ (NM_006826) were purchased from Ambion Inc. as 19-mer complementary RNA duplexes with UU overhangs at their 3′-ends. A scrambled sequence with no homology in the human genomic data base was used as a negative control. U2OS cells were seeded in 6-well plates and transfected 24 h later (day 1) with the corresponding siRNAs (20 nm) by the calcium phosphate method. The cells were retransfected 24 h later (day 2) following the same protocol, and on day 3, the cells were transfected with 2.0 μg of pCMV-FLAG-KSR1 using the FuGENE 6 reagent. On day 5, the cells were processed for Western blot and immunofluorescence. Immunofluorescence—Transfected cells grown on coverslips in 6-well plates were formalin-fixed, permeabilized with PBS plus 0.2% Triton X-100, and stained either singly or doubly with a mouse monoclonal FLAG antibody and a rabbit polyclonal Myc antibody followed by an Alexa 488-conjugated goat antimouse antibody and a rhodamine-conjugated goat anti-rabbit antibody. Isolation of 14-3-3 Dimers Bound to KSR1—Total cell lysates (3.5 mg) prepared from 293 cells coexpressing FLAG-KSR1 and Myc-14-3-3γ were immunoprecipitated with anti-FLAG beads as described above and washed sequentially with NP-40 buffer (five times) and Tris-buffered saline (twice). Bound FLAG-KSR1 was eluted twice by incubation at 15 °C for 15 min in 35 μl of 0.1 mg/ml FLAG peptide (Sigma-Aldrich) diluted in Tris-buffered saline. The combined eluates were diluted 1:2 in NP-40 buffer. One-half was immunoprecipitated with 5.6 μg of a Myc polyclonal antibody, and the other half was immunoprecipitated with 5.6 μg of a non-immune rabbit IgG for 2 h at 4 °C followed by a 1-h incubation with 30 μl of protein A-Sepharose. Immunoprecipitates were washed three times with NP-40 buffer, resuspended in 70 μl of Laemmli's sample buffer, and fractionated by SDS-PAGE followed by Western blot with specific anti-14-3-3 antibodies. A 10 μl-aliquot was loaded per well. As a control, we also immunoprecipitated sequentially, with FLAG and Myc antibodies, a 293 lysate expressing FLAG-KSR1 alone. Cross-linking Experiments—COS-7 lysates expressing either FLAG-14-3-3γ or FLAG-14-3-3γ-S59D were subjected to immunoprecipitation with an anti-FLAG affinity resin. Beads were washed three times with NP-40 buffer and twice with phosphate buffer. Dimers in the immunoprecipitates were chemically cross-linked with 50 μg/ml bis(sulfosuccinimidyl) suberate (Sigma-Aldrich) as described (27Woodcock J.M. Murphy J. Stomski F.C. Berndt M.C. Lopez A.F. J. Biol. Chem. 2003; 278: 36323-36327Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Molecular Modeling—Interactions of KSR1 phosphopeptides with residues in the 14-3-3γ basic pocket were modeled with the program Mutmodel (28Shih H.H. Brady J. Karplus M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1697-1700Crossref PubMed Scopus (65) Google Scholar) using the crystal structure of human 14-3-3γ bound to mode 1 phosphopeptide RAIpSLP (Protein Data Bank ID: 2B05) as template. Water accessibility for 14-3-3γ protein was calculated with the DSSP program (29Carter P. Andersen C.A. Rost B. Nucleic Acids Res. 2003; 31: 3293-3295Crossref PubMed Scopus (85) Google Scholar). The DALI program (30Holm L. Sander C. Science. 1996; 273: 595-603Crossref PubMed Scopus (1289) Google Scholar) was used to superimpose the different 14-3-3 proteins complexes retrieved from the Protein Data Bank and the two modeled KSR1 phosphopeptides, using the 2B05 coordinates as the fixed structural reference. The three-dimensional images were rendered with VMD (Visual Molecular Dynamics) (30Holm L. Sander C. Science. 1996; 273: 595-603Crossref PubMed Scopus (1289) Google Scholar). KSR1 Interacts Preferentially with 14-3-3γ in Vitro—The seven human 14-3-3 isoforms are highly similar at the amino acid level, sharing an average identity of 70% and having strictly conserved residues directly involved in pSer recognition (9Gardino A.K. Smerdon S.J. Yaffe M.B. Semin. Cancer Biol. 2006; 16: 173-182Crossref PubMed Scopus (222) Google Scholar). To gain insight into the functional role of 14-3-3 proteins in KSR1 regulation, we first searched for any isotype-related difference in binding. The seven GST-tagged human 14-3-3 isoforms were purified to near homogeneity (supplemental Fig. 1A) and used as baits in pulldown experiments with FLAG-KSR1-expressing cell lysates. We could not perform direct interaction assays because production of full-length recombinant KSR1 was not possible in our laboratory. This problem has also been reported by other groups (31Hartsough M.T. Morrison D.K. Salerno M. Palmieri D. Ouatas T. Mair M. Patrick J. Steeg P.S. J. Biol. Chem. 2002; 277: 32389-32399Abstract Full Text Full Text PDF PubMed Scopus (" @default.
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• W2037449911 title "The Functional Interaction of 14-3-3 Proteins with the ERK1/2 Scaffold KSR1 Occurs in an Isoform-specific Manner" @default.
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