Novel brain 14-3-3 interacting proteins involved in neurodegenerative disease Shaun Mackie* and Alastair Aitken University of Edinburgh, School of Biomedical and Clinical Laboratory Sciences, Edinburgh, Scotland, UK Dimeric 14-3-3 proteins have important functions in diverse biological processes [1–3]. An optimal motif for 14-3-3 binding was identified as R(S)XpSXP [4] where Sp is phosphoserine. This was later refined to include a second motif, mode 2, RXXXpSXP [5]. A number of proteins also bind to 14-3-3 at their C-terminus where the presence of a proline residue may be unnecessary as the peptide backbone would not be required to loop out again from the binding pocket. Thus in addition to the well-characterized nonphosphorylated binding motifs, there may be a third phospho-dependent 14-3-3- binding motif, -pS ⁄ pT (X 1-2 )-CO 2 H, referred to by Ganguly and colleagues as ‘mode III’ [6]. This motif has also been characterized structurally in plant proton ATPases [7]. The motif in b-COP (RRSpSV-CO 2 H) may also come into this category [8]. Unphosphory- lated motifs that interact with 14-3-3 at high affinity have also been characterized [1,2]. Structures of 14-3-3 and the binding site of the phospho- and unphosphorylated motifs have been determined [2,5,6]. Phosphorylation of specific 14-3-3 isoforms can also regulate interactions [9]. 14-3-3 isoforms are involved in neurodegenerative disorders including Alzheimer’s [10] and Parkinson’s disease [11]. We identified four of the seven mamma- lian 14-3-3 isoforms (b, c, e and g) in the spinal fluid (CSF) of patients with Creutzfeldt–Jakob disease (CJD) [12]. 14–3-3 g alone was also present in all patients with other dementias, including Alzheimer’s. Changes in the localization of 14-3-3 isoforms were Keywords 14-3-3; d-catenin; IRSp53; neurodegenerative diseases; yeast two- hybrid Correspondence A. Aitken, University of Edinburgh, School of Biomedical and Clinical Laboratory Sciences, George Square, Edinburgh, EH8 9XD, Scotland, UK Fax: +44 131 6503725 Tel: +44 131 6503721 E-mail: alastair.aitken@ed.ac.uk *Present address University of Edinburgh, Psychiatric Genet- ics Section, Medical Genetics Section, Western General Hospital, Edinburgh, Scotland (Received 31 January 2005, revised 13 May 2005, accepted 22 June 2005) doi:10.1111/j.1742-4658.2005.04832.x We isolated two novel 14-3-3 binding proteins using 14-3-3 f as bait in a yeast two-hybrid screen of a human brain cDNA library. One of these encoded the C-terminus of a neural specific armadillo-repeat protein, d-catenin (neural plakophilin-related arm-repeat protein or neurojungin). d-Catenin from brain lysates was retained on a 14-3-3 affinity column. Mutation of serine 1072 in the human protein and serine 1094 in the equiv- alent site in the mouse homologue (in a consensus binding motif for 14-3- 3) abolished 14-3-3 binding to d-catenin in vitro and in transfected cells. d-catenin binds to presenilin-1, encoded by the gene most commonly mutated in familial Alzheimer’s disease. The other clone was identified as the insulin receptor tyrosine kinase substrate protein of 53 kDa (IRSp53). Human IRSp53 interacts with the gene product implicated in dentatoru- bral-pallidoluysian atrophy, an autosomal recessive disorder associated with glutamine repeat expansion of atrophin-1. Abbreviations CJD, Creutzfeldt–Jakob disease; CSF, cerebrospinal fluid; DRPLA, dentatorubral-pallidoluysian atrophy; IRSp53, insulin receptor tyrosine kinase substrate protein of 53 kDa; SCA1, spinocerebellar Ataxia Type 1. 4202 FEBS Journal 272 (2005) 4202–4210 ª 2005 FEBS observed during neurodegeneration in mice as a result of scrapie infection [13]. 14-3-3 isoforms play a key role in neurodegeneration in the polyglutamine-repeat disease spinocerebellar ataxia type 1 (SCA1) [14]. SCA1 is characterized by ataxia, progressive motor deterioration and loss of cerebellar Purkinje cells caused by the expansion of a region of the ataxin-1 gene that produces an abnormally long stretch of glu- tamine. In SCA1, 14-3-3 f and e bind to and stabilize ataxin-1, after phosphorylation by Akt, thus slowing its normal degradation. A number of other inherited neurodegenerative diseases, including Huntington’s disease and Dentatorubral-pallidoluysian atrophy (DRPLA) are caused by proteins that undergo a simi- lar pathogenic polyGln expansion. 14-3-3 and a-synuc- lein colocalize with the perinuclear inclusions of huntingtin protein [15]. To identify novel 14-3-3 binding partners in mamma- lian brain, we performed a two-hybrid screen with human 14-3-3 f as bait and isolated clones for two pro- teins involved in distinct neurodegenerative diseases. Results Identification of d-catenin as a 14-3-3 interacting protein The yeast two-hybrid screen of a human brain cDNA library was carried out with a GAL4 binding domain 14-3-3 fusion protein as bait. From 2.54 · 10 6 trans- formants screened, 35 diploid colonies (D1–D35) grew up under selective conditions. 2 out of 35 colonies specified in-frame coding region cDNAs. BLAST searches showed that one of these, D16, was homologous to the C-terminal region of delta catenin (Primary accession number Q9UQB3 in Swiss-Prot, also known as neural plakophilin related armadillo protein, NPRAP or neurojungin) that is almost exclu- sively expressed in the nervous system [16]. d-Catenin is a member of the p120-catenin (p120ctn) subfamily, defined as proteins with 10 armadillo (ARM) repeats in characteristic spacing with diverse N- and C-ter- minal flanking sequences [17,18] see Fig. 1A. The 42-residue repeated Arm motif was originally described in the Drosophila segment polarity gene, armadillo [19]. The ARM domains of d-catenin are necessary and sufficient for adherens junction targeting and for direct interaction with cadherin (Fig. 1B). Clone D16 encoded a putative protein product of 386 amino acids (839–1125) which included 4 of the ARM repeats, a potential 14-3-3 binding site and a PDZ binding motif (Fig. 1A). Both northern blot and in situ hybridization studies indicate that delta-catenin is almost exclusively expressed in the nervous system [16,20]. d-Catenin has a structure similar to that of p0071 and is considered to be a neural isoform of p0071, which is expressed ubiquitously [21]. β or γ− catenin p120ctn or δ -catenin α -catenin β or γ− catenin p120ctn or δ -catenin Intracellular Extracellular α -catenin Actin Filaments Cadherin Receptor Ca ++ PDZ binding motif 532 840 1225 RSApSAP N A B 1 C 1225 1013 D16 2265bp 3’ UTR 14-3-3 phospho-binding siteARM domain Fig. 1. d-Catenin domains. (A) Alignment of d-catenin cDNA and protein domains with clone D16. PCR amplification of the pACT2 D16 clone identified a  2200 nucleotide insert. Sequence analysis established that clone D16 encoded the C-terminus of human d-catenin (GenBank accession num- ber U96136) and  1kbof3¢-untranslated region. The alignment of this fragment is shown below the full-length human d-catenin. The armadillo, ARM, domains, predicted 14-3-3 binding site and a PDZ binding motif are indicated. (B) d-catenin complexes in adherens junction targeting and interaction with cadherin. S. Mackie and A. Aitken 14-3-3 in neurodegenerative disease FEBS Journal 272 (2005) 4202–4210 ª 2005 FEBS 4203 14-3-3 Binds endogenous d-catenin from brain lysates To determine whether endogenous d-catenin associated in brain tissue, we passed sheep brain homogenate over GST-14-3-3 and control GST affinity columns. d-Catenin specifically bound to a GST-14-3-3 f column but not to a control GST column (Fig. 2A). We typic- ally detected a doublet by western blot that may be due to in vivo phosphorylation. A doublet has been observed previously [22] and a splice variant of d-cate- nin is known, although both forms include the 14-3-3 motif, which is not present in other, more widely expressed catenins, suggesting that this interaction may be functionally restricted to neuro-epithelial cells. We also performed immunoprecipitation assays from sheep and mouse brain homogenates using recombinant GST-14-3-3 and detected a 160 kDa doublet band with anti-d-catenin sera, consistent with the expected M r of full-length d-catenin. The immunoprecipitations were also probed with a phospho-specific antibody (New England Biolabs) against the consensus RSXpSXP 14-3-3 binding motif (Fig. 2B). This specifically detec- ted a 160 kDa polypeptide at the same position as the d-catenin antibody suggesting that phosphorylation at this site may be functionally important in vivo. Other species were evident which may represent partially degraded, phosphorylated forms of d-catenin. A 14-3-3 binding site on d-catenin Human and mouse d-catenin cDNAs encode proteins of 1225 and 1247 residues, respectively, and share 95% identity at the amino acid level [22]. A predicted 14-3-3 binding motif (RSApSAP) comprises phosphoSer1072 and 1094 and neighbouring residues, respectively. Therefore to establish the mode of binding of 14-3-3 to d-catenin, the 386 residue proteins encoded by the wild-type d-catenin clone D16 and the S1072A mutant were expressed as 35 S-labelled proteins in IVTT. 14-3-3 interacted efficiently with wild-type d-catenin but not with the S fi A mutant (Fig. 3). This indicates that the interaction is phosphorylation dependent at this site. We also used a synthetic phosphopeptide that AB Fig. 2. 14-3-3 Binds endogenous d-catenin from brain lysates. Sheep brain homogenate was lysed in NaCl ⁄ P i buffer (including protease inhibitors) containing 1% Triton-100 or NaCl ⁄ P i ⁄ 1% TX100 plus 0.1% SDS to aid solubilization of brain d-catenin. The extract was clarified by centrifugation at 40 000 g and the supernatant passed through a GST-affinity column. The flow-through was applied to a GST 14-3-3 f column. After extensive washing, bound proteins were eluted by directly boiling of the glutathione–Seph- arose beads in SDS ⁄ PAGE sample buffer and analysed by 6% SDS ⁄ PAGE and blotting with rabbit anti-(d catenin) Ig (Ab62, from K.S. Kosik, Harvard Medical School, Boston, USA). (A) Lane 1, con- trol GST column (TX100, no SDS); Lane 2, lysate prepared in 1%TX100) and affinity purified on the GST zeta column; Lane 3, lysate prepared in 1% TX100 plus 0.1% SDS to aid solubilization and affinity purified on the GST zeta column. (B) Samples prepared as in lanes 1 and 3 above then probed with anti-phospho 14-3-3 BS monoclonal (NEB, Cell Signaling Technology). A B Fig. 3. Mutation of Ser1072 of d-catenin abolishes binding to 14-3- 3. (A) Wild-type d-catenin clone D16 (S1072) and the S1072A mutant were expressed as 35 S-labelled proteins in IVTT. Lanes 1, 2: Input (2%) of the two constructs; 3 and 4: immunoprecipitation of wild-type D16 by GST and by GST-14-3-3. 5 and 6: immunopre- cipitation of the D16 S1072A by GST and by GST-14-3-3. (B) A simi- lar experiment was carried out in the presence of phosphorylated and unphosphorylated peptides. Lanes 1, immunoprecipitation of wild-type D16 by GST-14-3-3; 2, immunoprecipitation of wild-type D16 by GST; 3, immunoprecipitation of D16 S1072A mutant by GST-14-3-3; 4, immunoprecipitation of wild-type D16 by GST-14-3-3 in the presence of Raf-phosphopeptide (300 l M); 5, immunoprecipi- tation of wild-type D16 by GST-14-3-3 in the presence of the same concentration of unphosphorylated peptide. 14-3-3 in neurodegenerative disease S. Mackie and A. Aitken 4204 FEBS Journal 272 (2005) 4202–4210 ª 2005 FEBS corresponds to c-Raf1 amino acids 252–264, a canon- ical 14-3-3 binding motif previously shown to dissoci- ate Raf ⁄ 14-3-3 complexes [4]. By contrast, as a control, the unphosphorylated version of this peptide did not interfere with binding. 14-3-3 binds to d-catenin expressed in MDCK cells As cDNA encoding full-length human d-catenin was unavailable, we used a mouse cDNA clone for sub- sequent studies [16]. Classical adherens junctions hold epithelial cells together via cadherin-catenin protein complex linkages and d-catenin interacts with adhesive junction proteins both in transfected cells and mouse brain [22], Fig. 1B. Cadherins are Ca 2+ -dependent cell-cell adhesion receptors involved in a variety of bio- logical processes including development, morpho- genesis and tumour metastasis. Cadherins on adjacent cells contact one another through their extracellular domains. The intracellular domains anchor the junc- tional complex or adherens junction to the actin cyto- skeleton via the cytoplasmic catenins. Therefore to characterize 14-3-3 ⁄ d–catenin inter- actions in a defined culture system where adhesive junctions are prominent, we used Madin–Darby canine kidney (MDCK) epithelial cells. Lysates were prepared from cells transfected with untagged (as a control) and FLAG-tagged d-catenin. GST-14-3-3 f immunoprecipi- tations immunoblotted with anti-FLAG Ig, detected a 160 kDa doublet which bound specifically to GST- 14-3-3 f. Specific interaction was observed with wild- type full-length d-catenin in both Cos7 and MDCK cells and was ablated by mutation of serine 1094 to alanine (Fig. 4A,B). To establish the site of binding of 14-3-3 to d-cate- nin in MDCK cells, we performed binding assays in the presence of competitor peptides. We again used the synthetic c-Raf1 phosphopeptide (and the unphosphory- lated version of this peptide as control, not shown) and a nonphosphorylated peptide inhibitor of 14-3-3 binding, R18 (FHCVPRDLSWLDLEANMCLP). R18 was originally isolated from a phage display library with high affinity for the phosphoserine-binding pocket of 14-3-3 and which disrupts binding of 14-3-3 to tar- get proteins such as Raf, Ask1 [23] and EXO-S [24]. Both peptides efficiently prevented 14-3-3 ⁄ d–catenin complex association in vitro in cell extracts (Fig. 5). These results verified that the interaction between d-catenin and 14-3-3 is mediated through the phospho- binding pocket of 14-3-3. The 14-3-3 binding motif is not present in members of the p120ctn sequence family, which are more ubi- quitously expressed, suggesting that this interaction may be functionally restricted to neuro-epithelial cells. Interaction of IRSp53 with 14-3-3 We also identified a full-length clone of a 53 kDa SH3 domain-containing adaptor protein originally identified as a substrate of the insulin receptor kinase (IRSp53). IRSp53 interacts with Rho GTPases to regulate the organization of the actin cytoskeleton and is a compo- A B Fig. 4. 14-3-3 interacts with full-length d-catenin in cells. Cos7 (A) and MDCK cells (B) were transiently transfected with either empty vector (A) or untagged pcDNA wild type delta catenin (B) and Flag tagged delta catenin wild type and Flag tagged d-catenin with a Ser to Ala substitution at residue 1094 (S1094A). Transfected cell extracts were split and incubated with 20 lg GST and 20 lg GST- 14-3-3 for 120 min at 4 °C. Upper panels: lysate loading controls. Lanes: 1, untagged d-catenin; 2, Flag S1094 d-catenin; 3, Flag S1094A d-catenin. Lower panels: Immunoprecipitation of Flag tagged d-catenin (western blot with a-Flag). Lanes 1,3,5, GST immunoprecipitation; lanes 2,4,6, GST-14-3-3 f immunoprecipita- tion; Lanes 1,2, Vector (in Cos cells) or no flag tag (MDCK cells); 3,4, Flag S1094 d-catenin; 5,6, Flag S1094A d-catenin. S. Mackie and A. Aitken 14-3-3 in neurodegenerative disease FEBS Journal 272 (2005) 4202–4210 ª 2005 FEBS 4205 nent of signaling pathways that control the formation of lamellipodia and filopodia [25]. A number of splice variants of human and mouse IRSp53 are known, comprising mainly of a 12 residue longer C-terminus and a 40 residue insertion around residue 300 [26]. In this study we isolated the longer form that is mainly expressed in brain. A ‘Scansite’ search [27] revealed a number of potential suboptimal 14-3-3 binding motifs in IRSp53 conserved across mammalian species (Fig. 6). The best motif was the medium stringency site at Ser512, RSVS 512 SG, which would explain loss of binding of construct 1–366 but motif(s) near the N-terminus must also be important (e.g. RYLS 117 AA and ⁄ or RKKS 148 QG). A nonphos- phorylated Ser immediately following Arg within the first mode and a Pro two residues C-terminal to the phosphorylated Ser or Thr in both motifs is strongly favoured, but not an essential requirement for binding to 14-3-3 [3]. As it was not clear which region or potential motif(s) in IRSp53 might be involved in 14-3-3 associ- ation we attempted to identify the site(s) of interaction by deletion analysis. The constructs depicted in Fig. 7 A were coexpressed with HA-14–3-3f in Cos7 cells. The results in Fig. 7B clearly indicate that deletion of either the C-terminal region or the N- terminus caused loss of 14-3-3 interaction. This may be due to a requirement for binding through two sites to a 14-3-3 dimer and this type of tandem 14-3-3 binding has been clearly shown to be functionally important in cases such as Raf kinase [3,28] and the Forkhead transcrip- tion factor FOXO4 [29]. It is also probable that the interaction between 14-3-3 and IRSp53 is not phosphorylation dependent as treatment with lambda phosphatase of Cos7 cell lysates, into which Flag-IRSp53 and HA-14-3-3 zeta had been co transfected, did not reduce interaction (Fig. 8A). Immunoprecipitation experiments with a construct with mutations in essential residues of the phospho- peptide binding pocket, HA-14-3-3 zeta (R56A, R60A), that was transfected in Cos7 cells showed that much less IRSp53 was immunoprecipitated. This veri- fied that IRSp53 interacts with 14-3-3 in the binding pocket. Discussion One of the 14-3-3 interacting clones that we identified in the 2-hybrid analysis encoded the armadillo repeat protein named delta-catenin, NPRAP (neural plako- philin-related arm-repeat protein) or neurojungin [16]. Fig. 5. 14-3-3 Binding peptides prevent 14-3-3 ⁄ d–catenin associ- ation. MDCK cells expressing d-catenin constructs were lysed and extracts incubated in the absence or presence of 300 m M Raf phos- phopeptide or nonphosphorylated peptide, R18, at 4 °C for 60 min 20 lg GST-14-3-3 was added for 120 min. GST fusions were recov- ered on GSH–Sepharose beads and washed four times with 0.5 mL lysis buffer. Samples were separated by 6% SDS PAGE and assayed for associated d-catenin by anti-Flag immunoblots. Lanes 1–4, lysate loading controls. Lane 1, no Flag; 2–4, Flag d-catenin; 5, no Flag, no peptide; 6, Flag d-catenin, no peptide; 7, Flag d-catenin, plus Raf phosphopeptide; 8, Flag d-catenin, plus R18. Fig. 6. Alignment of IRSp53 cDNA and domains in the yeast two-hybrid clone, D6. (A) Domain alignment of full-length human IRSp53 cDNA. The SH3 domains (residues 377–435) which bind atrophin-1; an autoinhibitory region (AIR) that regulates Cdc42 binding to the CRIB motif and a Cdc42 binding motif (residues 238–292) and potential 14-3-3 binding sites are indicated. (B) PCR amplification of the pACT2 D6 clone identified a 2.4 kb insert that encoded the complete IRSp53 cDNA insert and  1kbof3¢-untranslated region. 14-3-3 in neurodegenerative disease S. Mackie and A. Aitken 4206 FEBS Journal 272 (2005) 4202–4210 ª 2005 FEBS d-Catenin was originally identified by its ability to bind to the loop region of presenilin-1, encoded by the gene most commonly mutated in familial Alzheimer’s disease [30]. Presenilin-1 interacts with complexes including d-catenin to modulate Wnt signaling which is respon- sible for a variety of signaling events that lead to neural plate formation and patterning decisions ⁄ development in the embryonic nervous system [20]. Although there is no evidence that 14-3-3 zeta bind- ing plays a role in this pathway, it nevertheless suggests another link between 14-3-3 and neurodegenerative disease. Wnt signaling also regulates neuronal cytoskeleton structure, cerebellar synaptic differentia- tion, apoptosis and degenerative processes in the aging brain. The latter establishes a link to pathogenesis in Alzheimer’s disease. Mutations in presenilin 1 (PS1) gene are the most common cause of early onset familial Alzheimer’s disease. d-Catenin expression is decreased in presenilin-1 deficient mice [30]. The other novel 14-3-3 interacting protein in our study was identified from a full-length clone of the insulin receptor tyrosine kinase substrate protein of 53 kDa (IRSp53). IRSp53 is an SH3 domain-contain- ing adaptor protein originally identified as a substrate of the insulin receptor kinase [25]. IRSp53 interacts with Rho GTPases to regulate the organization of the actin cytoskeleton and is a component of signaling pathways that control the formation of lamellipodia and filopodia [31]. Human IRSp53 was isolated as a protein which interacts with the gene product implicated in DRPLA, an autosomal recessive disorder caused by CAG ⁄ glutamine repeat expansion of atro- phin-1 [32]. While the DRPLA gene is ubiquitously expressed, neuron death occurs in specific anatomical areas of the brain. In a yeast two-hybrid screen of a human foetal brain cDNA library with a fragment of atrophin-1 (residues 335–1185, containing 10 CAG repeats) clones isolated included IRSp53, hDVL1, d-Catenin and 14-3-3 [33]. A proline rich region near the polyGln tract of atro- phin-1 bound to the SH3 domain of IRSp53 in vitro. Our results therefore expand the range of interacting proteins and diversity of neurodegenerative disorders A B Fig. 7. Domains of IRSp53 interacting with 14-3-3. (A) Schematic of the constructs of flag tagged IRSp53. (B) The ability of the N- and C-terminal constructs of flag tagged IRSp53 to be immunoprecipi- tated by HA-tagged 14-3-3 f. The constructs of flag tagged IRSp53 depicted in A were cotransfected with HA-14-3-3 f in Cos7 cells and immunoprecipitated with anti-HA-Ig as described in Experimen- tal procedures. Lane 1, HA-14-3-3 f + IRSp53; 2, HA-14-3-3 f + IRSp53-FLAG; 3, HA-14-3-3 f + D 1–125 IRSp53-FLAG; 4, HA-14-3- 3 f + D 1–179 IRSp53-FLAG; 5, HA-14-3-3 f + D 1–366 IRSp53- FLAG. Upper panel, expression levels of the constructs. Middle panel, expression levels of HA-14-3-3 f. Lower panel, western blot of IP with anti-Flag Ig. A B Fig. 8. IRSp53 interacts with 14-3-3 in the binding pocket but may do so in a nonphospho-dependent manner. (A) Flag-IRSp53 and HA-14-3-3 zeta were co transfected into Cos7 cells as described in Fig. 4 and Experimental procedures. The cell lysates were treated with lambda phosphatase and immunoprecipitated with anti-HA Ig. The IPs were western blotted with anti-Flag Ig. Lane 1, input; 2, no phosphatase treatment; 3, treatment with lambda phosphatase. (B) Flag-IRSp53 and HA-14-3-3 zeta constructs were co transfected into Cos7 cells as described in Fig. 4 and Experimental procedures. The cell lysates were immunoprecipitated with anti-HA Ig. The pellets were western blotted with anti-Flag Ig. Lane 1, input; 2, immunoprecipitation with wild-type HA-14-3-3 zeta; 3, immunopre- cipitation with HA-14-3-3 zeta (R56A, R60A) mutant construct. S. Mackie and A. Aitken 14-3-3 in neurodegenerative disease FEBS Journal 272 (2005) 4202–4210 ª 2005 FEBS 4207 in which isoforms of 14-3-3 are implicated, including another polyglutamine expansion disease, DRPLA. The key feature of all these diseases is the accumula- tion in specific areas of the brain of abnormal forms of proteins which results in neurodegeneration. The proteins that accumulate (due to their misfolding and ⁄ or genetic mutation) are specific to each disease. However, a common feature that is now emerging is the involvement of specific isoforms of 14-3-3. Deter- mining the component proteins and role of 14-3-3 complexes, may lead to advances in understanding of how these protein complexes regulate brain functions. Experimental procedures Two-Hybrid Screen cDNA encoding the 14-3-3 f ORF was cloned into the NdeI ⁄ Bam HI sites of the vector pGBKT7 (Clontech, Basingstoke, UK) to create an in-frame fusion with the DNA binding domain of GAL4. Plasmid pGBKT7 ⁄ 14-3-3 f was transformed into yeast strain SFY526 and combined with a pretransformed Matchmaker cDNA library (Clontech) using standard yeast mating pro- cedures. Diploid colonies were selected for activation of his- tidine (His) and adenine (Ade) reporter genes by growth on SD medium lacking Ade, His, Leu and Trp for 7–10 days. Clones that survived repeated auxotrophic selection were assayed for b-galactosidase activity by use of 5-bromo- 4-chloro-3-indolyl-b-d-galactopyranoside (X-gal) as a sub- strate. Plasmid DNA was isolated from 39 positive clones and library inserts were amplified by PCR using pACT2 vector specific primers. Plasmids and constructs Mouse d-catenin in pcDNA3.1 was from W. Franke (Ger- man Cancer Research Center, Heidelberg, Germany). Plasmids pGEX-2T 14-3-3 f and HA tagged 14-3-3 f (pcDNA.1 Zeo) have been described previously [35] The d-catenin ORF was amplified by PCR and the product inser- ted into Not1 ⁄ Xho1 cut pCMV TAG4A (Invitrogen, Paisley, UK) to generate a C-terminal FLAG tagged construct. Site- specific mutations were introduced into the d-catenin ORF using the QuikChange Site Directed Mutagenesis System (Stratagene, Cleveland, OH, USA) according to the manu- facturer’s instructions and confirmed by DNA sequencing. PCR amplification of DNA fragments was carried out using Pfu Turbo DNA polymerase (Stratagene) and integrity of cloned inserts were confirmed by DNA sequencing. The construct with mutations in essential residues of the phosphopeptide binding pocket, HA-14-3-3 zeta (R56A, R60A) was generated by Stratagene Quickchange site direc- ted mutagenesis according to the manufacturers instructions. In vitro transcription and translation (IVTT) was carried out using TnT expression kits (Promega) as described [34]. Transfection of cultured cells Cos7 and MDCK cells (ATCC) were maintained in high glu- cose Dulbecco’s modified Eagle’s medium (Sigma) supple- mented with 10% foetal bovine serum (Life Technologies), 1· nonessential amino acid supplement, 1· glutamine, peni- cillin and streptomycin (Life Technologies) in air plus 5% CO 2 with constant humidity. Cells were transfected at 80–90% confluence using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions and harvested 24–30 h later. Protein extracts Transfected cells (100 mm plates) were washed once with NaCl ⁄ Pi, scraped into 1.8 mL lysis buffer (50 mm Tris-Cl pH 7.5, 150 mm NaCl, 1% TX-100, I mm EDTA, 1 mm dithiothreitol, 1 mm NaVO 4 ,10mm NaF and protease inhibitor cocktail without EDTA (Roche Molecular Bio- chemicals), incubated on ice for 15 min and clarified by centrifugation for 20 mins at 16 000 g in a refrigerated microfuge. For brain extract preparation, adult sheep brain was briefly rinsed in lysis buffer (50 mm Tris ⁄ Cl pH 7.5, 150 mm NaCl, 1% TX-100, 2 mm EDTA, 10% glycerol, 2mm dithiotreitol, 2 mm NaVO 4 ,50mm NaF, 20 mm b-glycerophosphate, 1 mm PMSF and 2x protease inhibitor cocktail without EDTA) and homogenized in the same buffer. Lysates were precleared at 13 000 g for 30 min at 4 °C. Supernatant from the lysates was further clarified by centrifugation at 40 000 g for 60 min at 4 °C. Supernatants were filtered through 0.2 lm syringe filters (Nalgene) before application to GST or GST 14-3-3 f affinity columns. Treatment of cell lysates with lambda phosphatase (New England Biolabs) was with 400 units phosphatase for 60 min at 30 °C, according to the manufacturers instructions. Immunoprecipitation Equal amounts of GST or GST 14-3-3 f fusion protein (20 lg) were incubated overnight at 4 °C on a rotary wheel with lysates prepared from 10 cm dishes of confluent Cos7 or MDCK cells. Complexes were captured by incubation with glutathione Sepharose beads for 2 h at 4 °C. After centrifugation, beads were washed four times with lysis buf- fer. Bound proteins were eluted with SDS sample buffer and subjected to SDS PAGE and immunoblotting. For immunoprecipitations, 5 lg anti-HA7 monoclonal Ig (Sig- ma) was incubated for 4 h or overnight at 4 °C with con- trol or HA expressing lysates. Immunocomplexes were incubated with protein A ⁄ G beads (Pierce) for 2 h at 4 °C and captured by centrifugation. Immunocomplexes were washed as above before immunoblot analysis. SDS PAGE and western blotting were performed by standard methods. 14-3-3 in neurodegenerative disease S. Mackie and A. Aitken 4208 FEBS Journal 272 (2005) 4202–4210 ª 2005 FEBS Anti-FLAG M2 peroxidase conjugate and Anti-HA7 Igs were from Sigma and signals were detected using ECL, chemiluminescence detection (Amersham Pharmacia Bio- tech, Buckinghamshire, UK). Recombinant protein purification Protein expression was induced in E. coli strain BL21(DE3) (Novagen, Merck Biosciences, Nottingham, UK) carrying plasmid pGEX-2T or pGEX-2T 14-3-3 f. Briefly, cultures were grown overnight at 37 °C in Liquid Broth medium (Life Technologies, Inc., Paisley, UK) containing 50 lgÆmL )1 ampicillin and diluted the following day (1 ⁄ 10) in the same medium. Culture growth continued at 30 °C until the absorb- ance (600 nm) reached 0.8 to 1.0. Expression of the tagged protein was induced by the addition of 0.5 mm isopropyl b- d-thiogalactopyranoside for 3 h at 25 °C. The fusion proteins were purified by affinity chromatography on gluta- thione-Sepharose beads (Amersham Pharmacia Biotech.). For large-scale preparation of GST and GST 14-3-3 f affinity columns, fusion protein lysates prepared from 2.5 L of induced bacterial culture ( 7 mg fusion protein) were used to saturate 2 mL columns of glutathione-Sepharose beads. Peptide competition studies: dissociation of 14-3-3 ⁄ d-catenin complexes in vitro Cell extracts were incubated with 300 l m synthetic phos- phopeptide corresponding to a c-Raf1 14-3-3 binding motif (residues 252–264, SQRQRSTpSTPNVH) as well as with the control peptide of the same sequence but unphosphory- lated or with 300 lm of a nonphosphorylated peptide (R18, FHCVPRDLSWLDLEANMCLP [23]. Acknowledgements The work was funded by a MRC programme grant to AA. We thank Bengt Hallberg for the R18 peptide. Mouse d-catenin in pcDNA3.1 was a kind gift from the laboratory of Dr W. Franke; Rabbit anti-(d cate- nin) Ig (Ab62), raised against residues 434–530 was a kind gift from the laboratory of Dr Kenneth Kosik. HA tagged 14–3-3f (pcDNA.1 Zeo) was from Preeti Kerai and Thierry Dubois. References 1 Aitken A (2002) Functional specificity in 14-3-3 isoform interactions through dimer formation and phosphoryla- tion. Chromosome location of mammalian isoforms and variants. Plant Mol Biol 50, 993–1010. 2 Fu H, Subramanian RR & Masters SC (2000) 14-3-3 proteins: structure, function and regulation. Annu Rev Pharmacol Toxicol 40, 617–647. 3 Yaffe MB (2002) How do 14-3-3 proteins work? - Gate- keeper phosphorylation and the molecular anvil hypo- thesis. 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Phosphorylation at this site in vivo regulates Raf ⁄ 14-3-3 interaction. J Biol Chem 272, 28882–28888. 14-3-3 in neurodegenerative disease S. Mackie and A. Aitken 4210 FEBS Journal 272 (2005) 4202–4210 ª 2005 FEBS . Novel brain 14-3-3 interacting proteins involved in neurodegenerative disease Shaun Mackie* and Alastair Aitken University of Edinburgh, School. a-synuc- lein colocalize with the perinuclear inclusions of huntingtin protein [15]. To identify novel 14-3-3 binding partners in mamma- lian brain, we performed