Interaction between a domain of a negative ...

JBC Papers in Press. Published on December 3, 2015 as Manuscript M115.703710 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.703710

Effect of mutations on SPRED1-EVH and NF1-GRD interaction

Interaction between a domain of a negative regulator of the RAS-ERK pathway, SPRED1, and the GTPase-Activating Protein-Related Domain of neurofibromin is implicated in Legius Syndrome and Neurofibromatosis Type 1. Yasuko Hirata1, Hilde Brems2, Mayu Suzuki1, Mitsuhiro Kanamori1, Masahiro Okada1, Rimpei Morita1, Isabel Llano-Rivas3, Toyoyuki Ose4, Ludwine Messiaen5, Eric Legius2, and Akihiko Yoshimura1 1

Running title: Effect of mutations on SPRED1-EVH1 and NF1-GRD interaction To whom correspondence should be addressed: Prof. Akihiko Yoshimura, Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 1608582, Japan Tel: +81-3-5363-3483 Fax: +81-3-5360-1508 Email: [email protected] Keywords: growth factors, GAP, human genetics, protein domain, Ras protein, negative regulation mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome.

ABSTRACT Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple café-au-lait macules, axillary freckling, learning disabilities and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the RAS-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids (aa) and the C-terminal 20 aa of the GTPase-Activating Protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing α-helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120GAP. Several

Introduction Spred (Sprouty-related protein with an EVH1 domain) family proteins, initially discovered as ckit- and c-fms-binding proteins, have been shown to suppress the Ras-ERK pathway. Spreds form a subfamily of the Sprouty/Spred family, which is characterized by the Sprouty-related C-terminal cysteine-rich (SPR) domain. In mammals, four Sprouty homologues and three members of the Spred family of proteins, Spred1, Spred2, and 1

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Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, 2 Department of Human Genetics, Catholic University of Leuven, Leuven, Belgium 3 Department of Genetics, Hospital Universitario Cruces, BioCruces Health Research Institute, Biscay, Spain 4 Pharmaceutical Sciences, Hokkaido University, N12W6, Sapporo 060-0812, Japan 5 Medical Genomics Laboratory, Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, 35294, USA


negative form against endogenous Spred1 and augments serum and Nerve Growth Factorinduced ERK activation, suggesting that the EVH1 domain binds to a factor necessary for Ras inhibition (17). However, despite lengthy and extensive screening, no binding partner of the EVH1 domain has been found so far. Recently, a molecular link between neurofibromin and SPRED1 was discovered. Stowe et al. demonstrated that SPRED1 protein binds to neurofibromin, the NF1 gene product, resulting in the plasma membrane localization of neurofibromin, which subsequently downregulates Ras-GTP levels (18). This model explains why Legius syndrome resembles NF1 and how SPREDs suppress the Ras-ERK pathway. However, the molecular details of the Spredneurofibromin interaction and the effects of mutations in SPRED1 and NF1 remain to be clarified. In this study, we found that the SPRED1 EVH1 domain interacts with the N-terminal and C-terminal extended region of the GTPase activating protein (GAP)-related domain (GRD) of neurofibromin. Some mutations in these Nterminal and C-terminal regions of the GRD identified in NF1 patients reduced the binding of the GRD to the EVH1 domain. Furthermore, SPRED1 EVH1 mutations lost the ability to bind to the GRD, leading to reduced ERK suppression activity. Our data provide molecular details on the function of the EVH1 domain and the GRD, which contributes to the pathogenesis of NF1 and Legius syndrome features and the potential development of Ras-pathway inhibitors for cancer therapy. Experimental procedures Patient SPRED1 cDNA SPRED1 mutation analysis was performed in the Department of Human Genetics, Catholic University of Leuven, Belgium, and in the UAB Medical Genomics Laboratory, University of Alabama at Birmingham, Birmingham, USA, in individuals with a Legius syndrome phenotype (19). Some samples were sent by clinical geneticists from other centers. Individuals showed a phenotype compatible with Legius syndrome; specifically, the presence of CALM and/or freckling and the absence of neurofibromas, and comprehensive NF1 mutation analysis was performed in all patients with no NF1 mutations found. All patients carrying missense variants 2


Spred3, have been discovered. Mammalian Spreds can negatively regulate the Ras-Raf-ERK pathway (1,2); however, compared with Spred1 and Spred2, Spred3 has much weaker ERK suppression activity (3). The SPR domain has been shown to be palmitoylated, causing Sprouty and Spred to localize in the membrane fraction (4,5). Spred1 and Spred2 are composed of an N-terminal enabled/vasodilator-stimulated protein homology 1 homology 1 (EVH1) domain, a central c-kitbinding domain (KBD), and a cysteine-rich Cterminal SPR domain, while Spred3 lacks a functional KBD. Germline loss-of-function mutations in the SPRED1 gene have been identified in patients fulfilling the NIH clinical diagnostic criteria for neurofibromatosis type 1 (NF1) where no NF1 mutation could be identified. SPRED1 mutations account for at least 2% of the pathogenic mutations in patients clinically diagnosed with NF1 (6-11). The phenotype exhibited by such patients is known as NF1-like syndrome or Legius syndrome (OMIM 611431), and consists of multiple café-au-lait macules (CALM), axillary freckling, macrocephaly and sometimes mild neurocognitive impairment, as well as a lack of certain features that are common in NF1, such as neurofibromas, iris Lisch nodules and NF1-related malignancies (11). However there might be an increased risk for leukaemia in children with Legius syndrome (12). The similarities between NF1 and Legius syndrome, as well as that between the biochemical functions of neurofibromin and those of SPRED1, suggest that these two syndromes both result in part from hyperactive RAS-ERK signalling, as are Noonan syndrome, Noonan syndrome with lentigines (previous LEOPARD syndrome), cardio-faciocutaneous syndrome, and Costello syndrome (13). Furthermore, Spreds are putative tumour suppressors. It has been reported that SPRED1 and SPRED2 expression is reduced in human hepatocellular carcinoma (HCC) (14), and SPRED1 mutation and reduced expression are also found in acute myeloblastic leukaemia (AML) (15). Overexpression of SPRD1 in tumour cells resulted in reduced tumorigenicity in nude mice (16). Moreover, the bi-allelic inactivation of SPRED1 has been demonstrated in melanocytes cultured from a café-au-lait macule in a patient with Legius syndrome (8). It was revealed that the C-terminal deletion mutant of Spred1 functions as a dominant


Yeast two-hybrid assay The yeast two-hybrid assay was performed as described (17,23,24). Briefly, DNA fragments encoding the human SPRED1 EVH1 domain (amino acid numbers 1 to 136) or the substitution mutants were subcloned into the pGBKT7 vector as fusions to the LexA DNA-binding domain. Human NF1 cDNAs or GRD cDNAs were subcloned into the pGADT7 vector as a fusion to GAL4 DNA-activating domain. We have used Y187 strain in which interaction of bait and prey induces both α-galactosidase and β-galactosidase. To detect interaction on the filter paper, yeasts were grown in Leu and Trp double dropout plates containing X-α-Gal for in situ staining, as described in (23). Quantitative β-galactosidase assay for yeast two-hybrid systems was performed using o-nitrophenyl β-galactopilanoside (Nacalai Tesque) as a substrate according to a previously described method (25).

Patient NF1 cDNA NF1 mutation analysis was performed in the UAB Medical Genomics Laboratory, University of Alabama at Birmingham in Birmingham, USA, using an RNA-based approach complemented by DNA-based dosage analyses, essentially as previously described (21) (22). NF1 mutations are described following recommendations of the Human Genome Variation Society using NM_001042492.2 as the reference sequence. Phenotypic data of all individuals carrying an NF1 constitutional missense mutation affecting the amino acids (aa) 1202-1217 and aa1511-1530, as provided by the referring physicians at the time of submission of the sample for clinical testing using a standardized phenotypic checklist, were summarized.

Cell culture and transfection Human embryonic kidney HEK293 cells were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) in 10 cm dishes. Immunoprecipitation and immunoblotting were performed using anti-Myc (9E10), anti-HA (Takara-Clontech, rabbit polyclonal), and anti-FLAG (M2) antibodies as described (26). Briefly, the HEK293 cells were transfected with 1 to 2 µg plasmid of SPRED1 or GRD expression vectors using the polyethylenimine (PEI)-method as described (27). Cells were lysed in 0.5 ml of TNE lysis buffer containing 150 mM Tris-HCl (pH 7.6), 50 mM NaCl, 1 mM EDTA, and 0.5% NP40 supplemented with protease inhibitor cocktail (Nacalai Tesque)(28). After lysis, cellular debris was removed by centrifugation at 15,000 g for 10 min. Protein from cell lysates was precipitated using 2 µg of antibody and 25 µL of TrueBlot Anti-Mouse Ig IP Beads or Protein G-Sepharose (GE Healthcare) for 2 h at 4°C. The immune complex was washed three times with a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl and 1% NP40. For Western blotting, the immunoprecipitates or whole cell lysates were resolved using SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P membranes (Millipore). The membranes were blotted with the indicated antibodies, and the bound antibodies were

Ethics information This study was approved by the local institutional review board (IRB) of Catholic university of Leuven. The set of anonymous samples of individuals having multiple café-au-lait spots but lacking NF1 mutations was provided by the Medical Genomics Laboratory at the University of Alabama at Birmingham. Only limited clinical data were available on these samples, and IRB approval was obtained for further analysis of these samples to determine the genetic cause of the phenotype.

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affecting the SPRED1 EVH1 domain, except one (p.Gly62Arg, described in the ARUP database), are summarized in the SPRED1 LOVD database, including references therein for those patients previously reported (LOVD https://grenada.lumc.nl/LOVD2/mendelian_genes/ home.php?select_db=SPRED1). Some patients were previously described (6,7,9,19,20). Mutation numbering was based on the cDNA sequence with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence (GenBank accession code: NM_152594.2). For protein numbering, the initiation codon was codon 1 (NP_689807.1). Human SPRED1 cDNAs were cloned in pcDNA3 with a six-repeated Myc-tag or pCMV2 with a FLAG-tag at the N-terminus. Mutant SPRED1 construction in a pMax vector (Amaxa Biosystems, Gaithersburg, MD, USA) was performed by polymerase chain reaction (PCR)directed mutagenesis as described by (2).


visualized using horseradish peroxidaseconjugated antibodies against goat, rabbit, or mouse IgG, and Chemi-Lumi One L Westernblotting detection reagents (Nacalai Tesque). ERK-reporter assay and luciferase assay The Elk-1 activation was measured by the GAL4 DNA-binding domain (DB)/Elk-1 fusion system according to the manufacturer’s instructions (PathDetect in-vivo signal transduction pathway trans-reporting system, Agilent Technologies) as described (3). HEK293 cells were transfected with 50 ng Elk-1 consisting of GAL4 DB and Elk-1, 50 ng pFR-Luc carrying the GAL4 UAS-fused luciferase gene, 50 ng pCH110 encoding the βgalactosidase gene under the control of the SV40 promoter, and FLAG-tagged SPRED1 expression vectors. In some experiments, HA-tagged GRD expression vectors were included. After 24 h, cells were treated with 50 ng/ml Epidermal Growth Factor (EGF) for 6 h and then collected and lysed with a PicaGene Reporter lysis buffer (TOYO Ink). The activity of luciferase and βgalactosidase was analysed using beetle luciferin (Promega) and o-nitrophenyl β-galactopilanoside (Nacalai Tesque) as substrates. In all reporter assays, 2 × 105 HEK293 cells were plated on 12well dishes and transfected according to the calcium-phosphate method. Docking model The docking model of the SPREDl EVH1 domain and the NF1 GRD domain was calculated using ClusPro (29). Human SPREDl EVH1 domain (PDB ID: 3SYX) and human NF1 GRD domain (PDBID: 1NF1) were used for the computation. Out of the 30 clusters resulting from ClusPro, there are several models that can explain EVH1 missense mutations. A model came as cluster 10, which harbors 30 members, can explain the result of our Yeast two-hybrid assay (Fig. 5F). Results Interaction between the EVH1 domain and the GRD detected by a yeast two-hybrid system Neurofibromin is an approximately 300 kDa large protein consisting of several domains, including the GRD (Fig.1A). To identify the neurofibromin domain that interacts with the EVH1 domain of SPRED1, we performed a yeast two-hybrid assay. The human SPRED1 EVH1 domain was fused to the LexA-DNA binding domain (Bait), and six

Mutations of the N-terminal and C-terminal extended regions of the GRD observed in NF1 patients reduced EVH1 binding affinity The extended N- and C-terminal peptides of the 4


different segments of human neurofibromin were fused to the GAL4-transactivating domain (Prey). We found that only an internal region containing a tubulin-binding domain (TBD) and a GRD can strongly interact with the EVH1 domain within yeasts. This interaction was not observed when EVH1 contained a Thr102Arg (T102R) mutation, which disrupts the ERK suppression activity (7) (Fig.1A). Further deletion mutation analysis revealed that the GRD region binds to the EVH1 domain. Fine deletion mutation analysis also revealed that the N-terminal 16 aa and C-terminal 20 aa outside the GAP domain are essential for the interaction between EVH1 and GRD (Fig.1B). We noticed that EVH1 did not interact with p120RasGAP (Fig.1B). The NF1 GRD possesses extensions of the N- and C-terminal peptides that are not present in p120-RasGAP (Fig.1B, yellow and red regions). These regions correspond exactly to the regions we have identified as the EVH1 binding sites. Previously, a 483-residue GRD of neurofibromin had been described as the Ras-GAP domain (30,31). However, smaller fragments of p120(GAP) (GAP-273, residues Met714-His986) and neurofibromin (NF1-230, residues Asp1248-Phe1477) have also been shown to possess full Ras-GAP activity (32). Thus, extra Nand C-peptides unique to NF1 GRD are not necessary for the GAP function, but are essential for the binding to the EVH1 domain. We confirmed the binding of EVH1 and WTGRD in HEK293 cells by means of an immunoprecipitation (IP)-Western blotting assay (Fig.1C). GRD lacking these N- and C-terminal extensions could not bind to EVH1 (Fig.1C, ΔN and ΔC). To examine whether these extensions were important for suppression of the Ras-ERK pathway, WT-GRD or ΔN-GRD, which lacked Nterminal 16 aa, were overexpressed together with a moderate amount of SPRED1, the concentration of which reduced EGF-induced ERK activity to approximately 50% (Fig.1D). We found that WTGRD overexpression in HEK293 cells enhanced the SPRED1-mediated suppression of ERK activity, while ΔN-GRD did not (Fig.1D). These data confirmed that SPRED1 recruits neurofibromin to suppress Ras activation through the interaction between EVH1 and GRD.


for EGF-induced ERK activation was much lower than that of WT in HEK293 cells (Fig.2E). These data suggest that pathogenicity of the mutations outside GAP domain can be explained by the disruption of GRD-EVH1 interaction, thereby reduced Ras-ERK suppression activity. We also introduced mutations into putative phosphorylation sites, Tyr1521, Ser1523, and Ser1524; however, there were no apparent effects on the binding (data not shown). Since Glu1206 is a conserved residue with negative charge, we substituted this residue to Ala; however, we did not see any effects on the interaction between EVH1 and GRD (data not shown). As shown through crystal structural analysis (NF1-333; residue 1198-1530, which does not contain a 21 aa insertion) (36), these two extensions form two α-helix tubes that cross over each other, and severely affected mutations were mostly present in the surface of N-terminal α-helix (Fig.2F). Leu1511 is located on the same side of Leu1208, Met1215, and Asp1217, thus, we suspect that these residues form the binding site for the EVH1 domain (Fig.2F). EVH1-GRD interaction is essential for the suppression of ERK by neurofibromin To show the functional importance of the EVH1GRD interaction in the Ras-ERK inactivation in mammalian cells, we overexpressed a GRD mutant (R1276P) that lacked GAP activity (37). Since this mutant GRDR1276P still possessed full EVH1 binding activity (Fig.3A), it should function as a dominant negative form against the endogenous full-length neurofibromin. As expected, we found that the SPRED1-dependent suppression of EGF-induced ERK activation was reverted by the GRDR1276P mutant (Fig.3A bottom panel). This strongly supports the idea that the binding of the EVH1 of SPRED1 to GRD is critical for the suppressive function of neurofibromin. Since it has been shown that GRD binds to the Ras-GTP form through a Ras-binding groove (36), we investigated whether EVH1 binding modulates the GRD-Ras interaction or if Ras modulates the EVH1-GRD interaction. As shown in Fig.3B, V12-Ras (oncogenic Ras), but not WT-Ras, bound to GRD; however, the V12-Ras-GRD interaction was weaker than the EVH1-GRD interaction. When WT or V-12-Ras was co-expressed, the EVH1-GRD interaction was not affected (Fig.3C). We also found that the overexpression of EVH1 5


GRD of neurofibromin are highly conserved among species (Fig.2A). Since we have used the type II NF1 gene that contains a 21 aa insertion within the GRD (33), the C-terminal region corresponds to Leu1511-Ala1530. Among the mutations reported in NF1 patients, we noticed that two previously reported point mutations, p.Arg1204Gly (R1204G) and p.Arg1204Trp (R1204W), were localized in the N-terminal EVH1 interaction region (34). We also analysed eight additional mutations in these regions that were detected in NF1 patients (Fig.2A)(Patient information is shown in Supplementary Table 1). All of these amino acids were highly conserved among various species (Fig.2A). All NF1 missense mutations were classified for pathogenicity according to the criteria of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (35). As shown in Fig.2B and summarized in Fig.2A, the two-hybrid assay revealed that p.Arg1204Trp (R1204W), p.Arg1204Leu (R1204L), p.Leu1208Trp (L1208W), p.Met1215del (M1215Δ), p.Asp1217Gly (D1217G), and p.Leu1511Pro (L1511P) severely reduced the GRD-EVH1 interaction, while p.Ala1202Ser (A1202S), p.Arg1204Gly (R1204G), p.Leu 1211Arg (L1211R), p.Gln1515Arg (Q1515R), and p.Gly1519Glu (G1519E) did not significantly affect the GRD binding activity to the EVH1 domain (Fig.2B). Three (p.Met1215del, p.Asp1217Gly, p.Leu1511Pro) from the 5 likely pathogenic NF1 missense mutations (p.Arg1204Gly, p.Met1212del, p.Asp1217Gly, p.Leu1511Pro, p.Gln1515Arg) profoundly reduced binding of GRD with the SPRED1 EVH1-domain. We observed 40 % reduction of the interaction between EVH1 and GRD mutant carrying p.Arg1204Gly (R1204G), which is likely pathogenic NF1 mutation by using quantitative β-galactosidase assay (Fig.2C), indicating that the pathogenic mechanism of these mutations may also related to an impaired binding of the EVH1 domain of SPRED1. Three (p.Arg1204Leu, p.Arg1204Trp, p.Leu1208Trp) of the five variants of uncertain significance also showed a severely reduced GRD-EVH1 interaction. We confirmed reduced binding between EVH1 and GRDL1511P, GRDD1217G, and GRDM1215Δ by means of IP-Western blotting in HEK293 cells (Fig.2D). The suppression activity of GRDL1511P


activity assay (Fig.4B). Disruption of the EVH1GRD interaction by such mutations was confirmed by IP-Western blotting assay in 293 cells (Fig.4C). All except one pathogenic and likely pathogenic mutations, such as G30R, W31C, W31L, V44D, G62R, T88K, W92R, G100D, T102R, T102K severely reduced the EVH1-GRD binding (Fig. 4A,B). The three benign mutations and one variant of uncertain significance (D9V, N10K, V42I, and C74F) did not affect the interaction and behaved as wild type EVH1 (Fig.4A,B). ERK suppression activity was then measured by Elk reporter assay in HEK293 cells (Fig.4D), and the ERK suppression activity versus the strength of the EVH1-GRD interaction was plotted (Fig.4E). The ERK suppression activity was well correlated to the EVH1-GRD binding activity. We noticed that the mutations fell into three classes: those with almost completely diminished GRD binding, those with partially reduced binding (R24Q, L80R, T102M), and those with unaltered binding (D9V, N10K, V42I, C74F). W31C, T102R, and V44D have been shown previously to be (likely) pathogenic missense mutations, while C74F was a sequence variant that had previously been shown to have no effect on SPRED1 function (11), suggesting a correlation between the EVH1-GRD interaction and pathogenicity. Therefore, overall, mutations which disrupt the EVH1-GRD interaction are pathogenic and less suppressive for ERK. However, there are a couple of exceptions. The likely pathogenic mutation R24Q showed only a mildly reduced EVH1-GRD binding (the pedigree and clinical phenotypes of a family carrying R24Q mutation are shown in Fig.5), and variant of uncertain significance I116N showed a severely reduced EVH1-GRD binding, but retained relatively higher ERK suppression activity (Fig.4E). The SPRED1 EVH-l domain has been suggested to bind to a ligand in a groove formed by beta-strands ßl, ß2, ß6, and ß7 (38). Missense mutations with a severe effect on GRD binding such as G30R, W31C/L, V44D, G62R, G100D, and T102K/R are probably present in the groove for the binding to the GRD or important for the structure of the binding groove. As V42I and T102M are substitutions of similar amino acids, they may not affect the entire structure of the EVH1 domain. C74 and L80 are located in an outer strand of the EVHl β-sandwich (ß4), and,

EVH1 missense mutations found in Legius syndrome reduce the EVH1-GRD interaction At least 45 different SPRED1 missense mutations in humans have been reported so far (SPRED1 LOVD and 1 selected from the ARUP database); however, the pathogenicity is not always predictable. Of these, we selected 18 mutations in the EVH1 domain (unpublished patient information is shown in Supplementary Table 2 and 3). Pathogenicity of the missense mutations was also classified according to the criteria of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (35). Mutations p.Asp9Val (D9V), p.Asn10Lys (N10K) and p.Val42Ile (V42I) were classified as benign. Mutations p.Cys74Phe (C74F), p.Leu80Arg (L80R), p.Thr102Met (T102M), and p.Ile116Asn (I116N) fell in the category of variants of uncertain significance. While, p.Arg24Gln (R24Q), p.Gly30Arg (G30R), p.Trp31Leu (W31L), p.Gly44Asp (G44D), p.Gly62Arg (G62R), p.Thr88Lys (T88K), p.Trp92Arg (W92R), p.Gly100Asp (G100D), p.Thr102Arg (T102R), and p.Thr102Lys (T102K) were identified as probably pathogenic, and p.Trp31Cys (W31C) was confidently classified as pathogenic. The EVH1-GRD interaction was assessed by in situ staining of yeast two-hybrid assay (Fig.4A) and the results were quantified by a β-gal enzyme 6


did not affect the GRD-V12-Ras interaction as observed by the IP-Western assay (Fig.3D). These data suggest that the EVH1-GRD interaction is independent of the GRD-Ras interaction. This is reasonable because the extended N- and Cterminal EVH1-binding peptides of the GRD distantly locate from the Ras binding groove of the GRD (see Fig.4F). Previously, we reported that mSpred3 has much weaker ERK suppression activity compared with mSpred1 and mSpred2 (3). Thus, we compared the binding activity among the Spred1/2/3 EVH1 domains to the GRD. As shown in Fig.3E, the binding of the EVH1 domain of mSpred2 to the GRD was similar to that of mSpred1, while the mSpred3 EVH1 domain interacted with the GRD with much weaker affinity than mSpred1 or mSpred2 (Fig.3E). Thus, the lower binding affinity of the Spred3 EVH1 domain may explain its lower Ras-ERK suppression activity compared with that of Spred1 and Spred2.


accordingly, the effects of these mutations on the EVH1-GRD interaction and ERK suppression are less drastic compared to those of other mutations. We used computer simulation to provide a model for the interaction between EVH1 and GRD (Fig.4F). These mutations are very likely to disrupt the EVH1 binding pocket for interaction with the GRD-N- and C-terminal regions. Several amino acids that severely affected binding, G30, W31, V44, G62, G100, and T102, are located in this pocket. Discussion In this study, we demonstrate that the SPRED EVH1 domain interacts with the N-terminal and C-terminal extended regions of the GRD of neurofibromin, which is essential for the function of SPREDs as well as neurofibromin. Most pathogenic and likely pathogenic missense mutations in EVH1 found in Legius syndrome and some NF1 GRD pathogenic missense mutations from NF1 patients severely disrupt this interaction and reduce suppression activity against the RasERK pathway. Using the data on the severely reduced EVH1-GRD binding in this manuscript we could potentially “upgrade” the classification of 3 NF1 missense mutations of uncertain significance to likely pathogenic mutations (p.Arg1204Leu, p.Arg1204Trp, p.Leu1208Trp), and of 3 likely pathogenic mutations to pathogenic mutations (p.Met1215del, p.Asp1217Gly, p.Leu1511Pro). Overall, there is a strong correlation among pathogenic mutations, disruption of the EVH1GRD interaction and ERK suppression activity are pathogenic. The likely pathogenic SPRED1 missense mutations G30R, W31L, G62R, T88K, W92R, G100D, T102R, and T102K in the EVH1 domain showed a severely reduced binding affinity to the GRD, while the three benign mutations and one mutation of uncertain significance (D9V, N10K, V42I and C74F) did not affect binding to the GRD. Thus the effect of the EVH1 missense mutations on the EVH1-GRD interaction explains the pathogenetic mechanism. However, some SPRED1 mutations (R24Q, L80R, T102M), especially R24Q, have only a modest effect on the GRD interaction, but clearly associated with a Legius syndrome phenotype with café-au-lait macules in three generations. In addition, we cannot explain at the moment why variant of uncertain significance I116N has a low EVH1-GRD interaction index but a relatively high

Acknowledgments We thank R. Komine, S. Tsuruta, N. Shiino, Y. Noguchi, and M. Asakawa for technical assistance; Dr. F. McCormick, University of California at San Francisco, for neurofibromin cDNA and discussion, Y. Ushijima and R. Mizoguchi for manuscript preparation. HB is a postdoctoral researcher of the Research Foundation Flanders (FWO) at the Catholic University of Leuven (KU Leuven). We thank MJ Martínez-González from the Neuropediatric unit, Pediatric Department, Cruces University Hospital, Biocruces Health Research Institute, and Mao Rong, ARUP Laboratories, University of Utah, Salt Lake City, Utah, for providing us patient information. This work was supported by special Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 25221305), Advanced Research & 7


ERK suppression index. Further study is necessary to define the pathogenetic mechanism of these mutations. The overlapping phenotypes between NF1 and Legius syndrome are explained by the model proposed by Stowe et al. (18), that is, SPREDs recruit neurofibromin to the Ras-enriched cell membrane. Since there are three functionally overlapping SPREDs in humans, the relatively benign phenotype of Legius syndrome can be explained by SPRED2/3 activity compensating for SPRED1 mutations in most cell types. It is notable that the EVH1 domains of mSpred1 and mSpred2 have similar GRD binding affinity and similar ERK suppression activity. Physiological redundancy of Spred1 and Spred2 were confirmed by gene-disruption studies in which Spred1/2double KO mice were embryonically lethal while Spred1 and Spred2 single KO mice were viable (39). In conclusion, pathogenic and likely pathogenic mutations in the EVH1 domain of SPRED1 cause loss of binding affinity to neurofibromin GRD, and our yeast two-hybrid assay correlates excellently with the Ras-ERK suppression activity of SPRED1. The information obtained in this study will be helpful for the classification of missense mutations in Legius syndrome and NF1. This unique EVH1-GRD interaction may also be useful in the development of agents that could induce Ras inactivation in cancer.


Development Programs for Medical Innovation (AMED-CREST), the Takeda Science Foundation, the Uehara Memorial Foundation. This work was also supported by the “Opening the Future” grant of KU Leuven to EL.

Author Contributions Conceived and designed the experiments: HB, LM, EL, AY. Performed the experiments: YH, MK, MO, TO. Analyzed the data: HB, MS, MK, MO, RM. Contributed reagents/materials/analysis tools: HB, ILR, LM, EL. Wrote the paper: HB, LM, EL, AY.

Conflict of interest The authors have no conflicting financial interests.


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Genes to cells : devoted to molecular & cellular mechanisms 10, 887-895 5.

Impagnatiello, M. A., Weitzer, S., Gannon, G., Compagni, A., Cotten, M., and Christofori, G. (2001) Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells. The Journal of cell biology 152, 1087-1098

6.

Brems, H., Chmara, M., Sahbatou, M., Denayer, E., Taniguchi, K., Kato, R., Somers, R., Messiaen, L., De Schepper, S., Fryns, J. P., Cools, J., Marynen, P., Thomas, G., Yoshimura, A., and Legius, E. (2007) Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1-like phenotype. Nature genetics 39, 1120-1126

7.

Messiaen, L., Yao, S., Brems, H., Callens, T., Sathienkijkanchai, A., Denayer, E., Spencer, E., Arn, P., Babovic-Vuksanovic, D., Bay, C., Bobele, G., Cohen, B. H., Escobar, L., Eunpu, D., Grebe, T., Greenstein, R., Hachen, R., Irons, M., Kronn, D., Lemire, E., Leppig, K., Lim, C., McDonald, M., Narayanan, V., Pearn, A., Pedersen, R., Powell, B., Shapiro, L. R., Skidmore, D., Tegay, D., Thiese, H., Zackai, E. H., Vijzelaar, R., Taniguchi, K., Ayada, T., Okamoto, F., Yoshimura, A., Parret, A., Korf, B., and Legius, E. (2009) Clinical and mutational spectrum of neurofibromatosis type 1-like syndrome. JAMA : the journal of the American Medical Association 302, 2111-2118

8.

Pasmant, E., Sabbagh, A., Hanna, N., Masliah-Planchon, J., Jolly, E., Goussard, P., Ballerini, P., Cartault, F., Barbarot, S., Landman-Parker, J., Soufir, N., Parfait, B., Vidaud, M., Wolkenstein, P., Vidaud, D., and France, R. N. (2009) SPRED1 germline mutations caused a neurofibromatosis type 1 overlapping phenotype. Journal of medical genetics 46, 425-430

9.

Spurlock, G., Bennett, E., Chuzhanova, N., Thomas, N., Jim, H. P., Side, L., Davies, S., Haan, E., Kerr, B., Huson, S. M., and Upadhyaya, M. (2009) SPRED1 mutations (Legius syndrome):

9


localizes in a lipid raft/caveola and inhibits ERK activation in collaboration with caveolin-1.


another clinically useful genotype for dissecting the neurofibromatosis type 1 phenotype. Journal of medical genetics 46, 431-437 10.

Muram-Zborovski, T. M., Stevenson, D. A., Viskochil, D. H., Dries, D. C., Wilson, A. R., and Rong, M. (2010) SPRED 1 mutations in a neurofibromatosis clinic. Journal of child neurology 25, 1203-1209

11.

Brems, H., and Legius, E. (2013) Legius syndrome, an Update. Molecular pathology of mutations in SPRED1. The Keio journal of medicine 62, 107-112

12.

Pasmant, E., Gilbert-Dussardier, B., Petit, A., de Laval, B., Luscan, A., Gruber, A., Lapillonne, H., Deswarte, C., Goussard, P., Laurendeau, I., Uzan, B., Pflumio, F., Brizard, F., Vabres, P., Naguibvena, I., Fasola, S., Millot, F., Porteu, F., Vidaud, D., Landman-Parker, J., and

is a tumour suppressor downregulated in paediatric acute myeloblastic leukaemia. Oncogene 34, 631-638 13.

Rauen, K. A. (2013) The RASopathies. Annual review of genomics and human genetics 14, 355-369

14.

Yoshida, T., Hisamoto, T., Akiba, J., Koga, H., Nakamura, K., Tokunaga, Y., Hanada, S., Kumemura, H., Maeyama, M., Harada, M., Ogata, H., Yano, H., Kojiro, M., Ueno, T., Yoshimura, A., and Sata, M. (2006) Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors. Oncogene 25, 6056-6066

15.

Pasmant, E., Gilbert-Dussardier, B., Petit, A., de Laval, B., Luscan, A., Gruber, A., Lapillonne, H., Deswarte, C., Goussard, P., Laurendeau, I., Uzan, B., Pflumio, F., Brizard, F., Vabres, P., Naguibvena, I., Fasola, S., Millot, F., Porteu, F., Vidaud, D., Landman-Parker, J., and Ballerini, P. (2014) SPRED1, a RAS MAPK pathway inhibitor that causes Legius syndrome, is a tumour suppressor downregulated in paediatric acute myeloblastic leukaemia. Oncogene

16.

Miyoshi, K., Wakioka, T., Nishinakamura, H., Kamio, M., Yang, L., Inoue, M., Hasegawa, M., Yonemitsu, Y., Komiya, S., and Yoshimura, A. (2004) The Sprouty-related protein, Spred, inhibits cell motility, metastasis, and Rho-mediated actin reorganization. Oncogene 23, 55675576

17.

Wakioka, T., Sasaki, A., Kato, R., Shouda, T., Matsumoto, A., Miyoshi, K., Tsuneoka, M., Komiya, S., Baron, R., and Yoshimura, A. (2001) Spred is a Sprouty-related suppressor of Ras signalling. Nature 412, 647-651

18.

Stowe, I. B., Mercado, E. L., Stowe, T. R., Bell, E. L., Oses-Prieto, J. A., Hernandez, H.,

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Ballerini, P. (2015) SPRED1, a RAS MAPK pathway inhibitor that causes Legius syndrome,


Burlingame, A. L., and McCormick, F. (2012) A shared molecular mechanism underlies the human rasopathies Legius syndrome and Neurofibromatosis-1. Genes & development 26, 1421-1426 19.

Denayer, E., Chmara, M., Brems, H., Kievit, A. M., van Bever, Y., Van den Ouweland, A. M., Van Minkelen, R., de Goede-Bolder, A., Oostenbrink, R., Lakeman, P., Beert, E., Ishizaki, T., Mori, T., Keymolen, K., Van den Ende, J., Mangold, E., Peltonen, S., Brice, G., Rankin, J., Van Spaendonck-Zwarts, K. Y., Yoshimura, A., and Legius, E. (2011) Legius syndrome in fourteen families. Human mutation 32, E1985-1998

20.

Brems, H., Pasmant, E., Van Minkelen, R., Wimmer, K., Upadhyaya, M., Legius, E., and Messiaen, L. (2012) Review and update of SPRED1 mutations causing Legius syndrome.

21.

Messiaen, L. M., Callens, T., Mortier, G., Beysen, D., Vandenbroucke, I., Van Roy, N., Speleman, F., and Paepe, A. D. (2000) Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects. Human mutation 15, 541-555

22.

Messiaen, L., and Wimmer, K. (2008) NF1 Mutational Spectrum, Monogr Hum Genet. Basel, Karger

23.

Sasaki, A., Yasukawa, H., Suzuki, A., Kamizono, S., Syoda, T., Kinjyo, I., Sasaki, M., Johnston, J. A., and Yoshimura, A. (1999) Cytokine-inducible SH2 protein-3 (CIS3/SOCS3) inhibits Janus tyrosine kinase by binding through the N-terminal kinase inhibitory region as well as SH2 domain. Genes to cells : devoted to molecular & cellular mechanisms 4, 339-351

24.

Endo, T., Sasaki, A., Minoguchi, M., Joo, A., and Yoshimura, A. (2003) CIS1 interacts with the Y532 of the prolactin receptor and suppresses prolactin-dependent STAT5 activation. Journal of biochemistry 133, 109-113

25.

Schneider, S., Buchert, M., and Hovens, C. M. (1996) An in vitro assay of beta-galactosidase from yeast. BioTechniques 20, 960-962

26.

Sasaki, A., Taketomi, T., Wakioka, T., Kato, R., and Yoshimura, A. (2001) Identification of a dominant negative mutant of Sprouty that potentiates fibroblast growth factor- but not epidermal growth factor-induced ERK activation. The Journal of biological chemistry 276, 36804-36808

27.

Taniguchi, K., Ishizaki, T., Ayada, T., Sugiyama, Y., Wakabayashi, Y., Sekiya, T., Nakagawa, R., and Yoshimura, A. (2009) Sprouty4 deficiency potentiates Ras-independent angiogenic signals and tumor growth. Cancer science 100, 1648-1654

11


Human mutation 33, 1538-1546


28.

Lorenz, S., Lissewski, C., Simsek-Kiper, P. O., Alanay, Y., Boduroglu, K., Zenker, M., and Rosenberger, G. (2013) Functional analysis of a duplication (p.E63_D69dup) in the switch II region of HRAS: new aspects of the molecular pathogenesis underlying Costello syndrome. Human molecular genetics 22, 1643-1653

29.

Kozakov, D., Beglov, D., Bohnuud, T., Mottarella, S. E., Xia, B., Hall, D. R., and Vajda, S. (2013) How good is automated protein docking? Proteins 81, 2159-2166

30.

Martin, G. A., Viskochil, D., Bollag, G., McCabe, P. C., Crosier, W. J., Haubruck, H., Conroy, L., Clark, R., O'Connell, P., Cawthon, R. M., and et al. (1990) The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell 63, 843-849

31.

Xu, G. F., Lin, B., Tanaka, K., Dunn, D., Wood, D., Gesteland, R., White, R., Weiss, R., and

stimulates ras GTPase and complements ira mutants of S. cerevisiae. Cell 63, 835-841 32.

Ahmadian, M. R., Wiesmuller, L., Lautwein, A., Bischoff, F. R., and Wittinghofer, A. (1996) Structural differences in the minimal catalytic domains of the GTPase-activating proteins p120GAP and neurofibromin. The Journal of biological chemistry 271, 16409-16415

33.

Andersen, L. B., Ballester, R., Marchuk, D. A., Chang, E., Gutmann, D. H., Saulino, A. M., Camonis, J., Wigler, M., and Collins, F. S. (1993) A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity. Molecular and cellular biology 13, 487-495

34.

Thomas, L., Richards, M., Mort, M., Dunlop, E., Cooper, D. N., and Upadhyaya, M. (2012) Assessment of the potential pathogenicity of missense mutations identified in the GTPaseactivating protein (GAP)-related domain of the neurofibromatosis type-1 (NF1) gene. Human mutation 33, 1687-1696

35.

Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., Grody, W. W., Hegde, M., Lyon, E., Spector, E., Voelkerding, K., and Rehm, H. L. (2015) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 17, 405-424

36.

Scheffzek, K., Ahmadian, M. R., Wiesmuller, L., Kabsch, W., Stege, P., Schmitz, F., and Wittinghofer, A. (1998) Structural analysis of the GAP-related domain from neurofibromin and its implications. The EMBO journal 17, 4313-4327

37.

Klose, A., Ahmadian, M. R., Schuelke, M., Scheffzek, K., Hoffmeyer, S., Gewies, A., Schmitz, F., Kaufmann, D., Peters, H., Wittinghofer, A., and Nurnberg, P. (1998) Selective

12


Tamanoi, F. (1990) The catalytic domain of the neurofibromatosis type 1 gene product


disactivation of neurofibromin GAP activity in neurofibromatosis type 1. Human molecular genetics 7, 1261-1268 38.

Harmer, N. J., Sivak, J. M., Amaya, E., and Blundell, T. L. (2005) 1.15 A crystal structure of the X. tropicalis Spred1 EVH1 domain suggests a fourth distinct peptide-binding mechanism within the EVH1 family. FEBS letters 579, 1161-1166

39.

Taniguchi, K., Kohno, R., Ayada, T., Kato, R., Ichiyama, K., Morisada, T., Oike, Y., Yonemitsu, Y., Maehara, Y., and Yoshimura, A. (2007) Spreds are essential for embryonic lymphangiogenesis by regulating vascular endothelial growth factor receptor 3 signaling. Molecular and cellular biology 27, 4541-4550


13


Figure 2. GRD mutations in the N-terminal extended region reduce EVH1 binding affinity. (A) Amino acid sequences of the N- and C-terminal EVH1 binding region of GRDs from various species. Red letters indicate amino acids whose mutations severely affected binding to the EVH1 domain. Blue letters show amino acids whose mutations had little effect on the binding. (B) In situ yeast two-hybrid assay of the interaction between EVH1 and NF1 GRD mutants. Yeast strains carrying pGBKT7hEVH1 and the indicated GRD mutants in pGADT7 were restreaked on a filter paper and stained for an in situ galactosidase assay. (C) Quantitative β-gal assay of yeast strains carrying pGBKT7-hEVH1 and pGADT7-GRD with indicated mutations. (D) Binding of EVH1 and GRD mutants in HEK293 cells. HEK293 cells were transiently transfected with HA-tagged GRD mutants and FLAG-tagged SPRED1. The immunoprecipitates with anti-FLAG antibody were immunoblotted with anti-HA and anti-FLAG antibodies. (E) GRDL1511P showed a weaker effect on SPRED1-mediated ERK suppression than WT-GRD. HEK293 cells were transfected with Elk-1 reporter plasmid and hSPRED1 expression vector, together with indicated amounts of WT-GRD or GRDL1511P expression vectors. After 24 h, cells were treated with 50 ng/ml EGF for 6 h and luciferase activity was then measured. The Elkluciferase activity of cells with EGF stimulation without transfecting SPRED1 and GRD plasmids is standardized as 100%. (F) Ribbon drawing structures of GRDs of p120GAP and NF1. Blue: Nterminal region. Flesh colour: C-terminal region. The positions of L1511, L1208, M1212, and D1217 are shown. Figure 3. The GRD-EVH1 interaction is independent of the GRD-Ras interaction. (A) Effect of the GAP-null mutant of GRD on SPRED1 suppression activity. (upper panels) In situ yeast two-hybrid assay and IP-Western blotting assay for the interaction between WT EVH1 and R1276P mutant GRD. (lower panel) Reversal of the effect of hSPRED1 on R1276P GRD. HEK293 cells were transfected with or without 10 ng hSPRED1 cDNA in the presence of increased concentrations of R1276P GRD cDNA. EGF-induced Elk-1 reporter activity was measured one day after transfection. (B) Comparison of the binding of GRD between SPRED1 and Ras. HEK293 cells were transfected with FLAG-tagged SPRED1 (2µg), WT (0.5µg), or V12-H-Ras (0.25µg ) and Myc-GRD (2µg) expression plasmids. Cell extracts were immunoprecipitated with anti-FLAG antibody and then blotted with anti-FLAG or antiMyc antibodies. (C) Effect of the overexpression of Ras on the SPRED1-GRD interaction. HEK293 cells were transfected with FLAG-SPRED1 cDNA (3µg) and Myc-GRD cDNA (0.1µg) together with 14


Figure legends Figure 1. Yeast two-hybrid assay to detect EVH1 and neurofibromin interacting domains. (A) Yeast strains carrying pGBKT7-hEVH1 (1-136) and the indicated neurofibromin domains cloned in pGADT7 were restreaked on a filter paper and stained for an in situ galactosidase assay. (B) Interaction between the EVH1 domain and deletion mutants of the GRD. Left; Schematic view of the structural comparison between the NF-1 GRD and the GAP domain of p120GAP. The green box in the GRD indicates that it is minimum domain with full GTPase-activating activity (36). R and K in the GRD and GAP indicate conserved residues for Ras-GTP binding (40).Right; cDNAs of p120-RasGAP (705-1047) or neurofibromin GRD deletion mutants were subcloned into pACT2. N13 means fulllength GRD (1170-1570). Yeasts were restreaked on a filter paper and stained for an in situ galactosidase assay. (C) Binding of EVH1 and GRD mutants in HEK293 cells. HEK293 cells grown in 10 cm dishes were transiently transfected with HA-tagged deletion mutants of GRD plasmids (1.5 µg/dish) together with FLAG-tagged SPRED1 expression plasmid (1 µg /dish). The immunoprecipitates with anti-FLAG antibody were immunoblotted with anti-HA and anti-FLAG antibodies. (D) Enhancement of SPRED1-dependent ERK suppression by GRD. HEK293 cells grown in 12-well plates were transfected with Elk-1 reporter plasmid, hSPRED1 expression vector (10 ng/well), together with indicated amounts of WT-GRD or ΔN-GRD expression vectors. After 24 h, cells were treated with 50 ng/ml EGF for 6 h and luciferase activity was then measured. The Elkluciferase activity of cells with EGF stimulation without transfecting SPRED1 and GRD plasmids is standardized as 100%.


increased concentrations of FLAG-tagged WT or V12 H-Ras cDNA plasmids. One day after transfection, cell extracts were immunoprecipitated with anti-Myc antibody and then blotted with antiMyc or anti-FLAG antibodies. (D) Effect of the overexpression of the SPRED1-EVH1 domain on the V12-HRas-GRD interaction. HEK293 cells were transfected with 500 ng FLAG-Ras cDNA and 1.5 µg Myc-GRD cDNA together with increased concentrations of HA-tagged SPRED1 (EVH1) cDNA plasmids. One day after transfection, cell extracts were immunoprecipitated with anti-FLAG antibody and then blotted with anti-Myc or anti-FLAG antibodies. (E) Yeast strains carrying pGBKT7-EVH1s from mSpred1, 2 , 3 (mS1, mS2, mS3) and pGADT7-GRD were restreaked on a filter paper and stained for an in situ galactosidase assay (upper panels). A quantitative β-galactosidase (β-gal) assay was performed and normalized against the GRD-hSPRED1 EVH1 interaction.

Figure 5. Information of the family carrying Arg24Gln mutation. (upper panel) Pedigree of the family Open symbols indicate unaffected individuals, lined symbols indicate affected individuals. (lower Table). Clinical phenotypes of individuals carrying Arg24Gln mutation. CALM; café-au-lait spot , -; not present, u; unknown

15


Figure 4. The effects of EVH1 mutations on the EVH1-GRD interaction and ERK suppression activity. (A,B) Yeast strains carrying pGBKT7-hEVH1 with indicated mutations and pGADT7-GRD were restreaked on a filter paper and stained for an in situ galactosidase assay (A) and a quantitative βgal assay (B). Error bars denote the mean + standard deviation (S.D.) of triplicate experiments. *p < 0.05, **p < 0.01 (Student’s t-test). (C) Lack of interaction of the GRD with mutant EVH1 domains in 293 cells. Cells were transfected with Myc-tagged GRD and indicated FLAG-tagged SPRED1 mutants. Immunoprecipitates with anti-FLAG antibody were blotted with anti-Myc antibody. (D) ERK suppression activity by mutant SPREDs. HEK293 cells were transfected with the Elk-1 reporter system and 30 ng mutant SPRED1 cDNAs. One day after transfection, HEK293 cells were stimulated with 50 ng/ml EGF for 6 h and luciferase activity was then measured. (E) Correlation between ERK suppression activity and GRD-binding activity of mutant EVH1 domains. Plotting of the EVH1-GRD interaction index and ERK suppression activity index is shown. The EVH1-GRD interaction index is calculated from (B). WT EVH1 β-gal activity is standardized as 100%. ERK suppression index (calculated from (D)) = [(Elk-1 reporter with mutant Spred1) - (Elk-1 reporter with WT Spred1)] / (Elk-1 reporter without Spred1). (F) An in silico modelling of the EVH1 domain (green) and GRD (light blue). The side chains of the arginine residues in charge of binding to Ras (Arg1276 and Arg1416) in GRD are shown in magenta. The N-terminal and C-terminal helices in the GRD which are important for EVH1 binding are covered with a gray surface. Plausible residues in EVH1 for interaction with GRD suggested from the results of yeast-two-hybrid assay and ERK suppression assay (Gly30, Trp31, Gly62, Thr88, Gly100, and Thr102) are shown in yellow.

JBC


JBC


JBC


JBC


JBC


Supplementary Table 1

frontal bossing

c.3611G>T c.3611G>T

p.Arg1204Leu p.Arg1204Leu

UAB-R3347 UAB-R0782R

Y

0 - ≤8 years ≥36 years

F M

-

>5 >5

n n

n n

n n

n n

? NS

? ?

n n

c.3611G>T

p.Arg1204Leu

UAB-R0782

Y

9 - ≤18 years

F

+

>5

BA&I

?

NS

NS

NS

c.3611G>T

p.Arg1204Leu

UAB-R3164

Y

9 - ≤18 years

M

+

>5

LI

?

NS

c.3610C>G

p.Arg1204Gly

UAB-R5354R

Y

27 - ≤35 years

F

-

>5

n

?

?

n

c.3610C>G

p.Arg1204Gly

UAB-R5354

Y

0 - ≤8 years

M

-

1-5

RI&LA

?

n

n

?

?

relative macrocephaly

c.3610C>G c.3610C>G c.3610C>G c.3610C>G

p.Arg1204Gly p.Arg1204Gly p.Arg1204Gly p.Arg1204Gly

UAB-R9937 UAB-R6191 UAB-R7021 UAB-R0684

Y Y Y Y

0 - ≤8 years 9 - ≤18 years 9 - ≤18 years 0 - ≤8 years

M M F M

+ + -

>5 >5 >5 >5

n BA&I BA&I n

? ? B n

NS 1 cNF n n

NS NS n n suspecte d on R knee

NS ? ? ?

NS no by MRI ? ?

NS scoliosis n n

normal

RS

ADD, LD, speech delay normal normal

familial CALMs RS F

also found in the father who carries only 1 very small CAL

EA: 0.02%

absent

absent

tolerated (0.22)

Disease Causing (p:1.0)

absent

absent

absent

deleterious (0.02)

absent

present (Krkljus, 1998)

3x; called pathogenic by curator

absent

present (Ars, 2000)

absent

Functional assay

n

no by MRI

Interpretation Pathogenicity

?

?

Grantham Distance

?

n

PolyPhen

n

n

MutTaster

n

n

SIFT

n

n

LOVD

LA

>5

Human Genome Mutation Database (Cardiff)

>5 irregular borders

-

ESP, ExAC, 1000G

Spinal NF

+

F

Comments

PNF

F

9 - ≤18 years

Family History

Lisch

cNFs & sdNFs

0 - ≤8 years

Y

Development

Freckling

Osseous Lesion

CALMs

Y

UAB-R7357

OPG

Gender

UAB-R9745

p.Arg1204Leu

Age Group

p.Ala1202Ser

c.3611G>T

Proband

c.3604G>T

Patient ID#

NIH Criteria

mutation - DNA

Mutation - Protein

Supplementary Table 1: Phenotypic information on patients with missense mutations affecting the N- and C-terminal extended region of the NF1 GRD and evaluation of the pathogenicity of the missense variants

benign (0.346)

99

class 1 (benign)

normal interaction

Disease Causing probably (p:1.0) damaging (0.992)

102

class 3 (variant of uncertain clinical significance)

disrupted interaction

deleterious (0.01)


125

class 4 (likely pathogenic)

normal interaction

absent

deleterious (0.03)


101

class 3 (variant of uncertain clinical significance


absent

absent

deleterious (0.0)


61



absent

absent

absent

deleterious (0.01)


102


normal interaction

absent

present (Lee, 2006)

5x; called likely pathogenic by curator

NA

NA



absent

absent

absent

tolerated (0.1)


94



absent

present: NF??


deleterious (0.01)


98



absent

present (Van Minkelen, 2014)


deleterious (0.02)


43


normal interaction

absent

present (Ars, 2003)

absent

tolerated (0.23)


98


normal interaction

N-terminal extended region of the NF1-GRD

c.3610C>G

p.Arg1204Gly

UAB-R3947

Y

c.3610C>G

p.Arg1204Gly

UAB-R583

Y

c.3610C>G c.3610C>G c.3610C>G c.3610C>G c.3610C>G

p.Arg1204Gly p.Arg1204Gly p.Arg1204Gly p.Arg1204Gly p.Arg1204Gly

UAB-R0690 UAB-R2353 UAB-R0512 UAB-R3252 UAB-R3631

Y Y Y Y Y

c.3610C>T c.3610C>T c.3610C>T

p.Arg1204Trp p.Arg1204Trp p.Arg1204Trp

UAB-R8035R1 UAB-R8035R2 UAB-R8035

Y

9 - ≤18 years

n

n

F

+

>5

BA

?

n

F

+

>5

NS

?

yes

n

≥36 years 27 - ≤35 years 19 to ≤26 years 9 - ≤18 years 19 to ≤26 years 9 - ≤18 years

F M M M M

+ + + + +

>5 >5 >5 >5 >5

BA BI NS n BA&I

n ? B n ?

n 2-6 sdNFs 1 sd NF

0 - ≤8 years 0 - ≤8 years ≥36 years

M F F

+ + +

>5 >5 >5

n BA 1RI RA

? n n

n n n

c.3610C>T

p.Arg1204Trp

UAB-R7214

Y

0 - ≤8 years

c.3623T>G c.3623T>G

p.Leu1208Trp p.Leu1208Trp

UAB-R2671R UAB-R2671

Y

c.3623T>G

p.Leu1208Trp

UAB-R7785

Y

?

?

?

n

thoracic MRI: several areas of density wi spinal cord; extra axial cyst in posterior fossa

NS

NS

n n

? n

? no by MRI

n pectus excavatum

n

C6-C7

n

n short stature

n n n

? ? ?

? ? ?

n n n winging of scapulae

?

NS

F

ADD, LD

RS

LD LD, delayed early motor and speech milestones LD NS abnormal LD, autism, PDD

F

normal LD normal DD NS normal normal gross motor issues; speech therapy, macrocephalic

F unknown unknown unknown unknown

n

unknown

27 - ≤35 years 0 - ≤8 years

F F

+ -

>5 >5

2 faint n

? ?

n n

n n

? ?

? ?

scoliosis n

normal normal

F F

0 - ≤8 years

M

-

>5

n

n

n

n

?

?

n

hyperactivity, LD

unknown

F

c.3632T>G

p.Leu1211Arg

UAB-R7694R2

M

+

>5

n

?

2-6 cNFs

n

?

no by MRI

n

c.[3632T>G];[2728G>C]

p.[Leu1211Arg];[Gly910Arg]

UAB-R7694

Y

0 - ≤8 years

F

-

3-4

n

n

n

n

NS

?

macrocephaly

abnormal development

F

c.3639_3641delAAT

p.Met1215del

UAB-R0757

Y

0 - ≤8 years

F

+

>5

LI

?

NS

NS

NS

NS

NS

RS

c.3639_3641delAAT

p.Met1215del

UAB-R8947

Y

0 - ≤8 years

F

+

>5

1-2 RA

?

NS

NS

NS

NS

NS

RS

c.3639_3641delAAT

p.Met1215del

UAB-R3694

Y

9 - ≤18 years

F

+

>5

BI&A

?

n

?

?

n

LD

RS

c.3639_3641delAAT

p.Met1215del

UAB-R3912

Y

M

+

>5

BA&I

B

L leg

NS

NS

NS

NS

F

27 - ≤35 years

M

+

>5

A

?

NS

NS

NS

NS

NS

M

+

>5

LI&A

?

s

NS

NS

NS

NS

0 - ≤8 years 19 to ≤26 years 0 - ≤8 years 9 - ≤18 years 9 - ≤18 years 0 - ≤8 years

M F M F F F

+ + + +

>5 3 >5 >5 >5 >5

I BA&I n BA&I BA&I n

? ? n ? ? ?

NS NS n n n NS

n NS n n n NS

? NS ? ? ? NS

no by MRI NS ? ? ? NS

9 - ≤18 years

M

+

>5

BA&I

?

n

n

?

NS

n NS n NS NS NS mild assymmetry spine

NS abnormal development hyperactivity

F

0 - ≤8 years

F

+

>5

BA&I

?

n

NS

?

F

c.3650A>G

p.Asp1217Gly

UAB-R3763R

c.3650A>G

p.Asp1217Gly

UAB-R3763

c.3650A>G c.3650A>G c.3650A>G c.3650A>G c.3650A>G c.3650A>G

p.Asp1217Gly p.Asp1217Gly p.Asp1217Gly p.Asp1217Gly p.Asp1217Gly p.Asp1217Gly

UAB-R3532R2 UAB-R3532R1 UAB-R3532 UAB-R2381R6 UAB-R2381R5 UAB-R2381R2

c.3650A>G

p.Asp1217Gly

UAB-R2381R3

c.3650A>G

p.Asp1217Gly

UAB-R2381R1

c.3650A>G

p.Asp1217Gly

UAB-R2381

Y

+

>5

bil ax

?

n

?

c.3650A>G

p.Asp1217Gly

UAB-R9707

Y

0 - ≤8 years

M

-

>5

n

n

n

n

?

?

n

normal

S

c.3650A>G

p.Asp1217Gly

UAB-R1971

Y

9 - ≤18 years

F

+

>5

BA&I

?

NS

NS

NS

NS

NS

NS

unknown

≥36 years

M

+

>5

NS

?

6-99 cNFs

NS

normal

F

+

>5

n

n

n

n

?

? present by MRI, symptomatic, chiasm

n

M

n

normal

F

n

n

?

?

scoliosis

normal

unknown

n

n

?

?

normal

PrS

abnormal development, ADD, hyperactivity, LD, speech delay

F

Y

27 - ≤35 years 27 - ≤35 years

~7 subdermal 1 subdermal

n

?

?

mild scoliosis

her 8-yo daughter has >6 CALMS and a LD, no mutation possible Noonan pulmonic stenosis

F F F

had remedial reading when younger

Y

possible Noonan (low set ears)

unknown

>5

≥36 years

father also CALs

PrS

-

1-2 possible cNFs n >100 cNFs and 6-99 sdNFs

Noonan phenotype: short stature, hypertelorism, webbed neck

F

PrS F PrS PrS

M

27 - ≤35 years

pineal cyst

Noonan-like features; downslanting palpebral fissures, broad nasal bridge, bulbous nasal tip,

probably damaging (1)

melanoma on the back Noonan phenotype present: low set ears, midface hypoplasia, hypertelotism

proband carries two NF1 variants in trans; p.Leu1211Arg was found in the affected father; p.Gly910Arg was found in the unaffected mother and is a likely benign rare variant

possible Juvenile Xanthogranuloma

NA

NA

F

normal normal normal NS

F F F F F F

normal

F

normal

F

short stature, Noonan??

F proven to be de novo, but father with 3 large CALs and mild scoliosis

C-terminal extended region of the NF1-GRD c.4469T>C/c.4532T>C

p.Leu1490Pro/p.Leu1511Pro

UAB-R6344R

c.4469T>C/c.4532T>C


UAB-R6344

Y ≥36 years

c.4469T>C/c.4532T>C


UAB-R4702

Y

9 - ≤18 years

F

-

>5

n

c.4481A>G; c.4544A>G

p.Gln1494Arg/p.Gln1515Arg

UAB-R5602

Y

9 - ≤18 years

M

-

>5

n

n

c.4493G>A; 4556G>A

p.Gly1498Glu/p.Gly1519Glu

UAB-R5527R1

M

+

>5

n

?

2 sdNFs

n

?

c.4493G>A; 4556G>A c.4493G>A; 4556G>A c.4493G>A; 4556G>A

p.Gly1498Glu/p.Gly1519Glu p.Gly1498Glu/p.Gly1519Glu p.Gly1498Glu/p.Gly1519Glu

UAB-R5527 UAB-R6312 UAB-R3811

Y Y Y

27 - ≤35 years 0 - ≤8 years 0 - ≤8 years 0 - ≤8 years

M F M

+ + -

>5 >5 >5

n yes ?

n n ?

n n NS

? n NS

? ? NS

? ? NS

n n NS

normal learning disability

F unknown

c.4493G>A; 4556G>A

p.Gly1498Glu/p.Gly1519Glu

UAB-R314

Y

0 - ≤8 years

F

+

>5

yes

?

NS

NS

NS

NS

NS

mild speech delay

familial CALMs

Legend: + present; - absent; ? Unknown; NS not specified; M male; F female; + NIH criteria fulfilled; - NIH criteria not fulfilled I : Inguinal; A axillary; L left; R right; B bilateral cNF cutaneous neurofibroma; sdNF subdermal neurofibroma; LD learning difficulties; RS: reported as sporadic, parents not available for testing; PrS: proven de novo by testing both unaffected parents, but no paternity/maternity testing performed; F familial Classification: 5: pathogenic; 4: likely pathogenic; 3: variant of unknown significance; 2: likely benign; 1: benign

probably damaging (1)

intermittent pericarditis

VSD, Noonan possibe (short stature)

probably damaging (0.99)

Other Findings

Family History

?

-

?

c.26A>T

p.Asp9Val

UAB-SPR941

36/F

>6

bilateral axillary and inguinal freckling ?

?

proven pathogenic NF1 mutation, OPG, neurofibromas, SPRED1 mutation in unaffected brother

familial

c.26A>T

p.Asp9Val

UAB-SPR881

10/F

8

right axillary

?

?

-

?

c.26A>T

p.Asp9Val

UAB-SPR491

37/F

-

-

?

?

diffuse histological confirmed gastric neurofibromas, bone cysts on wrist and scoliosis

?

c.30C>A

p.Asn10Lys

UAB-S84

10/M

>6

axillary and inguinal

?

+

likely pathogenic missense mutation in NF1 (K263E), also in affected father both NF1 and SPRED1 mutations present familial

c.30C>A

p.Asn10Lys

UAB-S133

2/M

2 CALMs R upper quadrant, 7 cm 4-5 freckles R hyperpigmented neck, R axillary region on R neck freckling

c.71G>A c.71G>A c.71G>A c.71G>A

p.Arg24Gln p.Arg24Gln p.Arg24Gln p.Arg24Gln

BISC-I2 BISC-II3 BICS-III1 BISC-III3

u/F u/M new born/F 9 months/M

2 3 8 9

axillar

c.71G>A

p.Arg24Gln

UAB-S52

20/F

>6

c.71G>A c.71G>A

p.Arg24Gln p.Arg24Gln

UAB-S119 UAB-S432

37/M 8/F

>6 >6

Brems et al. 2012, dbSNP

newly described

newly described

segmental distribution of pigmentary abnormalities, abnormal development

?

-

scoliosis -

familial familial familial familial

bilateral axillary ?

?

left congenital ventricular aneurism

?

bilateral axillary ? bilateral -

? -

2-6 skin nodules maternal family history of CALM

? familial

c.88G>A

p.Gly30Arg

UAB-S46

?

?

?

?

?

?

c.92G>T

p.Trp31Leu

UAB-S129

5,5/M

8

?

?

?

mild thoracic scoliosis, abnormal development with heart murmur, possible Noonan phenotype with short stature and midface hypoplasia familial

c.93G>T c.93G>T

p.Trp31Cys p.Trp31Cys

Denayer-family 3: I1 Net-16

15/M M

>5 multiple

?

? ?

language delay, ADHD ?

hypertelorism, downslanting palpebral fissues, ptosis, low implanted postriorly rotated ears, low posterior hairline, mild pterygium colli, pectus carinatum/excavatum, motor delay -

newly described newly described newly described newly described

deleterious (score 0)

disease causing (p:0.99)

benign (0,042)

94

class 1 (benign)

disease causing (p=1)

probably damaging (score 1)

43


Spencer et al. 2011

absent

Highly conserved amino acid, up to Fruitfly




125


newly described

absent





61


absent





215

class 5 (pathogenic)

ExAC SAS:0.4%; ESP AA:0.07%




probably damaging (score 0,999)

29

class 1 (benign)

Spurlock et al. 2009

absent

Highly conserved amino acid, up to Zebrafish




152


newly described here

absent





125


Messiaen et al. 2009

absent





205


Spencer et al. 2011

absent





102


newly described here - LOVDSPRED1 - Pasmant Paris

absent


deleterious (score 0.02)



78



absent





101


Brems et al. 2012

absent





94


Spencer et al. 2011

absent





81


absent





78


absent





71


absent

Highly conserved amino acid, up to Zebrafish




149


newly described

?

Denayer et al. 2011 de novo ? newly described here

UAB-SPR061

F

>6

UAB-S68 UAB-S77

8/u 9 months/M

8-10 >6

-

? ?

? ?

c.124G>A

p.Val42Ile

UAB-SPR451

8/F

>6

-

?

?

variant also present in unaffected mother familial

c.131T>A

p.Val44Asp

Spurlock-family 6

60-68/M

+

+

98th

?

phenotype of affected son and 2 grandchildren of proband described with same mutation familial

c.184G>C

p.Gly62Arg

Utah-1

3/F

+

-

+

?

-

familial

c.221G>T

p.Cys74Phe

UAB-S5/SB4

?

?

?

?

?

segmental distribution of pigmentary abnormalities

?

c.239T>G

p.Leu80Arg

UAB-S78

11/M

>6

-

-

-

-

sporadic

c.263C>A

p.Thr88Lys

Fr-7

7/M

multiple

axillary and inguinal

?

?

mutation also present in affected mother

familial

c.274T>C

p.Trp92Arg

UAB-S127

3/M

>6

?

?

-

also found in the proband's mother with 1 hyperpigmented region on the abdomen familial

c.299G>A

p.Gly100Asp

UAB-S104

62/F

>6

present

?

?

?

?

c.305C>T

p.Thr102Met

UAB-S83-I1

26/F

>6

-

-

+

short stature, headache

familial

c.305C>T

p.Thr102Met

UAB-S83-II1

11 months/F

>6

-

-

-

c.305C>A c.305C>A

p.Thr102Lys p.Thr102Lys

UAB-S64 UAB-S105

0,75/M 2/M

>6 >6

-

-

-

? ?

sporadic sporadic

Spencer et al. 2011 Brems et al. 2012

c.305C>G

p.Thr102Arg

UAB-S24

17/M

>6

+

?

?

large right temporal venous anomaly in brain, progressive dystonia of unexplained etiology

familial

Messiaen et al. 2009

c.347T>A

p.Ile116Asn

UAB-S110-I1

35/M

>6

+

?

-

familial


c.347T>A

p.Ile116Asn

UAB-S110-II1

21 months/M

>6

-

?

?

familial


4= likely pathogenic 5= pathogenic

Eur Am: 0.02%; Eur Moderately conserved nonamino acid Finnish: 0.16%


p.Val42Ile p.Val42Ile

1= benign 2= likely benign 3= uncertain significance

class 1 (benign)


p.Trp31Cys

Legend: M male F female y years u unknown - not present + present pathogenicity: score according to Richards et al., Genetics in Medicine, 17:405-424, 2015.

125

absent

c.124G>A c.124G>A

-

disease causing benign (0,0002) (p=1)

Spencer et al. 2011

c.93G>T

spinal tumors and bilateral vestibular schwannomas, pathogenic NF2 mut

deleterious (score 0,01)

Brems et al. 2012 dbSNP

bilateral axillary and inguinal

+

ESP: 0.02%; ExAC: Moderately conserved 0.14-1.32% amino acid (Latino and South Asian)

newly described

newly described

>97th 97th 50th

Pathogenicity

Learning Disabilities

?

Grantham Distance

Macrocephaly

-

Polyphen

Freckling

>6

Mutation Taster

CALM

5,5/M

SIFT

Age (yr)/ Gender

UAB-S103

Evolutionary Conservation

patient ID#

p.Asp9Val

ESP, ExAC, 1000G

mutation - protein

c.26A>T

Reference

mutation - DNA

Supplementary Table 2: Phenotypic information on patients with missense mutations in the EVH1 domain of SPRED1

?

? ?

Spencer et al. 2011, dbSNP newly described here

familial

Interaction between a domain of a negative regulator of the RAS-ERK pathway, SPRED1, and the GTPase-Activating Protein-Related Domain of neurofibromin is implicated in Legius Syndrome and Neurofibromatosis Type 1. Yasuko Hirata, Hilde Brems, Mayu Suzuki, Mitsuhiro Kanamori, Masahiro Okada, Rimpei Morita, Isabel Llano-Rivas, Toyoyuki Ose, Ludwine Messiaen, Eric Legius and Akihiko Yoshimura J. Biol. Chem. published online December 3, 2015

Access the most updated version of this article at doi: 10.1074/jbc.M115.703710 Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts

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