Mutations Impairing GSK3-Mediated MAF Phosphorylation Cause Cataract, Deafness, Intellectual Disability, Seizures, and a Down Syndrome-like Facies
2015; Elsevier BV; Volume: 96; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2015.03.001
ISSN1537-6605
AutoresMarcello Niceta, Emilia Stellacci, Karen W. Gripp, Giuseppe Zampino, Maria Kousi, Massimiliano Anselmi, Alice Traversa, Andrea Ciolfi, Deborah L. Stabley, Alessandro Bruselles, Viviana Caputo, Serena Cecchetti, Sabrina Prudente, Maria Teresa Fiorenza, Carla Boitani, Nicole Philip, Dmitriy Niyazov, Chiara Leoni, Takaya Nakane, Kim M. Keppler‐Noreuil, Stephen R. Braddock, Gabriele Gillessen‐Kaesbach, Antonio Palleschi, Philippe M. Campeau, Brendan Lee, Célio Pouponnot, Lorenzo Stella, Gianfranco Bocchinfuso, Nicholas Katsanis, Katia Sol‐Church, Marco Tartaglia,
Tópico(s)Cerebrovascular and genetic disorders
ResumoTranscription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development. Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development. Dual sensory impairment due to cataracts and sensorineural hearing loss is a well-recognized consequence of infectious teratogenic exposure (i.e., fetal rubella syndrome), but only rarely observed as a developmental defect in genetic disease phenotypes. In 1996, Gripp and co-workers described two unrelated subjects with congenital cataracts and sensorineural deafness associated with intellectual disability, short stature, brachycephaly, and a distinctive flat facial appearance and considered this trait to represent a previously unrecognized syndrome (MIM 601088).1Gripp K.W. Nicholson L. Scott Jr., C.I. Apparently new syndrome of congenital cataracts, sensorineural deafness, Down syndrome-like facial appearance, short stature, and mental retardation.Am. J. Med. Genet. 1996; 61: 382-386Crossref PubMed Scopus (17) Google Scholar In their clinical report,2Aymé S. Philip N. Fine-Lubinsky syndrome: a fourth patient with brachycephaly, deafness, cataract, microstomia and mental retardation.Clin. Dysmorphol. 1996; 5: 55-60Crossref PubMed Scopus (15) Google Scholar Aymé and Philip discussed on the similarities between the clinical features exhibited by their case and others previously reported by other authors4Aymé S. Philip N. Apparently new syndrome of congenital cataracts, sensorineural deafness, Down syndrome-like facial appearance, short stature, and mental retardation.Am. J. Med. Genet. 1997; 70: 333-335Crossref PubMed Google Scholar and those of the patients reported by Gripp and co-authors.1Gripp K.W. Nicholson L. Scott Jr., C.I. Apparently new syndrome of congenital cataracts, sensorineural deafness, Down syndrome-like facial appearance, short stature, and mental retardation.Am. J. Med. Genet. 1996; 61: 382-386Crossref PubMed Scopus (17) Google Scholar Aymè and Philip concluded that all these cases were clinically related to the patient originally described by Fine and Lubinsky.3Fine B.A. Lubinsky M. Craniofacial and CNS anomalies with body asymmetry, severe retardation, and other malformations.J. Clin. Dysmorphol. 1983; 1: 6-9PubMed Google Scholar For this reason, the authors proposed the term "Fine-Lubinsky syndrome" to define this developmental disorder. Since then, a few additional cases exhibiting features fitting or partially overlapping this condition(s) have been reported,5Keppler-Noreuil K. Welch J. Baker-Lange K. Syndrome of congenital cataracts, sensorineural deafness, Down syndrome-like facial appearance, short stature, and mental retardation: two additional cases.Am. J. Med. Genet. A. 2007; 143A: 2581-2587Crossref PubMed Scopus (9) Google Scholar, 6Nakane T. Mizobe N. Hayashibe H. Nakazawa S. A variant of Fine-Lubinsky syndrome: a Japanese boy with profound deafness, cataracts, mental retardation, and brachycephaly without craniosynostosis.Clin. Dysmorphol. 2002; 11: 195-198Crossref PubMed Scopus (7) Google Scholar, 7Holder A.M. Graham B.H. Lee B. Scott D.A. Fine-Lubinsky syndrome: sibling pair suggests possible autosomal recessive inheritance.Am. J. Med. Genet. A. 2007; 143A: 2576-2580Crossref PubMed Scopus (3) Google Scholar, 8Schoner K. Bald R. Fritz B. Rehder H. Fetal manifestation of the Fine-Lubinsky syndrome. Brachycephaly, deafness, cataract, microstomia and mental retardation syndrome complicated by Pierre-Robin anomaly and polyhydramnios.Fetal Diagn. Ther. 2008; 23: 228-232Crossref PubMed Scopus (6) Google Scholar, 9Corona-Rivera J.R. López-Marure E. García-Cruz D. Romo-Huerta C.O. Rea-Rosas A. Orozco-Alatorre L.G. Ramírez-Valdivia J.M. Further clinical delineation of Fine-Lubinsky syndrome.Am. J. Med. Genet. A. 2009; 149A: 1070-1075Crossref PubMed Scopus (2) Google Scholar and whether these phenotypes represent variable manifestations of a single nosologic entity remained unresolved. Autosomal recessive inheritance was suggested, based on affected siblings.7Holder A.M. Graham B.H. Lee B. Scott D.A. Fine-Lubinsky syndrome: sibling pair suggests possible autosomal recessive inheritance.Am. J. Med. Genet. A. 2007; 143A: 2576-2580Crossref PubMed Scopus (3) Google Scholar Here, whole-exome sequencing (WES) on a single affected individual and Sanger sequencing on a selected cohort of subjects with phenotype suggestive of FLS were used to identify a narrow spectrum of missense mutations in v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF [MIM 177075]) as the molecular cause underlying this previously poorly understood multisystem disorder, and delineate its clinical phenotype. The provided biochemical and functional data demonstrate that the mutations identified in this study specifically affect the phosphorylation of MAF promoted by the protein GSK3, which is a serine/threonine kinase that requires a specific recognition motif for its action—i.e., the presence of a proline residue adjacent to the serine/threonine residue that is substrate of its action. The impaired phosphorylation at those sites affects MAF ubiquitination, which, in turn, impairs degradation of the mutated (unphosphorylated and unubiquitinilated) protein, generally mediated by the proteasome complex. Finally, these mutations are able to induce neurodevelopmental defects in vivo (zebrafish), thus representing dominant-acting mutations. Thirteen subjects were included in this study. All individuals were clinically assessed by experienced clinical geneticists. Among them, nine subjects had previously been reported.1Gripp K.W. Nicholson L. Scott Jr., C.I. Apparently new syndrome of congenital cataracts, sensorineural deafness, Down syndrome-like facial appearance, short stature, and mental retardation.Am. J. Med. Genet. 1996; 61: 382-386Crossref PubMed Scopus (17) Google Scholar, 2Aymé S. Philip N. Fine-Lubinsky syndrome: a fourth patient with brachycephaly, deafness, cataract, microstomia and mental retardation.Clin. Dysmorphol. 1996; 5: 55-60Crossref PubMed Scopus (15) Google Scholar, 5Keppler-Noreuil K. Welch J. Baker-Lange K. Syndrome of congenital cataracts, sensorineural deafness, Down syndrome-like facial appearance, short stature, and mental retardation: two additional cases.Am. J. Med. Genet. A. 2007; 143A: 2581-2587Crossref PubMed Scopus (9) Google Scholar, 6Nakane T. Mizobe N. Hayashibe H. Nakazawa S. A variant of Fine-Lubinsky syndrome: a Japanese boy with profound deafness, cataracts, mental retardation, and brachycephaly without craniosynostosis.Clin. Dysmorphol. 2002; 11: 195-198Crossref PubMed Scopus (7) Google Scholar, 7Holder A.M. Graham B.H. Lee B. Scott D.A. Fine-Lubinsky syndrome: sibling pair suggests possible autosomal recessive inheritance.Am. J. Med. Genet. A. 2007; 143A: 2576-2580Crossref PubMed Scopus (3) Google Scholar, 8Schoner K. Bald R. Fritz B. Rehder H. Fetal manifestation of the Fine-Lubinsky syndrome. Brachycephaly, deafness, cataract, microstomia and mental retardation syndrome complicated by Pierre-Robin anomaly and polyhydramnios.Fetal Diagn. Ther. 2008; 23: 228-232Crossref PubMed Scopus (6) Google Scholar Clinical features are described in detail in Table S1. Clinical data and biological material collection and storage were attained from the participating families after written informed consent was secured, following procedures in accordance with the ethical standards of the responsible committees on human experimentation (institutional and national). Genomic DNA was isolated from peripheral blood leukocytes, skin fibroblasts, hair bulb cells, and/or buccal mucosal epithelial cells, using standard protocols. We performed WES on genomic DNA extracted from circulating leukocytes of a single affected subject (case 8) (Figure 1A, CaGi_UCSC). Exome capture was performed using NimbleGen SeqCap EZ Exome V. 3.0 (Roche) and sequencing by a HiSeq2000 instrument (Illumina). WES data analysis was performed using an in-house implemented pipeline.10Cordeddu V. Redeker B. Stellacci E. Jongejan A. Fragale A. Bradley T.E. Anselmi M. Ciolfi A. Cecchetti S. Muto V. et al.Mutations in ZBTB20 cause Primrose syndrome.Nat. Genet. 2014; 46: 815-817Crossref PubMed Scopus (63) Google Scholar For sequencing statistics, see Table S2. Data annotation predicted 11,168 high-quality variants having functional impact (i.e., non-synonymous and splice site changes). Among them, 259 private, rare (minor allele frequency < 0.001), or clinically associated changes were retained for further analyses. After excluding the presence of variants compatible with autosomal recessive transmission (Table S3), we reasoned that the clinical symptomatology might be caused by a de novo event. Candidates were stratified through a mixed filtering/prioritization strategy taking into account the predicted impact of each variant and the functional relevance of individual genes on the developmental processes altered in the disorder. Only changes (private, clinically associated, or having unknown frequency or minor allele frequency < 0.001) predicted to be deleterious by the Combined Annotation Dependent Depletion (CADD)11Kircher M. Witten D.M. Jain P. O'Roak B.J. Cooper G.M. Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants.Nat. Genet. 2014; 46: 310-315Crossref PubMed Scopus (3670) Google Scholar (score > 15.0) or Database for Nonsynonymous SNPs' Functional Predictions (dbNSFP) Support Vector Machine (SVM)12Liu X. Jian X. Boerwinkle E. dbNSFP v2.0: a database of human non-synonymous SNVs and their functional predictions and annotations.Hum. Mutat. 2013; 34: E2393-E2402Crossref PubMed Scopus (467) Google Scholar (radial score > 0.0) algorithm were retained and prioritized on the basis of the functional relevance of genes using GeneDistiller. Genes were ranked based on combinations of terms from the OMIM clinical synopsis for MIM 601088 and 601353 (i.e., cataract, deafness, mental retardation, facial dysmorphism, short stature, and seizure) as keywords, using similarity of expression patterns and protein-protein interactions as major weights. We obtained the highest score for MAF, a gene whose mutations had previously been reported to cause autosomal dominant congenital cataracts and lens abnormalities (MIM 610202).13Jamieson R.V. Perveen R. Kerr B. Carette M. Yardley J. Heon E. Wirth M.G. van Heyningen V. Donnai D. Munier F. Black G.C. Domain disruption and mutation of the bZIP transcription factor, MAF, associated with cataract, ocular anterior segment dysgenesis and coloboma.Hum. Mol. Genet. 2002; 11: 33-42Crossref PubMed Scopus (226) Google Scholar, 14Lyon M.F. Jamieson R.V. Perveen R. Glenister P.H. Griffiths R. Boyd Y. Glimcher L.H. Favor J. Munier F.L. Black G.C. A dominant mutation within the DNA-binding domain of the bZIP transcription factor Maf causes murine cataract and results in selective alteration in DNA binding.Hum. Mol. Genet. 2003; 12: 585-594Crossref PubMed Scopus (51) Google Scholar Sanger sequencing confirmed heterozygosity for the c.161C>T (p.Ser54Leu) change in the proband, and sequencing of parental DNAs revealed only the reference allele, evidence for its de novo origin (Figure S1). STR genotyping (AmpFlSTR Identifiler Plus [Life Technologies]) confirmed paternity. The variant was documented in the proband's skin fibroblasts as well as hair bulb and buccal epithelial cell specimens, strongly arguing against the possibility of a somatic event (Figure S1). All the other candidate variants turned out to be inherited from one of the unaffected parents (Table S4). To confirm the causal involvement of MAF, we scanned the entire coding sequence of the gene (NM_005360.4 and NM_001031804.2) for mutations in DNA samples from 12 additional subjects (Table S1), including a sib pair, with features overlapping the conditions delineated by Gripp et al.1Gripp K.W. Nicholson L. Scott Jr., C.I. Apparently new syndrome of congenital cataracts, sensorineural deafness, Down syndrome-like facial appearance, short stature, and mental retardation.Am. J. Med. Genet. 1996; 61: 382-386Crossref PubMed Scopus (17) Google Scholar and Aymé and Philip,2Aymé S. Philip N. Fine-Lubinsky syndrome: a fourth patient with brachycephaly, deafness, cataract, microstomia and mental retardation.Clin. Dysmorphol. 1996; 5: 55-60Crossref PubMed Scopus (15) Google Scholar by direct sequencing. Primer pairs designed to amplify the MAF coding exons and their intron boundaries (NC_000016.10, 79593848..79600725) are listed in Table S5. We identified heterozygous missense mutations in seven unrelated individuals (Table 1 and Figure S1). Two different changes affected Thr58 and Pro59, while others involved adjacent residues, including the previously identified c.161C>T substitution. In all family trios for which parental DNA samples were available, genotyping documented the de novo origin of each mutation, and STR analysis confirmed paternity (Table 1). Sanger sequencing of DNA from available oral mucosal epithelial cells (case 11-1), or epithelial cells and fibroblasts (case 4-1) supported the germline origin of mutations. All changes were predicted to impact protein function by dbNSFP and/or CADD (Table 1), and affected residues conserved among orthologs and paralogs (Figure S2).Table 1MAF Mutations Identified in Individuals with Aymé-Gripp SyndromeSubjectReferenceNucleotide ChangeaNucleotide and amino acid positions refer to transcript variant 1 and protein isoform a (longer isoform) (NM_005360.4, NP_005351.2).Amino Acid ChangeaNucleotide and amino acid positions refer to transcript variant 1 and protein isoform a (longer isoform) (NM_005360.4, NP_005351.2).Protein DomainInheritanceFunctional Impact (Radial SVM Score/ CADD Score)1 (19474)2c.161C>Tp.Ser54LeuTDDe novo1.10/16.272 (11-1)1c.172A>Gp.Thr58AlaTDDe novo, germline0.83/16.073 (4-1)1c.206C>Gp.Pro69ArgTDDe novo, germline0.98/15.494 (ICN_ICW)5c.173C>Tp.Thr58IleTDNot availablebParental DNAs were not available for molecular analyses.0.93/15.385 (14-1)4c.176C>Ap.Pro59HisTDDe novo1.10/17.695 (10-1)p.s.c.176C>Tp.Pro59LeuTDDe novo0.98/9.087 (962112)p.s.c.185C>Gp.Thr62ArgTDDe novo1.10/17.888 (CaGi_UCSC)p.s.c.161C>Tp.Ser54LeuTDDe novo, germline1.10/16.27p.s., present study; TD, transactivation domain.a Nucleotide and amino acid positions refer to transcript variant 1 and protein isoform a (longer isoform) (NM_005360.4, NP_005351.2).b Parental DNAs were not available for molecular analyses. Open table in a new tab p.s., present study; TD, transactivation domain. MAF is a basic leucine zipper (bZIP)-containing transcription factor of the AP1 superfamily.15Blank V. Andrews N.C. The Maf transcription factors: regulators of differentiation.Trends Biochem. Sci. 1997; 22: 437-441Abstract Full Text PDF PubMed Scopus (220) Google Scholar, 16Eychène A. Rocques N. Pouponnot C. A new MAFia in cancer.Nat. Rev. Cancer. 2008; 8: 683-693Crossref PubMed Scopus (148) Google Scholar It is important for lens and eye development17Kim J.I. Li T. Ho I.-C. Grusby M.J. Glimcher L.H. Requirement for the c-Maf transcription factor in crystallin gene regulation and lens development.Proc. Natl. Acad. Sci. USA. 1999; 96: 3781-3785Crossref PubMed Scopus (196) Google Scholar, 18Ring B.Z. Cordes S.P. Overbeek P.A. Barsh G.S. Regulation of mouse lens fiber cell development and differentiation by the Maf gene.Development. 2000; 127: 307-317Crossref PubMed Google Scholar and controls multiple physiological processes, including mechanosensory function, and chondrocyte and T cell differentiation.19Ho I.C. Lo D. Glimcher L.H. c-maf promotes T helper cell type 2 (Th2) and attenuates Th1 differentiation by both interleukin 4-dependent and -independent mechanisms.J. Exp. Med. 1998; 188: 1859-1866Crossref PubMed Scopus (249) Google Scholar, 20Wende H. Lechner S.G. Cheret C. Bourane S. Kolanczyk M.E. Pattyn A. Reuter K. Munier F.L. Carroll P. Lewin G.R. Birchmeier C. The transcription factor c-Maf controls touch receptor development and function.Science. 2012; 335: 1373-1376Crossref PubMed Scopus (109) Google Scholar, 21MacLean H.E. Kim J.I. Glimcher M.J. Wang J. Kronenberg H.M. Glimcher L.H. Absence of transcription factor c-maf causes abnormal terminal differentiation of hypertrophic chondrocytes during endochondral bone development.Dev. Biol. 2003; 262: 51-63Crossref PubMed Scopus (71) Google Scholar Immunohistochemical analyses in mouse embryos documented wide Maf expression (Figure S3). Consistent with previous reports, we observed Maf staining in the lens, dorsal spinal cord, dorsal root ganglia, skin, kidney, hypertrophic chondrocytes of vertebrae, rib and limb cartilage, and the cartilage primordium of the basioccipital bone.22Sakai M. Imaki J. Yoshida K. Ogata A. Matsushima-Hibaya Y. Kuboki Y. Nishizawa M. Nishi S. Rat maf related genes: specific expression in chondrocytes, lens and spinal cord.Oncogene. 1997; 14: 745-750Crossref PubMed Scopus (83) Google Scholar, 23Ogata A. Shimizu T. Abe R. Shimizu H. Sakai M. Expression of c-maf and mafB genes in the skin during rat embryonic development.Acta Histochem. 2004; 106: 65-67Crossref PubMed Scopus (15) Google Scholar In line with the sensorineural hearing loss occurring in all mutation-positive subjects, we detected a specific and strong signal in cochlear cells of E14.5 embryos. Similar to other "large" MAF subfamily members (i.e., MAFA, MAFB, and NRL), MAF's structure is characterized by a C-terminal extended homology region and bZIP domain mediating DNA binding, and a N-terminal transactivation domain required for transcriptional activity and regulatory function (Figure 1B). The latter contains four GSK3 phosphorylation motifs, highly conserved among large MAF proteins (Figure 1C). In MAFA, the sequential phosphorylation of these serine/threonine residues promotes ubiquitination and rapid degradation, but also increases transactivation potential.24Rocques N. Abou Zeid N. Sii-Felice K. Lecoin L. Felder-Schmittbuhl M.P. Eychène A. Pouponnot C. GSK-3-mediated phosphorylation enhances Maf-transforming activity.Mol. Cell. 2007; 28: 584-597Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 25Han S.I. Aramata S. Yasuda K. Kataoka K. MafA stability in pancreatic beta cells is regulated by glucose and is dependent on its constitutive phosphorylation at multiple sites by glycogen synthase kinase 3.Mol. Cell. Biol. 2007; 27: 6593-6605Crossref PubMed Scopus (62) Google Scholar Remarkably, all identified MAF mutations clustered within these motifs. Three affected residues, Ser54, Thr58, and Ser62, are known GSK3 phosphorylation target sites.26Herath N.I. Rocques N. Garancher A. Eychène A. Pouponnot C. GSK3-mediated MAF phosphorylation in multiple myeloma as a potential therapeutic target.Blood Cancer J. 2014; 4: e175Crossref PubMed Scopus (39) Google Scholar The remainder did not involve phosphorylatable residues, but were predicted to affect GSK3-mediated phosphorylation by altering proline residues adjacent to either a phosphorylation site (Thr58) or the C-terminal priming site (Ser70), whose phosphorylation is absolutely required for GSK3 function. To explore the impact of the p.Pro59His, p.Pro59Leu, and p.Pro69Arg changes, we performed molecular dynamics (MD) simulations on complexes formed by full-length GSK3 and ten residue-long peptides of MAF corresponding to the segment that interacts directly with the GSK3 binding cleft, encompassing both the GSK3 target and pSer/pThr primed residues (Table S6). The starting coordinates for the ATP-bound GSK3 were taken from the crystallographic structure of GSK3B complexed with AMP-PNP (PDB entry 1pyx).27Bertrand J.A. Thieffine S. Vulpetti A. Cristiani C. Valsasina B. Knapp S. Kalisz H.M. Flocco M. Structural characterization of the GSK-3beta active site using selective and non-selective ATP-mimetic inhibitors.J. Mol. Biol. 2003; 333: 393-407Crossref PubMed Scopus (272) Google Scholar Each decapeptide was set in an extended conformation along the catalytic cleft of GSK3 as specified in Table S6. The MD simulations were carried out according to the protocol previously described.10Cordeddu V. Redeker B. Stellacci E. Jongejan A. Fragale A. Bradley T.E. Anselmi M. Ciolfi A. Cecchetti S. Muto V. et al.Mutations in ZBTB20 cause Primrose syndrome.Nat. Genet. 2014; 46: 815-817Crossref PubMed Scopus (63) Google Scholar The Gromos 53a6 force field was used, with the exception of the partial charges of pSer/pThr,28Oostenbrink C. Villa A. Mark A.E. van Gunsteren W.F. A biomolecular force field based on the free enthalpy of hydration and solvation: the GROMOS force-field parameter sets 53A5 and 53A6.J. Comput. Chem. 2004; 25: 1656-1676Crossref PubMed Scopus (2955) Google Scholar, 29Hansson T. Nordlund P. Aqvist J. Energetics of nucleophile activation in a protein tyrosine phosphatase.J. Mol. Biol. 1997; 265: 118-127Crossref PubMed Scopus (73) Google Scholar and the parameters for ATP, obtained from quantum mechanical calculation of the molecular system reported in Figure S4. For p.Pro59His and p.Pro59Leu decamers, the conformation of the trimer comprised between the substrate and primed residues was rearranged considerably during the simulations (Figure 2A and Figure S5), with the correct orientation of the substrate residue in the GSK3 active site being destabilized (Figure 2A). p.Pro69Arg, introducing a cationic residue in the proximity of the positively charged GSK3 priming pocket formed by residues Arg96, Arg180, and Lys205, caused a general rearrangement of the adjacent pSer70, pulling it away from the binding pocket (Figure 2B). Overall, our simulations indicated consistently that all disease-causing MAF mutations inhibit GSK3-mediated phosphorylation through impaired association and/or catalysis, by perturbing the interaction with the priming site (p.Pro69Arg) or the active site (substitutions affecting Pro59). To explore the mutations' functional impact directly, we evaluated MAF phosphorylation status. The disease-causing p.Ser54Leu, p.Thr58Ala, p.Thr58Ile, p.Pro59Leu, p.Pro59His, and p.Pro69Arg (FLS-like disorder), and p.Arg288Pro (c.863G>C) changes, the latter considered as representative of lesions associated with isolated cataract,13Jamieson R.V. Perveen R. Kerr B. Carette M. Yardley J. Heon E. Wirth M.G. van Heyningen V. Donnai D. Munier F. Black G.C. Domain disruption and mutation of the bZIP transcription factor, MAF, associated with cataract, ocular anterior segment dysgenesis and coloboma.Hum. Mol. Genet. 2002; 11: 33-42Crossref PubMed Scopus (226) Google Scholar were introduced into the MAF cDNA cloned in pCS2+ vector using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). Consistent with previous reports,26Herath N.I. Rocques N. Garancher A. Eychène A. Pouponnot C. GSK3-mediated MAF phosphorylation in multiple myeloma as a potential therapeutic target.Blood Cancer J. 2014; 4: e175Crossref PubMed Scopus (39) Google Scholar Western blot analysis of transiently transfected COS1 cell lysates documented two MAF states: a slower-migrating, fully phosphorylated form, and a faster-migrating, unphosphorylated form. In cells expressing wild-type MAF, the phosphorylated protein (upper band) predominated, while unphosphorylated MAF (lower band) was barely detectable (Figure 3A, upper panel). Similarly, the mutant carrying the p.Arg288Pro substitution in the DNA binding domain, previously associated with isolated lens and eye defects, was efficiently phosphorylated. This was in sharp contrast to all MAF mutants identified in the present study, which accumulated in cells as unphosphorylated proteins. GSK3-mediated phosphorylation represents a regulatory mechanism promoting MAFA ubiquitination and degradation.24Rocques N. Abou Zeid N. Sii-Felice K. Lecoin L. Felder-Schmittbuhl M.P. Eychène A. Pouponnot C. GSK-3-mediated phosphorylation enhances Maf-transforming activity.Mol. Cell. 2007; 28: 584-597Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 25Han S.I. Aramata S. Yasuda K. Kataoka K. MafA stability in pancreatic beta cells is regulated by glucose and is dependent on its constitutive phosphorylation at multiple sites by glycogen synthase kinase 3.Mol. Cell. Biol. 2007; 27: 6593-6605Crossref PubMed Scopus (62) Google Scholar Based on the high conservation of the GSK3 recognition motif and MAF being a GSK3 substrate, we hypothesized that the amino acid changes in our affected subjects might mediate inefficient protein clearance. On Western blot analyses, we noted increased protein levels (Figure 3A, upper panel) and decreased ubiquitination (Figure 3A, middle panel) for the disease-causing MAF mutants when compared to the wild-type protein (Figures 3A and 3B). Treatment with cycloheximide (CHX), a protein synthesis inhibitor, showed that the half-life of wild-type MAF was much shorter than that of the mutants (Figure 3B). Indeed, a complete disappearance of the protein was observed upon 4 hr CHX treatment, while the steady-state level of the mutants was largely unchanged. Consistently, treatment with MG132, which specifically inhibit proteasomal function, stabilized the protein level of wild-type MAF, while it did not have any significant effects on mutants (Figure 3B). Taken together, these results showed that mutations prevented MAF degradation and enhanced their stability. Of note, a partial phosphorylation was apparent for the p.Pro69Arg MAF mutant, which was associated with increased degradation via proteasome, even though less efficiently compared to wild-type MAF. This finding suggests a milder perturbing role of the proline-to-arginine substitution on GSK3-mediated phosphorylation at Ser66 compared to the other disease-causing amino acid changes, possibly due to the peculiar effect of the introduced arginine residue, which was documented to primarily affect MAF interaction with the GSK priming site. Confocal microscopy of transfected COS1 cells confirmed the nuclear localization of all tested mutants and their higher abundance within cells (Figure 3C). Moreover, treatment with CSK buffer prior fixation indicated that the syndrome-causing mutants retained efficient interaction with chromatin suggesting that they bind to DNA, in contrast to the DNA binding-impaired cataract-associated p.Arg288Pro mutant (Figure 3C and Table S7). Transactivation assays using luciferase as reporter under control of the IL4 promoter documented that COS1 cells transiently expressing the cataract-causing mutant allele had barely detectable reporter induction (Figure 3D). In contrast, cells expressing the p.Ser54Leu, p.Thr58Ala, p.Thr58Ile, p.Pro59Leu, p.Pro59His, or p.Pro69Arg MAF coding alleles showed efficient induction of luciferase levels, though not reaching the levels of the wild-type protein, suggesting that, despite their stabilization and much higher levels, these mutants are less active, at least under these specific
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