G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
G00001299 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000014212 (Vega human gene)
ENSG00000096696 (Ensembl human gene)
1832 (Entrez Gene)
1059 (G2Cdb plasticity & disease)
DSP (GeneCards)
125647 (OMIM)
Marker Symbol
HGNC:3052 (HGNC)
Protein Sequence
P15924 (UniProt)

Synonyms (4)

  • DPI
  • DPII
  • KPPS2
  • PPKS2

Literature (65)

Pubmed - other

  • Lethal acantholytic epidermolysis bullosa.

    McGrath JA, Bolling MC and Jonkman MF

    St John's Institute of Dermatology, King's College London, Guy's Campus, London, UK. john.mcgrath@kcl.ac.uk

    Lethal acantholytic epidermolysis bullosa (LAEB) is an autosomal recessive disorder caused by mutations in the gene encoding the desmosomal protein, desmoplakin (DSP). It is recognized as a distinct form of suprabasal epidermolysis bullosa simplex, although only a single case has been reported. The phenotype comprises severe fragility of skin and mucous membranes with marked transcutaneous fluid loss. Other features include total alopecia, neonatal teeth, and anonychia. Skin biopsy reveals abnormal desmosomes with suprabasal clefting and acantholysis and disconnection of keratin intermediate filaments from desmosomes. The DSP abnormalities present in the affected individual involved expression of truncated DSP polypeptides that lacked the tail domain of the protein. This part of DSP has a vital role in binding to keratin filaments. The affected neonate died after 10 days because of heart failure with evidence of loss of epithelial integrity in the skin, lung, gastrointestinal tract, and bladder. This article provides a clinicopathologic overview of this unique desmosomal genodermatosis, set in the context of other DSP gene mutations, both dominant and recessive, that can cause a spectrum of skin, hair, and heart abnormalities.

    Dermatologic clinics 2010;28;1;131-5

  • Role of genetic testing in arrhythmogenic right ventricular cardiomyopathy/dysplasia.

    Barahona-Dussault C, Benito B, Campuzano O, Iglesias A, Leung TL, Robb L, Talajic M and Brugada R

    Institut de Cardiologie de Montréal, Montréal, Québec, Canada.

    In a cohort of patients with confirmed or suspected arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D), genetic testing is useful in confirming the diagnosis, particularly in individuals who do not completely fulfil Task Force criteria for the disease, thereby also enabling the adoption of preventive measures in family members. Due to the high percentage of novel mutations that are expected to be identified in ARVC/D, the use of genetic screening technology based on the identification of known mutations seems to have very restricted value. Our results support that the presence of certain genetic variations could play a role in the final phenotype of patients with ARVC/D, where single and compound mutation carriers would have more symptomatic forms of the disease and the polymorphism P366L could be associated to a more benign phenotype.

    Clinical genetics 2010;77;1;37-48

  • Comprehensive desmosome mutation analysis in north americans with arrhythmogenic right ventricular dysplasia/cardiomyopathy.

    den Haan AD, Tan BY, Zikusoka MN, Lladó LI, Jain R, Daly A, Tichnell C, James C, Amat-Alarcon N, Abraham T, Russell SD, Bluemke DA, Calkins H, Dalal D and Judge DP

    Department of Medicine/Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

    Background: Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited disorder typically caused by mutations in components of the cardiac desmosome. The prevalence and significance of desmosome mutations among patients with ARVD/C in North America have not been described previously. We report comprehensive desmosome genetic analysis for 100 North Americans with clinically confirmed or suspected ARVD/C.

    In 82 individuals with ARVD/C and 18 people with suspected ARVD/C, DNA sequence analysis was performed on PKP2, DSG2, DSP, DSC2, and JUP. In those with ARVD/C, 52% harbored a desmosome mutation. A majority of these mutations occurred in PKP2. Notably, 3 of the individuals studied have a mutation in more than 1 gene. Patients with a desmosome mutation were more likely to have experienced ventricular tachycardia (73% versus 44%), and they presented at a younger age (33 versus 41 years) compared with those without a desmosome mutation. Men with ARVD/C were more likely than women to carry a desmosome mutation (63% versus 38%). A mutation was identified in 5 of 18 patients (28%) with suspected ARVD. In this smaller subgroup, there were no significant phenotypic differences identified between individuals with a desmosome mutation compared with those without a mutation.

    Conclusions: Our study shows that in 52% of North Americans with ARVD/C a mutation in one of the cardiac desmosome genes can be identified. Compared with those without a desmosome gene mutation, individuals with a desmosome gene mutation had earlier-onset ARVD/C and were more likely to have ventricular tachycardia.

    Funded by: Intramural NIH HHS: ZIA CL090019-01, ZIA EB000072-01; NHLBI NIH HHS: HL088072, R21 HL088072-02; Unspecified: R21 HL088072

    Circulation. Cardiovascular genetics 2009;2;5;428-35

  • Altered desmoplakin expression at transcriptional and protein levels provides prognostic information in human oropharyngeal cancer.

    Papagerakis S, Shabana AH, Pollock BH, Papagerakis P, Depondt J and Berdal A

    Laboratoire de Biologie Orofaciale et Pathologie, INSERM UMRS872, Université Paris 7, Centre de Recherche des Cordeliers, Paris, 75006 France. silvanap@umich.edu

    Desmoplakin, a desmosomal component, is a key protein involved in cell-cell adhesion. Down-regulation of desmosomal proteins is associated with the invasive and metastatic ability of tumor cells. We examined 37 cases of human primary oropharyngeal squamous cell carcinomas lacking overt distant metastases to gain further insights on the potential role of desmoplakin in oral cancer. Desmoplakin expression was evaluated using reverse transcriptase-polymerase chain reaction and immunohistochemistry on frozen unfixed sections. Western blotting was performed to characterize the relative expression levels for each of the 2 desmoplakin protein isoforms, I and II. Desmoplakin expression was compared with histopathological grade, clinical stage, and patient outcome. Desmoplakin expression was prominent in highly differentiated tumors and reduced or absent in poorly differentiated tumors that developed distant metastases within the 3 years of follow-up period. Desmoplakin mRNA levels tracked with protein levels, suggesting that lack of desmoplakin protein expression is due to down-regulation of mRNA expression at the transcription level. Western blot analysis demonstrated that the 2 desmoplakin isoforms displayed different patterns of subcellular distribution in tumors, with the desmoplakin II detected only in patients in which desmoplakin immunoreactivity displayed an abnormal cytoplasmic localization. Our findings suggest that down-regulation of desmoplakin expression may represent a useful marker for evaluating the risk of distant metastasis formation in oropharyngeal squamous cell carcinomas. Interestingly, desmoplakin II was detected only in tumors associated with a poor clinical outcome, suggesting a potential specific function for this isoform in oral carcinogenesis. Characterizing DSP expression may improve evaluation risk of distant metastasis formation in oral cancer patients.

    Human pathology 2009;40;9;1320-9

  • Identification and quantification of preterm birth biomarkers in human cervicovaginal fluid by liquid chromatography/tandem mass spectrometry.

    Shah SJ, Yu KH, Sangar V, Parry SI and Blair IA

    Center for Excellence in Environmental Toxicology, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160, USA.

    Spontaneous preterm birth (PTB) before 37 completed weeks of gestation resulting from preterm labor (PTL) is a leading contributor of perinatal morbidity and mortality. Early identification of at-risk women by reliable screening tests could alleviate this health issue; however, conventional methods such as obstetric history and clinical risk factors, uterine activity monitoring, biochemical markers, and cervical sonography for screening women at risk for PTB have proven unsuccessful in lowering the rate of PTB. Cervicovaginal fluid (CVF) might prove to be a useful, readily available biological fluid for identifying diagnostic PTB biomarkers. Human columnar epithelial endocervical-1 (End1) and vaginal (Vk2) cell secretomes were employed to generate a stable isotope labeled proteome (SILAP) standard to facilitate characterization and relative quantification of proteins present in CVF. The SILAP standard was prepared using stable isotope labeling by amino acids in cell culture (SILAC) of End1 and Vk2 through seven passages. The labeled secreted proteins from both cell lines were combined and characterized by liquid-chromatography-tandem mass spectrometry (LC-MS/MS). In total, 1211 proteins were identified in the End1-Vk2 SILAP standard, with 236 proteins being consistently identified in each of the replicates analyzed. Individual proteins were found to contain <0.5% of the endogenous unlabeled forms. Identified proteins were screened to provide a set of 15 candidates that have either previously been identified as potential PTB biomarkers or could be linked mechanistically to PTB. Stable isotope dilution LC-multiple reaction monitoring (MRM/MS) assays were then developed for conducting relative quantification of the 15 candidate biomarkers in human CVF samples from term and PTB cases. Three proteins were significantly elevated in PTB cases (desmoplakin isoform 1, stratifin, and thrombospondin 1 precursor), providing a foundation for further validation in larger patient cohorts.

    Funded by: NCRR NIH HHS: UL1 RR024134, UL1 RR024134-02, UL1RR024134; NICHD NIH HHS: U01 HD050088, U01HD050088; NIEHS NIH HHS: P30 ES013508, P30 ES013508-03, P30ES013508

    Journal of proteome research 2009;8;5;2407-17

  • Novel truncating mutations in PKP1 and DSP cause similar skin phenotypes in two Brazilian families.

    Tanaka A, Lai-Cheong JE, Café ME, Gontijo B, Salomão PR, Pereira L and McGrath JA

    Division of Genetics and Molecular Medicine, Genetic Skin Disease Group, St John's Institute of Dermatology, King's College London, Guy's Hospital, London SE1 9RT, UK.

    Inherited mutations in components of desmosomes result in a spectrum of syndromes characterized by variable abnormalities in the skin and its appendages, including blisters and erosions, palmoplantar hyperkeratosis, woolly hair or hypotrichosis and, in some cases, extracutaneous features such as cardiomyopathy. We investigated the molecular basis of two Brazilian patients presenting with clinical features consistent with ectodermal dysplasia-skin fragility syndrome. In patient 1 we identified a homozygous nonsense mutation, p.R672X, in the PKP1 gene (encoding plakophilin 1). This particular mutation has not been reported previously but is similar to the molecular pathology underlying other cases of this syndrome. In patient 2 we found compound heterozygosity for two frameshift mutations, c.2516del4 and c.3971del4, in the DSP gene (encoding desm 1f40 oplakin). Although there was considerable clinical overlap in the skin and hair abnormalities in these two cases, patient 2 also had early-onset cardiomyopathy. The mutation c.3971del4 occurs in the longer desmoplakin-I isoform (which is the major cardiac transcript) but not in the more ubiquitous desmoplakin-II. In contrast, PKP1 is not expressed in the heart, which accounts for the lack of cardiomyopathy in patient 1. Collectively, these cases represent the first desmosomal genodermatoses to be reported from Brazil and add to genotype-phenotype correlation in this group of inherited disorders. Loss-of-function mutations in the DSP gene can result in a phenotype similar to ectodermal dysplasia-skin fragility syndrome resulting from PKP1 mutations but only DSP pathology is associated with cardiac disease.

    Funded by: Wellcome Trust

    The British journal of dermatology 2009;160;3;692-7

  • Arrhythmogenic right ventricular dysplasia: clinical characteristics and identification of novel desmosome gene mutations.

    Yu CC, Yu CH, Hsueh CH, Yang CT, Juang JM, Hwang JJ, Lin JL and Lai LP

    Department of Integrated Diagnostics and Therapeutics, National Taiwan University Hospital, Taipei, Taiwan.

    Desmosome gene mutations have been reported in patients with arrhythmogenic right ventricular dysplasia (ARVD). However, there are hardly any genetic studies in Asians. We studied the clinical characteristics, cardiac manifestations and desmosome gene mutations in ARVD patients in Taiwan.

    Methods: Medical records of five ARVD patients were reviewed and genomic DNA was obtained from peripheral blood samples. Mutation screening in desmoplakin (DSP), plakophilin-2, desmoglein-2 (DSG2) and desmocollin-2 genes was performed using polymerase chain reaction and DNA sequencing techniques.

    Results: Among the five patients, three presented with palpitations followed by loss of consciousness, and the other two had palpitations or chest tightness without loss of consciousness. Electrocardiogram (ECG), magnetic resonance imaging and signal averaged ECG results were similar to those reported in Western countries. Mutations in the desmosome genes were identified in four of the five patients (three with a DSG2 mutation and one with a DSP mutation). Five gene mutations were noted in four patients and all mutations were novel (one patient had a DSG2 double mutation). The mutation types were missense in four and splicing mutation in one.

    Conclusion: Patients with ARVD in Taiwan had similar clinical and cardiac manifestations as reported in the Western literature. More than half of the patients had desmosome gene mutations.

    Journal of the Formosan Medical Association = Taiwan yi zhi 2008;107;7;548-58

  • Arrhythmogenic right ventricular dysplasia.

    Calkins H

    Baltimore, Maryland, USA. hcalkins@jhmi.edu

    Background: Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD) is a genetic cardiomyopathy characterized by ventricular arrhythmias and structural abnormalities of the right ventricle (RV). The discovery of desmosomal mutations associated with arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) has led researchers to hypothesize equal right (RV) and left (LV) ventricular affliction in the disease process. The purpose of this paper is to provide an overview of ARVD and also to present the results of a new study of the morphological variants of ARVD METHODS AND RESULTS: Thirty-eight (age: 30+/-17 years; 18 male) family members of twelve desmosomal mutation-carrying ARVD probands underwent genotyping and cardiac magnetic resonance imaging (CMR). The CMR investigators were blinded to clinical and genetic data. Twenty-five individuals had mutations in PKP2, DSP, and/or DSG2 genes. RV abnormalities were associated with the presence of mutation(s) and with disease severity determined by criteria (minor=1; major=2) points for ARVD diagnosis. The only LV abnormality detected, the presence of intramyocardial fat, was present in four individuals. Each of these individuals was a mutation-carrier, while one had no previously described ARVD-related abnormality. On detailed CMR, a focal "crinkling' of the RV outflow tract and subtricuspid regions ("accordion sign") was observed in 60% of the mutation-carriers and none of the non-carriers (P<0.001). The sign was present in 0%, 37%, 71%, and 75% of individuals who met 1, 2, 3, and 4+ criteria points respectively (P<0.01).

    Conclusion: Despite a possible LV involvement in ARVD/C, the overall LV structure and function are well preserved. Independent LV involvement is of rare occurrence. The "accordion sign" is a promising tool for early diagnosis of ARVD. Its diagnostic utility should be confirmed in larger cohorts.

    Transactions of the American Clinical and Climatological Association 2008;119;273-86; discussion 287-8

  • Increased keratinocyte proliferation initiated through downregulation of desmoplakin by RNA interference.

    Wan H, South AP and Hart IR

    Centre for Tumour Biology, Institute of Cancer and CR-UK Clinical Centre, Barts and The London, Queen Mary's School of Medicine and Dentistry, John Vane Science Centre, Charterhouse Square, London, UK. hong.wan@cancer.org.uk

    The intercellular adhesive junction desmosomes are essential for the maintenance of tissue structure and integrity in skin. Desmoplakin (Dp) is a major obligate plaque protein which plays a fundamental role in anchoring intermediate filaments to desmosomal cadherins. Evidence from hereditary human disease caused by mutations in the gene encoding Dp, e.g. Dp haploinsufficiency, suggests that alterations in Dp expression result not only in the disruption of tissue structure and integrity but also could evoke changes in keratinocyte proliferation. We have used transient RNA interference (RNAi) to downregulate Dp specifically in HaCaT keratinocytes. We showed that this Dp downregulation also caused reduced expression of several other desmosomal proteins. Increased cell proliferation and enhanced G(1)-to-S-phase entry in the cell cycle, as monitored by colonial cellular density and BrdU incorporation, were seen in Dp RNAi-treated cells. These proliferative changes were associated with elevated phospho-ERK1/2 and phospho-Akt levels. Furthermore, this increase in phospho-ERK/1/2 and phospho-Akt levels was sustained in Dp RNAi-treated cells at confluence whereas in control cells there was a significant reduction in phosphorylation of ERK1/2. This study indicates that Dp may participate in the regulation of keratinocyte cell proliferation by, in part at least, regulating cell cycle progression.

    Funded by: Medical Research Council: G113/32

    Experimental cell research 2007;313;11;2336-44

  • The Ki67+ proliferation index correlates with increased cellular retinol-binding protein-1 and the coordinated loss of plakophilin-1 and desmoplakin during progression of cervical squamous lesions.

    Schmitt-Graeff A, Koeninger A, Olschewski M, Haxelmans S, Nitschke R, Bochaton-Piallat ML, Lifschitz-Mercer B, Gabbiani G, Langbein L and Czernobilsky B

    Institute of Pathology, University Hospital, Freiburg, Germany. annette.schmitt-graeff@uniklinik-freiburg.de

    Aims: To investigate the modulation of cellular retinol-binding protein (CRBP)-1 and the desmosomal plaque proteins plakophilin (PKP)-1 and desmoplakin (DP) in correlation with the Ki67+ proliferation index (PI) during the progression of cervical squamous intraepithelial lesions (SIL) to squamous cell carcinoma (SCC).

    Methods: Using in situ imaging by brightfield and confocal laser scanning microscopy, the expression of CRBP-1 protein and transcripts, PKP-1, DP and the Ki67 PI were analysed in 38 low-grade (L) SIL, 56 high-grade (H) SIL, 49 SCC, 30 control cervices and 10 human papillomavirus-positive condylomatous lesions.

    Results: CRBP-1+ cells increased from 11.4% in the normal cervix to 80.3% in LSILs, 92.3% in HSILs and slightly decreased to 78.3% in invasive SCCs (P = 0.0001) in close association with the Ki67 PI (r =0.41; P < 0.0001). PKP-1+ and DP+ cells were correlated (0.32; P < 0.0001) and decreased from normal (81% versus 92.3%) to LSIL (53.1% versus 85.3%), to HSIL (46.4% versus 67.5%) and SCC (35.1% versus 35.9%). The Ki67+ PI was inversely correlated with DP (-0.20, P = 0.0014) and PKP-1 (-0.19, P = 0.015). Condylomata retained low CRBP-1 and high expression of PKP-1 and DP.

    Conclusions: The gain of CRBP-1 and the loss of desmosomal proteins occur early in cervical carcinogenesis.

    Histopathology 2007;51;1;87-97

  • Large-scale mapping of human protein-protein interactions by mass spectrometry.

    Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T and Figeys D

    Protana, Toronto, Ontario, Canada.

    Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

    Molecular systems biology 2007;3;89

  • Desmosomal dysfunction due to mutations in desmoplakin causes arrhythmogenic right ventricular dysplasia/cardiomyopathy.

    Yang Z, Bowles NE, Scherer SE, Taylor MD, Kearney DL, Ge S, Nadvoretskiy VV, DeFreitas G, Carabello B, Brandon LI, Godsel LM, Green KJ, Saffitz JE, Li H, Danieli GA, Calkins H, Marcus F and Towbin JA

    Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.

    Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by progressive degeneration of the right ventricular myocardium, ventricular arrhythmias, fibrous-fatty replacement, and increased risk of sudden death. Mutations in 6 genes, including 4 encoding desmosomal proteins (Junctional plakoglobin (JUP), Desmoplakin (DSP), Plakophilin 2, and Desmoglein 2), have been identified in patients with ARVD/C. Mutation analysis of 66 probands identified 4 variants in DSP; V30M, Q90R, W233X, and R2834H. To establish a cause and effect relationship between those DSP missense mutations and ARVD/C, we performed in vitro and in vivo analyses of the mutated proteins. Unlike wild-type (WT) DSP, the N-terminal mutants (V30M and Q90R) failed to localize to the cell membrane in desomosome-forming cell line and failed to bind to and coimmunoprecipitate JUP. Multiple attempts to generate N-terminal DSP (V30M and Q90R) cardiac-specific transgenes have failed: analysis of embryos revealed evidence of profound ventricular dilation, which likely resulted in embryonic lethality. We were able to develop transgenic (Tg) mice with cardiac-restricted overexpression of the C-terminal mutant (R2834H) or WT DSP. Whereas mice overexpressing WT DSP had no detectable histologic, morphological, or functional cardiac changes, the R2834H-Tg mice had increased cardiomyocyte apoptosis, cardiac fibrosis, and lipid accumulation, along with ventricular enlargement and cardiac dysfunction in both ventricles. These mice also displayed interruption of DSP-desmin interaction at intercalated discs (IDs) and marked ultra-structural changes of IDs. These data suggest DSP expression in cardiomyocytes is crucial for maintaining cardiac tissue integrity, and DSP abnormalities result in ARVD/C by cardiomyocyte death, changes in lipid metabolism, and defects in cardiac development.

    Funded by: PHS HHS: P01-67155, U01-65652

    Circulation research 2006;99;6;646-55

  • Early death from cardiomyopathy in a family with autosomal dominant striate palmoplantar keratoderma and woolly hair associated with a novel insertion mutation in desmoplakin.

    Norgett EE, Lucke TW, Bowers B, Munro CS, Leigh IM and Kelsell DP

    Funded by: Wellcome Trust

    The Journal of investigative dermatology 2006;126;7;1651-4

  • Phosphoproteome analysis of the human mitotic spindle.

    Nousiainen M, Silljé HH, Sauer G, Nigg EA and Körner R

    Department of Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.

    During cell division, the mitotic spindle segregates the sister chromatids into two nascent cells, such that each daughter cell inherits one complete set of chromosomes. Errors in spindle formation can result in both chromosome missegregation and cytokinesis defects and hence lead to genomic instability. To ensure the correct function of the spindle, the activity and localization of spindle associated proteins has to be tightly regulated in time and space. Reversible phosphorylation has been shown to be one of the key regulatory mechanisms for the organization of the mitotic spindle. The relatively low number of identified in vivo phosphorylation sites of spindle components, however, has hampered functional analysis of regulatory spindle networks. A more complete inventory of the phosphorylation sites of spindle-associated proteins would therefore constitute an important advance. Here, we describe the mass spectrometry-based identification of in vivo phosphorylation sites from purified human mitotic spindles. In total, 736 phosphorylation sites were identified, of which 312 could be attributed to known spindle proteins. Among these are phosphorylation sites that were previously shown to be important for the regulation of spindle-associated proteins. Importantly, this data set also comprises 279 novel phosphorylation sites of known spindle proteins for future functional studies. This inventory of spindle phosphorylation sites should thus make an important contribution to a better understanding of the molecular mechanisms that regulate the formation, function, and integrity of the mitotic spindle.

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;14;5391-6

  • Loss of desmoplakin isoform I causes early onset cardiomyopathy and heart failure in a Naxos-like syndrome.

    Uzumcu A, Norgett EE, Dindar A, Uyguner O, Nisli K, Kayserili H, Sahin SE, Dupont E, Severs NJ, Leigh IM, Yuksel-Apak M, Kelsell DP and Wollnik B

    Background: Desmosomes are cellular junctions important for intercellular adhesion and anchoring the intermediate filament (IF) cytoskeleton to the cell membrane. Desmoplakin (DSP) is the most abundant desmosomal protein with 2 isoforms produced by alternative splicing.

    Methods: We describe a patient with a recessively inherited arrhythmogenic dilated cardiomyopathy with left and right ventricular involvement, epidermolytic palmoplantar keratoderma, and woolly hair. The patient showed a severe heart phenotype with an early onset and rapid progression to heart failure at 4 years of age.

    Results: A homozygous nonsense mutation, R1267X, was found in exon 23 of the desmoplakin gene, which results in an isoform specific truncation of the larger DSPI isoform. The loss of most of the DSPI specific rod domain and C-terminal area was confirmed by Western blotting and immunofluorescence. We further showed that the truncated DSPI transcript is unstable, leading to a loss of DSPI. DSPI is reported to be an obligate constituent of desmosomes and the only isoform present in cardiac tissue. To address this, we reviewed the expression of DSP isoforms in the heart. Our data suggest that DSPI is the major cardiac isoform but we also show that specific compartments of the heart have detectable DSPII expression.

    Conclusions: This is the first description of a phenotype caused by a mutation affecting only one DSP isoform. Our findings emphasise the importance of desmoplakin and desmosomes in epidermal and cardiac function and additionally highlight the possibility that the different isoforms of desmoplakin may have distinct functional properties within the desmosome.

    Funded by: Wellcome Trust

    Journal of medical genetics 2006;43;2;e5

  • Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin.

    Godsel LM, Hsieh SN, Amargo EV, Bass AE, Pascoe-McGillicuddy LT, Huen AC, Thorne ME, Gaudry CA, Park JK, Myung K, Goldman RD, Chew TL and Green KJ

    Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.

    The intermediate filament (IF)-binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell-cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 microm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.

    Funded by: NIAMS NIH HHS: AR41836, R01 AR041836, R01 AR043380, R01 AR43380, T32 AR007593, T32 AR07593; NIDCR NIH HHS: DE12328, P01 DE012328

    The Journal of cell biology 2005;171;6;1045-59

  • Loss of desmoplakin tail causes lethal acantholytic epidermolysis bullosa.

    Jonkman MF, Pasmooij AM, Pasmans SG, van den Berg MP, Ter Horst HJ, Timmer A and Pas HH

    Department of Dermatology, University Medical Centre Groningen, the Netherlands. m.f.jonkman@med.umcg.nl

    The cytoplasmic plaque protein desmoplakin (DP), which is located in desmosomes, plays a major role in epithelial and muscle cell adhesion by linking the transmembrane cadherins to the cytoplasmic intermediate filament network. Mutations of DP may cause striate palmoplantar keratoderma, arrhythmogenic right ventricular dysplasia, skin fragility/woolly hair syndrome, Naxos-like disease, and Carvajal syndrome. DP must be indispensable, because DP-/- mice are early abortive. Here, we report a patient with severe fragility of skin and mucous membranes caused by genetic truncation of the DP tail. The new phenotype is lethal in the neonatal period because of immense transcutaneous fluid loss. The phenotype also comprised universal alopecia, neonatal teeth, and nail loss. Histology showed suprabasal clefting and acantholysis throughout the spinous layer, mimicking pemphigus. Electron microscopy revealed disconnection of keratin intermediate filaments from desmosomes. Immunofluorescence staining of DP showed a distinct punctate intercellular pattern in the patient's skin. Protein analysis revealed expression of truncated DP polypeptides. Mutational analysis of the patient demonstrated compound heterozygosity for two DP mutations, 6079C-->T (R1934X) and 6370delTT, respectively. Aberrant mRNA transcripts that predict premature termination of translation with loss of the three intermediate filament-binding subdomains in the DP tail were detected by RT-PCR. The new dramatic phenotype, which we named "lethal acantholytic epidermolysis bullosa," underscores the paramount role of DP in epidermal integrity.

    American journal of human genetics 2005;77;4;653-60

  • Novel mutation in desmoplakin causes arrhythmogenic left ventricular cardiomyopathy.

    Norman M, Simpson M, Mogensen J, Shaw A, Hughes S, Syrris P, Sen-Chowdhry S, Rowland E, Crosby A and McKenna WJ

    Department of Cardiological Sciences, St George's Hospital Medical School, London, England.

    Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial heart muscle disease characterized by structural, electrical, and pathological abnormalities of the right ventricle (RV). Several disease loci have been identified. Mutations in desmoplakin have recently been isolated in both autosomal-dominant and autosomal-recessive forms of ARVC. Primary left ventricular (LV) variants of the disease are increasingly recognized. We report on a large family with autosomal-dominant left-sided ARVC.

    The proband presented with sudden cardiac death and fibrofatty replacement of the LV myocardium. The family was evaluated. Diagnosis was based on modified diagnostic criteria for ARVC. Seven had inferior and/or lateral T-wave inversion on ECG, LV dilatation, and ventricular arrhythmia, predominantly extrasystoles of LV origin. Three had sustained ventricular tachycardia; 7 had late potentials on signal-averaged ECG. Cardiovascular magnetic resonance imaging in 4 patients revealed wall-motion abnormalities of the RV and patchy, late gadolinium enhancement in the LV, suggestive of fibrosis. Linkage confirmed cosegregation to the desmoplakin intragenic marker D6S2975. A heterozygous, single adenine insertion (2034insA) in the desmoplakin gene was identified in affected individuals only. A frameshift introducing a premature stop codon with truncation of the rod and carboxy terminus of desmoplakin was confirmed by Western blot analysis.

    Conclusions: We have described a new dominant mutation in desmoplakin that causes left-sided ARVC, with arrhythmias of LV origin, lateral T-wave inversion, and late gadolinium enhancement in the LV on magnetic resonance images. Truncation of the carboxy terminus of desmoplakin and consequent disruption of intermediate filament binding may account for the predominant LV phenotype.

    Circulation 2005;112;5;636-42

  • Clinical profile of four families with arrhythmogenic right ventricular cardiomyopathy caused by dominant desmoplakin mutations.

    Bauce B, Basso C, Rampazzo A, Beffagna G, Daliento L, Frigo G, Malacrida S, Settimo L, Danieli G, Thiene G and Nava A

    Division of Cardiology, University of Padua Medical School, Padova, Italy.

    Aims: To characterize the clinical profile of patients belonging to families affected with autosomal dominant arrhythmogenic right ventricular cardiomyopathy (ARVC) due to mutations of the gene encoding for the cell-to-cell adhesion protein desmoplakin (DSP).

    Thirty-eight subjects belonging to four families showing different DSP mutations (three missense and one in the intron-exon splicing region) underwent clinical and genetic investigation, including annual 12-lead ECG, signal averaged ECG, 24 h Holter ECG, and two-dimensional echocardiography. Twenty-six family members (11 males and 15 females) were found to carry a DSP mutation. After a follow-up of 1-24 years, median 6, 14 (54%) fulfilled (mean age at diagnosis 33+/-15 years) and 12 (mean age 43+/-24 years at the last follow-up) did not fulfil the established diagnostic criteria of ARVC, although five of them had some cardiac abnormalities. Clinical presentations were palpitations in six, sudden death (SD) in three, syncope in one, and chest pain with increased myocardial enzymes in two. Abnormal 12-lead ECG findings were present in 15 cases (58%), ventricular arrhythmias in 12 (46%), and late potentials in 11 (42%). Fourteen (54%) had abnormal echocardiographic findings, with left ventricular involvement in seven of them. SD occurred in six subjects and in three it was the first symptom of the disease; moreover, one subject died due to heart failure. The annual disease-related death and SD/aborted SD were 0.028 and 0.023 patient/year, respectively.

    Conclusion: Familial ARVC caused by DSP mutations is characterized by a high occurrence of SD even as first clinical manifestation. Left ventricular involvement is not a rare feature of the disease, which frequently escapes clinical diagnosis by applying the currently available criteria. Genetic screening is mandatory for early identification of asymptomatic carriers and preventive strategies within a family with a genotyped index case.

    European heart journal 2005;26;16;1666-75

  • beta-Catenin and plakoglobin N- and C-tails determine ligand specificity.

    Solanas G, Miravet S, Casagolda D, Castaño J, Raurell I, Corrionero A, de Herreros AG and Duñach M

    Unitat de Biofísica, Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra.

    beta-Catenin and plakoglobin are related proteins involved in the regulation of adherens junctions and desmosomes. Moreover, by binding to Tcf-4, they can act as transcriptional modulators of genes involved in embryonic development and tumorigenesis. However, they associate to distinct Tcf-4 subdomains causing opposing effects on Tcf-4 binding to DNA: whereas beta-catenin does not affect this binding, plakoglobin prevents it. Both proteins are composed by two N- and C-tails and a central armadillo repeat domain. Interaction of Tcf-4, as well as other desmosomal or adherens junction components, with beta-catenin or plakoglobin takes place through the central armadillo domain. Here we show that, as reported for beta-catenin, plakoglobin terminal tails also interact with the central domain and regulate the ability of this region to bind to different cofactors. Moreover the specificity of the interaction of beta-catenin and plakoglobin with different subdomains in Tcf-4 or with other junctional components resides within the terminal tails and not in the armadillo domain. For instance, a chimeric protein in which the central domain of beta-catenin was replaced by that of plakoglobin presented the same specificity as wild-type beta-catenin. Therefore, the terminal tails of these proteins are responsible for discerning among binding of factors to the armadillo domain. These results contribute to the understanding of the molecular basis of the interactions established by these key regulators of epithelial tumorigenesis.

    The Journal of biological chemistry 2004;279;48;49849-56

  • Plakin proteins are coordinately cleaved during apoptosis but preferentially through the action of different caspases.

    Aho S

    Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA. sirpa.aho@jefferson.edu

    In epithelial cells, cell-cell and cell-matrix junctions, desmosomes and hemidesmosomes, provide anchorage sites for the keratin-intermediate filaments. The plakin proteins desmoplakin (DP), plectin, and periplakin represent intracellular constituents of these adhesion junctions. In staurosporine-treated apoptotic HaCaT cells, DP, plectin, and periplakin became cleaved coordinately with the elimination of keratins 10 and 14, while involucrin, actin, and keratin 18 displayed considerable stability. The caspase inhibitor zVAD-fmk prevented both the cell detachment and protein cleavage, indicating the function of caspases in these events. Closer examination in vitro revealed that while caspases 2 and 4 most effic 13eb iently cleaved DP, and plectin served as a target for caspases 3 and 7, periplakin as well as keratins were cleaved by caspase 6. The involvement of multiple caspases in the destruction of epithelial cell integrity ensures the efficient elimination of cytoskeleton, but also provides specificity for selectively targeting individual adhesion molecules.

    Funded by: PHS HHS: R0133588

    Experimental dermatology 2004;13;11;700-7

  • Naxos disease in an Arab family is not caused by the Pk2157del2 mutation. Evidence for exclusion of the plakoglobin gene.

    Stuhrmann M, Bukhari IA and El-Harith el-HA

    Institute of Human Genetics, Medical University Hannover, Hannover, Federal Republic of Germany. stuhrmann.manfred@mh-hannover.de

    Objective: Naxos disease is a rare hereditary disorder characterized by palmoplantar keratoderma, woolly hair and cardiomyopathy. This study aims to determine whether Naxos disease in a Saudi Arab family is caused by the Pk2157del2 mutation that was identified in Greek families from Naxos Island where the disease had originally been described.

    Methods: This study was undertaken at King Fahad Hospital of the University, Al-Khobar, and the Medical University of Hannover, in the spring of 2003. Naxos disease has been encountered in a 2-year-old girl and her 30-year-old aunt of a Saudi Arab family. Deoxyribonucleic acid samples of this family were analyzed by polymerase chain-reaction (PCR) amplification of the respective region of the plakoglobin gene, and direct nucleotide sequencing of the PCR-products. Segregation analysis was performed employing the newly detected IVS11+22G/A polymorphism.

    Results: Molecular genetic analysis of the DNA sample of the child diagnosed with Naxos disease showed absence of the Pk2157del2 mutation. In addition, the segregation analysis revealed heterozygosity for IVS11+22G/A in the affected girl.

    Conclusion: Absence of the Pk2157del2 frameshift in the affected child proved that Naxos disease in this Saudi Arab family is not caused by the same mutation that was identified in the Greek families. Furthermore, heterozygosity for the IVS11+22G/A polymorphism provided evidence for exclusion of the plakoglobin gene in this consanguineous family.

    Saudi medical journal 2004;25;10;1449-52

  • Large-scale characterization of HeLa cell nuclear phosphoproteins.

    Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Li J, Cohn MA, Cantley LC and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

    Funded by: NHGRI NIH HHS: HG00041, K22 HG000041, T32 HG000041; NIGMS NIH HHS: GM67945, GMS6203, R01 GM056203, R01 GM067945

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;33;12130-5

  • Desmoplakin is required for microvascular tube formation in culture.

    Zhou X, Stuart A, Dettin LE, Rodriguez G, Hoel B and Gallicano GI

    Department of Cell Biology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, DC 20007, USA.

    Desmoplakin (DP) is a key component of cellular adhesion junctions known as desmosomes; however, recent investigations have revealed a novel location for DP in junctions separate from desmosomes termed complexus adherens junctions. These junctions are found at contact sites between endothelial cells that line capillaries. Few studies have focused on the function of DP in de novo capillary formation (vasculogenesis) and branching (angiogenesis) during tumorigenesis, embryonic development, cardiovascular development or wound healing. Only recently have investigations begun to determine the effect the loss of DP has on capillaries during embryogenesis (i.e. in DP-/- mice). Evidence shows that the loss of desmoplakin in vivo results in leaky capillaries and/or capillary malformation. Consequently, the goal of this study was to determine the function of DP in complexus adherens junctions during capillary formation. To accomplish this goal, we used siRNA technology to knock down desmoplakin expression in endothelial cells before they were induced to form microvascular tubes on matrigel. DP siRNA treated cells sent out filopodia and came in close contact with each other when plated onto matrigel; however, in most cases they failed to form tubes as compared with control endothelial cells. Interestingly, after siRNA degradation, endothelial cells were then capable of forming microvascular tubes. In depth analyses into the function of DP in capillary formation were not previously possible because the tools and experimental approaches only recently have become available (i.e. siRNA). Consequently, fully understanding the role of desmoplakin in capillary formation may lead to a novel approach for inhibiting vasculo- and angiogenesis in tumor formation.

    Funded by: NCI NIH HHS: CA09686, CA51008-13; NHLBI NIH HHS: HL70204-01

    Journal of cell science 2004;117;Pt 15;3129-40

  • Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation.

    Brandenberger R, Wei H, Zhang S, Lei S, Murage J, Fisk GJ, Li Y, Xu C, Fang R, Guegler K, Rao MS, Mandalam R, Lebkowski J and Stanton LW

    Geron Corporation, Menlo Park, California 94025, USA. rbrandenberger@geron.com

    Human embryonic stem (hES) cells hold promise for generating an unlimited supply of cells for replacement therapies. To characterize hES cells at the molecular level, we obtained 148,453 expressed sequence tags (ESTs) from undifferentiated hES cells and three differentiated derivative subpopulations. Over 32,000 different transcripts expressed in hES cells were identified, of which more than 16,000 do not match closely any gene in the UniGene public database. Queries to this EST database revealed 532 significantly upregulated and 140 significantly downregulated genes in undifferentiated hES cells. These data highlight changes in the transcriptional network that occur when hES cells differentiate. Among the differentially regulated genes are several components of signaling pathways and transcriptional regulators that likely play key roles in hES cell growth and differentiation. The genomic data presented here may facilitate the derivation of clinically useful cell types from hES cells.

    Nature biotechnology 2004;22;6;707-16

  • A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway.

    Bouwmeester T, Bauch A, Ruffner H, Angrand PO, Bergamini G, Croughton K, Cruciat C, Eberhard D, Gagneur J, Ghidelli S, Hopf C, Huhse B, Mangano R, Michon AM, Schirle M, Schlegl J, Schwab M, Stein MA, Bauer A, Casari G, Drewes G, Gavin AC, Jackson DB, Joberty G, Neubauer G, Rick J, Kuster B and Superti-Furga G

    Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany. tewis.bouwmeester@cellzome.com

    Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.

    Nature cell biology 2004;6;2;97-105

  • The DNA sequence and analysis of human chromosome 6.

    Mungall AJ, Palmer SA, Sims SK, Edwards CA, Ashurst JL, Wilming L, Jones MC, Horton R, Hunt SE, Scott CE, Gilbert JG, Clamp ME, Bethel G, Milne S, Ainscough R, Almeida JP, Ambrose KD, Andrews TD, Ashwell RI, Babbage AK, Bagguley CL, Bailey J, Banerjee R, Barker DJ, Barlow KF, Bates K, Beare DM, Beasley H, Beasley O, Bird CP, Blakey S, Bray-Allen S, Brook J, Brown AJ, Brown JY, Burford DC, Burrill W, Burton J, Carder C, Carter NP, Chapman JC, Clark SY, Clark G, Clee CM, Clegg S, Cobley V, Collier RE, Collins JE, Colman LK, Corby NR, Coville GJ, Culley KM, Dhami P, Davies J, Dunn M, Earthrowl ME, Ellington AE, Evans KA, Faulkner L, Francis MD, Frankish A, Frankland J, French L, Garner P, Garnett J, Ghori MJ, Gilby LM, Gillson CJ, Glithero RJ, Grafham DV, Grant M, Gribble S, Griffiths C, Griffiths M, Hall R, Halls KS, Hammond S, Harley JL, Hart EA, Heath PD, Heathcott R, Holmes SJ, Howden PJ, Howe KL, Howell GR, Huckle E, Humphray SJ, Humphries MD, Hunt AR, Johnson CM, Joy AA, Kay M, Keenan SJ, Kimberley AM, King A, Laird GK, Langford C, Lawlor S, Leongamornlert DA, Leversha M, Lloyd CR, Lloyd DM, Loveland JE, Lovell J, Martin S, Mashreghi-Mohammadi M, Maslen GL, Matthews L, McCann OT, McLaren SJ, McLay K, McMurray A, Moore MJ, Mullikin JC, Niblett D, Nickerson T, Novik KL, Oliver K, Overton-Larty EK, Parker A, Patel R, Pearce AV, Peck AI, Phillimore B, Phillips S, Plumb RW, Porter KM, Ramsey Y, Ranby SA, Rice CM, Ross MT, Searle SM, Sehra HK, Sheridan E, Skuce CD, Smith S, Smith M, Spraggon L, Squares SL, Steward CA, Sycamore N, Tamlyn-Hall G, Tester J, Theaker AJ, Thomas DW, Thorpe A, Tracey A, Tromans A, Tubby B, Wall M, Wallis JM, West AP, White SS, Whitehead SL, Whittaker H, Wild A, Willey DJ, Wilmer TE, Wood JM, Wray PW, Wyatt JC, Young L, Younger RM, Bentley DR, Coulson A, Durbin R, Hubbard T, Sulston JE, Dunham I, Rogers J and Beck S

    The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. ajm@sanger.ac.uk

    Chromosome 6 is a metacentric chromosome that constitutes about 6% of the human genome. The finished sequence comprises 166,880,988 base pairs, representing the largest chromosome sequenced so far. The entire sequence has been subjected to high-quality manual annotation, resulting in the evidence-supported identification of 1,557 genes and 633 pseudogenes. Here we report that at least 96% of the protein-coding genes have been identified, as assessed by multi-species comparative sequence analysis, and provide evidence for the presence of further, otherwise unsupported exons/genes. Among these are genes directly implicated in cancer, schizophrenia, autoimmunity and many other diseases. Chromosome 6 harbours the largest transfer RNA gene cluster in the genome; we show that this cluster co-localizes with a region of high transcriptional activity. Within the essential immune loci of the major histocompatibility complex, we find HLA-B to be the most polymorphic gene on chromosome 6 and in the human genome.

    Nature 2003;425;6960;805-11

  • A recessive mutation in desmoplakin causes arrhythmogenic right ventricular dysplasia, skin disorder, and woolly hair.

    Alcalai R, Metzger S, Rosenheck S, Meiner V and Chajek-Shaul T

    Department of Medicine Mount Scopus, Jerusalem, Israel.

    Objectives: The goal of this study was to analyze the genetic disorder of a family with cardiomyopathy, skin disorder, and woolly hair.

    Background: Arrhythmogenic right ventricular dysplasia (ARVD) is a heart muscle disorder causing arrhythmia and sudden cardiac death. We report a patient with familial autosomal recessive ARVD, woolly hair, and a pemphigous-like skin disorder with a new mutation in the desmoplakin gene.

    Methods: Genomic deoxyribonucleic acid was extracted from the patient's blood and 12 first- and second-degree family members, and was amplified by polymerase chain reaction. Linkage analysis with polymorphic microsatellites was performed for 11 genes that code for structural desmosomal proteins. The genetic locus of the disease in this family was mapped to the chromosomal region 6p24 that contains the desmoplakin gene. Exons of the desmoplakin gene were analyzed by single-strand conformational polymorphism and direct sequencing. Confirmation of the mutation was carried out by restriction enzyme analysis.

    Results: We identified in the patient a homozygous missense mutation in exon 24 of the desmoplakin gene, leading to a Gly2375Arg substitution in the C-terminal of the protein where the binding site to intermediate filaments is located. Eight of 12 family members without hair or skin abnormalities were heterozygous for this mutation. The remaining 4, as well as 90 unrelated healthy control individuals of the same ethnic origin, were homozygous for the normal allele.

    Conclusions: We have described a new mutation in the desmoplakin gene that causes familial ARVD. These findings suggest that desmosomal proteins play an important role in the integrity and function of the myocardium. Dysfunction of these proteins can lead to the development of cardiomyopathies and arrhythmias.

    Journal of the American College of Cardiology 2003;42;2;319-27

  • Defining desmosomal plakophilin-3 interactions.

    Bonné S, Gilbert B, Hatzfeld M, Chen X, Green KJ and van Roy F

    Molecular Cell Biology Unit, Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology (VIB)-Ghent University, B-9000 Ghent, Belgium.

    Plakophilin 3 (PKP3) is a recently described armadillo protein of the desmosomal plaque, which is synthesized in simple and stratified epithelia. We investigated the localization pattern of endogenous and exogenous PKP3 and fragments thereof. The desmosomal binding properties of PKP3 were determined using yeast two-hybrid, coimmunoprecipitation and colocalization experiments. To this end, novel mouse anti-PKP3 mAbs were generated. We found that PKP3 binds all three desmogleins, desmocollin (Dsc) 3a and -3b, and possibly also Dsc1a and -2a. As such, this is the first protein interaction ever observed with a Dsc-b isoform. Moreover, we determined that PKP3 interacts with plakoglobin, desmoplakin (DP) and the epithelial keratin 18. Evidence was found for the presence of at least two DP-PKP3 interaction sites. This finding might explain how lateral DP-PKP interactions are established in the upper layers of stratified epithelia, increasing the size of the desmosome and the number of anchoring points available for keratins. Together, these results show that PKP3, whose epithelial and epidermal desmosomal expression pattern and protein interaction repertoire are broader than those of PKP1 and -2, is a unique multiprotein binding element in the basic architecture of a vast majority of epithelial desmosomes.

    Funded by: NIAMS NIH HHS: AR41836, AR43380, R01 AR041836, R01 AR043380, R37 AR043380; NIDCR NIH HHS: P01 DE012328, P01 DE12328

    The Journal of cell biology 2003;161;2;403-16

  • Targeting of p0071 to desmosomes and adherens junctions is mediated by different protein domains.

    Hatzfeld M, Green KJ and Sauter H

    Institute of Physiological Chemistry, Medical Faculty of the University of Halle, 06097 Halle/Saale, Germany. mechthild.hatzfeld@medizin.uni-halle.de

    p0071, a member of the armadillo protein family, is most closely related to p120(ctn) and the plakophilins 1-3. Whereas plakophilins are desmosomal plaque proteins, p120(ctn) localizes to adherens junctions and interacts with classical cadherins. In contrast, p0071 has been described as a protein with dual localization in adherens junctions and desmosomes depending on the cell type examined. Here we have analyzed the localization of p0071 and its domains in detail. Although by sequence analysis, p0071 is more closely related to the adherens junction proteins p120(ctn), ARVCF and delta-catenin, endogenous p0071 associated preferentially with desmosomes in MCF-7 epithelial cells. Overexpressed p0071 localized along cell borders and overlapped only partially with desmosomal markers but colocalized with non-desmosomal cadherins and recruited cadherins to the membrane. The head domain of p0071 was sufficient for desmosomal targeting, whereas the arm repeat domain associated with adherens junctions and enhanced membrane association of classical cadherins. The tail domain localized preferentially to the nucleus and associated with desmosomes. To examine the mechanism underlying this dual localization more closely we determined binding partners of p0071 by using yeast-two-hybrid and mom-targeting assays. These approaches show that the head domain interacted with desmosomal proteins desmocollin 3a and desmoplakin, whereas the armadillo repeat domain binds to non-desmosomal cadherins. Head and armadillo repeat domains both interacted with plakoglobin by binding to different sites. Our data suggest that, in addition to plakoglobin, p0071 is the second armadillo protein present in both types of adhesive junctions and may play a role in regulating crosstalk between adherens junctions and desmosomes.

    Funded by: NIAMS NIH HHS: AR43380; NIDCR NIH HHS: P01 DE12328

    Journal of cell science 2003;116;Pt 7;1219-33

  • The Armadillo family protein p0071 is a VE-cadherin- and desmoplakin-binding protein.

    Calkins CC, Hoepner BL, Law CM, Novak MR, Setzer SV, Hatzfeld M and Kowalczyk AP

    Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    p0071, a member of the armadillo protein family, localizes to both adherens junctions and desmosomes in epithelial cells and exhibits homology to the adherens junction protein p120 and the desmosomal protein plakophilin-1. p0071 is also present at dermal microvascular endothelial intercellular junctions and colocalizes with VE-cadherin, an endothelium-specific cadherin that associates with both actin and intermediate filament networks. To define the role of p0071 in junction assembly, p0071 was tested for interactions with other components of the endothelial junctional complex. In transient expression assays, p0071 colocalized with and formed complexes with both VE-cadherin and desmoplakin. Deletion analysis using the yeast two-hybrid system revealed that the armadillo repeat domain of p0071 bound directly to VE-cadherin. Site-directed mutagenesis experiments demonstrated that p0071 and p120 bound to the same region on the cytoplasmic tail of VE-cadherin and that overexpression of p0071 could displace p120 from intercellular junctions. In contrast to VE-cadherin, desmoplakin was found to associate with the non-armadillo head domain of p0071. Cotransfections and triple-label immunofluorescence analysis revealed that VE-cadherin colocalization with desmoplakin in transfected COS cells required p0071, suggesting that p0071 may couple VE-cadherin to desmoplakin. Based on previous findings that both VE-cadherin and desmoplakin play central roles in vasculogenesis, these new results suggest that p0071 may play an important role in endothelial junction assembly and in the morphogenic events associated with vascular remodeling.

    Funded by: NIAMS NIH HHS: HP30 AR042687, K01 AR002039, R01 AR48266-01, R03 AR47147, T32 AR007587

    The Journal of biological chemistry 2003;278;3;1774-83

  • Analysis of the interactions between BP180, BP230, plectin and the integrin alpha6beta4 important for hemidesmosome assembly.

    Koster J, Geerts D, Favre B, Borradori L and Sonnenberg A

    The Netherlands Cancer Institute, Division of Cell Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

    Hemidesmosomes (HDs) are multi-protein complexes that promote stable adhesion of epithelial cells to the underlying extracellular matrix. We assessed the interactions between different hemidesmosomal components with each other, mapped the binding sites and studied the importance of these interactions for HD assembly in yeast two-hybrid and cell-transfection assays. The results show that: (1) bullous pemphigoid antigen (BP) 180 binds not only to BP230, but also to plectin. The interactions between these proteins are facilitated by the Y subdomain in the N-terminal plakin domain of BP230 and plectin, and residues 145-230 of the cytoplasmic domain of BP180; (2) different, but overlapping, sequences on BP180 mediate binding to beta4, which, in turn associates with BP180 via its third fibronectin type III repeat; (3) sequences in the N-terminal extremity of BP230 mediate its binding to beta4, which requires the C-terminal end of the connecting segment up to the fourth FNIII repeat of the beta4 subunit. (4) Finally, cell-transfection studies showed that the localization of BP230 into hemidesmosome-like structures depends on its Z-Y subdomains as well as on the availability of BP180. By having further uncovered interactions between various hemidesmosomal components, mapped the involved binding sites and dissected a hierarchy of interactions relevant for their topogenic fate, our findings give novel insights into the molecular organization of hemidesmosomes.

    Journal of cell science 2003;116;Pt 2;387-99

  • Mutation in human desmoplakin domain binding to plakoglobin causes a dominant form of arrhythmogenic right ventricular cardiomyopathy.

    Rampazzo A, Nava A, Malacrida S, Beffagna G, Bauce B, Rossi V, Zimbello R, Simionati B, Basso C, Thiene G, Towbin JA and Danieli GA

    Department of Biology, University of Padua, Italy.

    Arrhythmogenic right ventricular cardiomyopathy (ARVD/C) is a genetically heterogeneous disease characterized by progressive degeneration of the right ventricular myocardium and increased risk of sudden death. Here, we report on a genome scan in one Italian family in which the disease appeared unlinked to any of the six different ARVD loci reported so far; we identify a mutation (S299R) in exon 7 of desmoplakin (DSP), which modifies a putative phosphorylation site in the N-terminal domain binding plakoglobin. It is interesting that a nonsense DSP mutation was reported elsewhere in the literature, inherited as a recessive trait and causing a biventricular dilative cardiomyopathy associated with palmoplantar keratoderma and woolly hairs. Therefore, different DSP mutations might produce different clinical phenotypes, with different modes of inheritance.

    Funded by: NHLBI NIH HHS: 1 U01 HL 65652, U01 HL065652

    American journal of human genetics 2002;71;5;1200-6

  • Unique role for the periplakin tail in intermediate filament association: specific binding to keratin 8 and vimentin.

    Kazerounian S, Uitto J and Aho S

    Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

    Plectin, desmoplakin, and the 230-kDa bullous pemphigoid antigen (BPAG1), members of the plakin family of proteins, are multifunctional cytolinkers, connecting the cytoskeletal structures to the cell adhesion complexes. Envoplakin and periplakin are components of the cornified envelope, but less is known about their role in tissues other than the stratified epithelium. Our tissue-wide survey utilizing RT-PCR revealed that periplakin, like plectin and desmoplakin, has a wide tissue distribution, but envoplakin expression is limited to certain tissues only, and BPAG1 is clearly specific for epidermal keratinocytes. Plectin, desmoplakin and BPAG1 are known to bind to the intermediate filaments through their C-terminal domains. The short C-terminal domain of periplakin is composed only of the linker domain, a region highly homologous between the plakin proteins. Here we demonstrate, through the use of yeast two-hybrid assay, a specific interaction of the periplakin linker domain with keratin 8 and vimentin. Co-expression of each plakin linker domain with keratin 8 revealed that periplakin and BPAG1 linkers co-localize with keratin signals in HaCaT cells, plectin and desmoplakin linkers were detected both in the nucleus and in cytoplasm together with the overexpressed keratin 8, while envoplakin linker localized independently into the nucleus. These results suggest that, in spite of its high homology and structural similarity with envoplakin, periplakin is functionally closer to the well-characterized plakin proteins plectin and desmoplakin, and thus may function tissue-wide as a scaffolding protein in intermediate filament assembly.

    Funded by: NIAMS NIH HHS: R01-AR44833

    Experimental dermatology 2002;11;5;428-38

  • Structures of two intermediate filament-binding fragments of desmoplakin reveal a unique repeat motif structure.

    Choi HJ, Park-Snyder S, Pascoe LT, Green KJ and Weis WI

    Department of Structural Biology, Stanford University School of Medicine, 299 Campus Drive West, Stanford, California 94305, USA.

    Desmosomes are intercellular junctions in which cadherin cell adhesion molecules are linked to the intermediate filament (IF) system. Desmoplakin is a member of the plakin family of IF-binding proteins. The C-terminal domain of desmoplakin (DPCT) mediates binding to IFs in desmosomes. The DPCT sequence contains three regions, termed A, B and C, consisting of 4.5 copies of a 38-amino acid repeat motif. We demonstrate that these regions form discrete subdomains that bind to IFs and report the crystal structures of domains B and C. In contrast to the elongated structures formed by other kinds of repeat motifs, the plakin repeats form a globular structure with a unique fold. A conserved basic groove found on the domain may represent an IF-binding site.

    Nature structural biology 2002;9;8;612-20

  • Differential expression and biodistribution of cytokeratin 18 and desmoplakins in non-small cell lung carcinoma subtypes.

    Young GD, Winokur TS, Cerfolio RJ, Van Tine BA, Chow LT, Okoh V and Garver RI

    Department of Medicine, University of Alabama at Birmingham, and Birmingham VAMC, 701 South 19th Street, LHRB 339, 35294, USA.

    Adenocarcinoma (AC), squamous cell carcinoma (SCC) and adenosquamous carcinoma (ASC) of the lung are morphologically distinguished in part by cyto-architectural features. However, little is known about the relative expression and distribution of cyto-architectural proteins among AC, SCC and ASC. Initial microarray analysis revealed significant differences in expression of two cyto-architectural genes in AC, SCC and ASC. Desmoplakin (DP) 1 and 2, which link desmosomes to intermediate filaments, was strongly expressed in SCC relative to AC and ASC. Cytokeratin 18 (CK18), an intermediate filament that is commonly linked to desmoplakin, was strongly expressed in AC and ASC relative to SCC. Western blot analysis demonstrated that AC and ASC had abundant CK18 protein, whereas CK18 was weakly detected in SCC. DP 1 and 2 are strongly expressed in SCC and minimally expressed in AC and ASC. However, the ratio of one to the other is the same in SCC and AC, but DP2 is lost in ASC. Microscopic analysis with fluorescence-labeled antibodies for CK18 and DP 1 and 2 revealed abundant membrane localization of DP and minimal perinuclear localization of CK18 in SCC. In contrast, in both AC and ASC, the CK18 protein was diffusely distributed within the cytoplasm, and DP showed both membranous and cytoplasmic localization. In conclusion, the data here shows that AC, SCC and ASC each have specific patterns of DP 1 and 2 and CK18 gene expression, protein content and biodistribution.

    Lung cancer (Amsterdam, Netherlands) 2002;36;2;133-41

  • Protein binding and functional characterization of plakophilin 2. Evidence for its diverse roles in desmosomes and beta -catenin signaling.

    Chen X, Bonne S, Hatzfeld M, van Roy F and Green KJ

    Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611, USA.

    Plakophilins are a subfamily of p120-related arm-repeat proteins that can be found in both desmosomes and the nucleus. Among the three known plakophilin members, plakophilin 1 has been linked to a genetic skin disorder and shown to play important roles in desmosome assembly and organization. However, little is known about the binding partners and functions of the most widely expressed member, plakophilin 2. To better understand the cellular functions of plakophilin 2, we have examined its protein interactions with other junctional molecules using co-immunoprecipitation and yeast two-hybrid assays. Here we show that plakophilin 2 can interact directly with several desmosomal components, including desmoplakin, plakoglobin, desmoglein 1 and 2, and desmocollin 1a and 2a. The head domain of plakophilin 2 is critical for most of these interactions and is sufficient to direct plakophilin 2 to cell borders. In addition, plakophilin 2 is less efficient than plakophilin 1 in localizing to the nucleus and enhancing the recruitment of excess desmoplakin to cell borders in transiently transfected COS cells. Furthermore, plakophilin 2 is able to associate with beta-catenin through its head domain, and the expression of plakophilin 2 in SW480 cells up-regulates the endogenous beta-catenin/T cell factor-signaling activity. This up-regulation by plakophilin 2 is abolished by ectopic expression of E-cadherin, suggesting that these proteins compete for the same pool of signaling active beta-catenin. Our results demonstrate that plakophilin 2 interacts with a broader repertoire of desmosomal components than plakophilin 1 and provide new insight into the possible roles of plakophilin 2 in regulating the signaling activity of beta-catenin.

    Funded by: NIAMS NIH HHS: AR41836, R01 AR43380; NIDCR NIH HHS: P01 DE12328

    The Journal of biological chemistry 2002;277;12;10512-22

  • Compound heterozygosity for non-sense and mis-sense mutations in desmoplakin underlies skin fragility/woolly hair syndrome.

    Whittock NV, Wan H, Morley SM, Garzon MC, Kristal L, Hyde P, McLean WH, Pulkkinen L, Uitto J, Christiano AM, Eady RA and McGrath JA

    Department of Cellular and Molecular Pathology, St John's Institute of Dermatology, The Guy's, King's College, and St Thomas' Hospitals' Medical School, London, UK. nwhittoc@hgmp.mrc.ac.uk

    The constitutive desmosomal plaque protein desmoplakin plays a vital part in keratinocyte adhesion in linking the transmembranous desmosomal cadherins to the cytoplasmic keratin filament network. Recently, mutations in desmoplakin have been shown to underlie some cases of the autosomal dominant disorder, striate palmoplantar keratoderma, as well as an autosomal recessive condition characterized by dilated cardiomyopathy, woolly hair, and keratoderma. Here, we describe two unrelated individuals with a new autosomal recessive genodermatosis characterized by focal and diffuse palmoplantar keratoderma, hyperkeratotic plaques on the trunk and limbs, varying degrees of alopecia, but no apparent cardiac anomalies. Mutation screening of desmoplakin demonstrated compound heterozygosity for a non-sense/mis-sense combination of mutations in both cases, C809X/N287K and Q664X/R2366C, respectively. Heterozygous carriers of any of these mutations displayed no phenotypic abnormalities. Immunohistochemistry of skin biopsies from both affected individuals revealed that desmoplakin was not just located at the cell periphery but there was also cytoplasmic staining. In addition, electron microscopy demonstrated acantholysis throughout all layers of the skin, focal detachment of desmosomes into the intercellular spaces, and perinuclear condensation of the suprabasal keratin intermediate filament network. Clinicopathologic and mutational analyses therefore demonstrate that desmoplakin haploinsufficiency can be tolerated in some cases, but that in combination with a mis-sense mutation on the other allele, the consequences are a severe genodermatosis with specific clinical manifestations.

    The Journal of investigative dermatology 2002;118;2;232-8

  • Directed proteomic analysis of the human nucleolus.

    Andersen JS, Lyon CE, Fox AH, Leung AK, Lam YW, Steen H, Mann M and Lamond AI

    Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark.

    Background: The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspects of their structure and function remain uncharacterized.

    Results: We report a proteomic analysis of human nucleoli. Using a combination of mass spectrometry (MS) and sequence database searches, including online analysis of the draft human genome sequence, 271 proteins were identified. Over 30% of the nucleolar proteins were encoded by novel or uncharacterized genes, while the known proteins included several unexpected factors with no previously known nucleolar functions. MS analysis of nucleoli isolated from HeLa cells in which transcription had been inhibited showed that a subset of proteins was enriched. These data highlight the dynamic nature of the nucleolar proteome and show that proteins can either associate with nucleoli transiently or accumulate only under specific metabolic conditions.

    Conclusions: This extensive proteomic analysis shows that nucleoli have a surprisingly large protein complexity. The many novel factors and separate classes of proteins identified support the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis. The data also show that the protein composition of nucleoli is not static and can alter significantly in response to the metabolic state of the cell.

    Current biology : CB 2002;12;1;1-11

  • The fate of desmosomal proteins in apoptotic cells.

    Weiske J, Schöneberg T, Schröder W, Hatzfeld M, Tauber R and Huber O

    Institute of Clinical Chemistry and Pathobiochemistry, University Hospital Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany.

    Activation of caspases results in the disruption of structural and signaling networks in apoptotic cells. Recent biochemical and cell biological studies have shown that components of the cadherin-catenin adhesion complex in epithelial adherens junctions are targeted by caspases during apoptosis. In epithelial cells, desmosomes represent a second type of anchoring junctions mediating strong cell-cell contacts. Using antibodies directed against a set of desmosomal proteins, we show that desmosomes are proteolytically targeted during apoptosis. Desmogleins and desmocollins, representing desmosome-specific members of the cadherin superfamily of cell adhesion molecules, are specifically cleaved after onset of apoptosis. Similar to E-cadherin, the desmoglein-3 cytoplasmic tail is cleaved by caspases. In addition the extracellular domains of desmoglein-3 and desmocollin-3 are released from the cell surface by a metalloproteinase activity. In the presence of caspase and/or metalloproteinase inhibitors, both cleavage reactions are almost completely inhibited. As reported previously, the desmosomal plaque protein plakoglobin is cleaved by caspase-3 during apoptosis. Our studies now show that plakophilin-1 and two other major plaque proteins, desmoplakin-1 and -2, are also cleaved by caspases. Immunofluorescence analysis confirmed that this cleavage results in the disruption of the desmosome structure and thus contributes to cell rounding and disintegration of the intermediate filament system.

    The Journal of biological chemistry 2001;276;44;41175-81

  • Recessive mutation in desmoplakin disrupts desmoplakin-intermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma.

    Norgett EE, Hatsell SJ, Carvajal-Huerta L, Cabezas JC, Common J, Purkis PE, Whittock N, Leigh IM, Stevens HP and Kelsell DP

    Centre for Cutaneous Research, St Bartholomews' and Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, 2 Newark Street, London E1 2AT, UK.

    Desmosomes are major cell adhesion junctions, particularly prominent in the epidermis and cardiac tissue and are important for the rigidity and strength of the cells. The desmosome consists of several proteins, of which desmoplakin is the most abundant. Here, we describe the first recessive human mutation, 7901delG, in the desmoplakin gene which causes a generalized striate keratoderma particularly affecting the palmoplantar epidermis, woolly hair and a dilated left ventricular cardiomyopathy. A number of the patients with this syndromic disorder suffer heart failure in their teenage years, resulting in early morbidity. All tested affected members of three families from Ecuador were homozygous for this mutation which produces a premature stop codon leading to a truncated desmoplakin protein missing the C domain of the tail region. Histology of the skin revealed large intercellular spaces and clustering of desmosomes at the infrequent sites of keratinocyte adhesion. Immunohistochemistry of skin from the patients showed a perinuclear localization of keratin in suprabasal keratinocytes, suggesting a collapsed intermediate filament network. This study demonstrates the importance of desmoplakin in the attachment of intermediate filaments to the desmosome. In contrast to null DESMOPLAKIN: mice which die in early development, the truncated protein due to the homozygous 7901delG mutation in humans is not embryonic lethal. This suggests that the tail domain of desmoplakin is not required for establishing tissue architecture during development.

    Human molecular genetics 2000;9;18;2761-6

  • Small proline-rich protein 1 is the major component of the cell envelope of normal human oral keratinocytes.

    Lee CH, Marekov LN, Kim S, Brahim JS, Park MH and Steinert PM

    Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, MD 20892-7252, USA.

    Oral keratinocytes of buccal and gingival tissues undergo a terminal differentiation program to form a protective epithelial barrier as non-keratinized or parakeratinized stratified cells. We have examined the protein composition of cell envelopes (CEs) from normal human buccal and gingival tissues as well as keratinocytes from normal human gingival cells grown in culture. Biochemical and sequencing analyses reveal that the CEs contain 60-70% small proline-rich protein 1a/b (SPR1a/b), together with smaller amounts of involucrin, annexin I and several other known CE proteins. The data imply a specialized role for SPR1 proteins in the unique barrier function requirements of oral epithelia.

    FEBS letters 2000;477;3;268-72

  • Platelet-endothelial cell adhesion molecule-1 (CD31), a scaffolding molecule for selected catenin family members whose binding is mediated by different tyrosine and serine/threonine phosphorylation.

    Ilan N, Cheung L, Pinter E and Madri JA

    Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

    Platelet-endothelial cell adhesion molecule (PECAM)-1 is a 130-kDa glycoprotein commonly used as an endothelium-specific marker. Evidence to date suggests that PECAM-1 is more than just an endothelial cell marker but is intimately involved in signal transduction pathways. This is mediated in part by phosphorylation of specific tyrosine residues within the ITAM domain of PECAM-1 and by recruitment of adapter and signaling molecules. Recently we demonstrated that PECAM-1/beta-catenin association functions to regulate beta-catenin localization and, moreover, to modulate beta-catenin tyrosine phosphorylation levels. Here we show that: 1) not only beta-catenin, but also gamma-catenin is associated with PECAM-1 in vitro and in vivo; 2) PKC enzyme directly phosphorylates purified PECAM-1; 3) PKC-derived PECAM-1 serine/threonine phosphorylation inversely correlates with gamma-catenin association; 4) PECAM-1 recruits gamma-catenin to cell-cell junctions in transfected SW480 cells; and 5) gamma-catenin may recruit PECAM-1 into an insoluble cytoskeletal fraction. These data further support the concept that PECAM-1 functions as a binder and modulator of catenins and provides a molecular mechanism for previously reported PECAM-1/cytoskeleton interactions.

    Funded by: NHLBI NIH HHS: R37-HL28373; NIDDK NIH HHS: P01-DK38979

    The Journal of biological chemistry 2000;275;28;21435-43

  • Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis.

    Hofmann I, Mertens C, Brettel M, Nimmrich V, Schnölzer M and Herrmann H

    Division of Cell Biology/A0100 and Protein Analysis Facility/R0800, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. i.hofmann@dkfz-heidelberg.de

    Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm-repeat protein family. They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they occur in the nuclei of cells and, in epithelial cells, at the plasma membrane within the desmosomal plaques. We have shown by solid phase-binding assays that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epithelial cells and, to some extent, to vimentin. In line with this we show that recombinant PKP1a binds strongly to IFs assembled in vitro from CKs 8/18, 5/14, vimentin or desmin and integrates them into thick (up to 120 nm in diameter) IF bundles extending for several microm. The basic amino-terminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation experiments. In particular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio. In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by co-immunoprecipitation and sucrose density fractionation. The significance of these interactions of PKP1a with IF proteins on the one hand and desmoplakin on the other is discussed in relation to the fact that PKP1a is not bound - and does not bind - to extended IFs in vivo. We postulate that (1) effective cellular regulatory mechanisms exist that prevent plakophilins from unscheduled IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recruitment of plakophilins to the desmosomal plaques.

    Journal of cell science 2000;113 ( Pt 13);2471-83

  • Shotgun sequencing of the human transcriptome with ORF expressed sequence tags.

    Dias Neto E, Correa RG, Verjovski-Almeida S, Briones MR, Nagai MA, da Silva W, Zago MA, Bordin S, Costa FF, Goldman GH, Carvalho AF, Matsukuma A, Baia GS, Simpson DH, Brunstein A, de Oliveira PS, Bucher P, Jongeneel CV, O'Hare MJ, Soares F, Brentani RR, Reis LF, de Souza SJ and Simpson AJ

    Ludwig Institute for Cancer Research, São Paulo 01509-010, Brazil.

    Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription-PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. Systematic, high-throughput sequencing of such products would result in an expressed sequence tag (EST) database consisting of central, generally coding regions of expressed genes. Such a database would add significant value to existing public EST databases, which consist mostly of sequences derived from the extremities of cDNAs, and facilitate the construction of contigs of transcript sequences. We tested our predictions, creating a database of 10,000 sequences from human breast tumors. The data confirmed the central distribution of the sequences, the significant normalization of the sequence population, the frequent extension of contigs composed of existing human ESTs, and the identification of a series of potentially important homologues of known genes. This approach should make a significant contribution to the early identification of important human genes, the deciphering of the draft human genome sequence currently being compiled, and the shotgun sequencing of the human transcriptome.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;7;3491-6

  • Epithelial structural proteins of the skin and oral cavity: function in health and disease.

    Presland RB and Dale BA

    Department of Oral Biology, University of Washington, Seattle 98195, USA. rp@u.washington.edu

    Epithelial tissues function to protect the organism from physical, chemical, and microbial damage and are essential for survival. To perform this role, epithelial keratinocytes undergo a well-defined differentiation program that results in the expression of structural proteins which maintain the integrity of epithelial tissues and function as a protective barrier. This review focuses on structural proteins of the epidermis and oral mucosa. Keratin proteins comprise the predominant cytoskeletal component of these epithelia. Keratin filaments are attached to the plasma membrane via desmosomes, and together these structural components form a three-dimensional array within the cytoplasm of epithelial cells and tissues. Desmosomes contain two types of transmembrane proteins, the desmogleins and desmocollins, that are members of the cadherin family. The desmosomal cadherins are linked to the keratin cytoskeleton via several cytoplasmic plaque proteins, including desmoplakin and plakoglobin (gamma-catenin). Epidermal and oral keratinocytes express additional differentiation markers, including filaggrin and trichohyalin, that associate with the keratin cytoskeleton during terminal differentiation, and proteins such as loricrin, small proline-rich proteins, and involucrin, that are cross-linked into the cornified envelope by transglutaminase enzymes. The importance of these cellular structures is highlighted by the large numbers of genetic and acquired (autoimmune) human disorders that involve mutations in, or autoantibodies to, keratins and desmosomal and cornified envelope proteins. While much progress has been made in the identification of the structural proteins and enzymes involved in epithelial differentiation, regulation of this process is less clear. Both calcium and retinoids influence epithelial differentiation by altering the transcription of target genes and by regulating activity of enzymes critical in epithelial differentiation, such as transglutaminases, proteinases, and protein kinases. These studies have furthered our understanding of how epithelial tissue and cell integrity is maintained and provide a basis for the future treatment of skin and oral disorders by gene therapy and other novel therapeutics.

    Funded by: NIAMS NIH HHS: P01 AR21557, R29 AR45276

    Critical reviews in oral biology and medicine : an official publication of the American Association of Oral Biologists 2000;11;4;383-408

  • Striate palmoplantar keratoderma resulting from desmoplakin haploinsufficiency.

    Whittock NV, Ashton GH, Dopping-Hepenstal PJ, Gratian MJ, Keane FM, Eady RA and McGrath JA

    Department of Cell and Molecular Pathology, St John's Institute of Dermatology, The Guy's, King's College, and St Thomas' Hospitals' Medical School, St Thomas' Hospital, London, UK. neil.2.whittock

    Recently, the first example of a human mutation in the gene encoding the desmosomal plaque protein, desmoplakin, has been described in a patient with autosomal dominant striate palmoplantar kerato-derma. We now report a further case of a desmoplakin mutation in a proband with striate palmoplantar keratoderma that also results in a null allele and haploinsufficiency. The mutation was a heterozygous G > A transition at the donor + 1 site of intron 7 of the desmoplakin gene (939 + 1 G > A; Genbank M77830). The aberrant splicing leads to retention of the entire intron 7, which contains a premature termination codon within the N-terminal domain of the peptide. Because the mutant null allele could not be identified on cDNA sequencing, we determined by polymerase chain reaction the exon-intron organization of the desmoplakin gene to facilitate analysis of genomic DNA. The gene spans approximately 45 kb of chromosome 6 and comprises 24 exons ranging in size from 51 bp to 3922 bp. We have also characterized fully the 3'UTR of the desmoplakin cDNA. This study demonstrates the relevance of haploinsufficiency for desmoplakin in the pathogenesis of this genodermatosis. Assessment of family members bearing the mutant allele also emphasizes the significance of an individual's age and exposure to skin trauma in manifesting full phenotypic expression of the disorder.

    Funded by: Wellcome Trust

    The Journal of investigative dermatology 1999;113;6;940-6

  • Haploinsufficiency of desmoplakin causes a striate subtype of palmoplantar keratoderma.

    Armstrong DK, McKenna KE, Purkis PE, Green KJ, Eady RA, Leigh IM and Hughes AE

    Department of Medical Genetics, The Queen's University of Belfast, Belfast City Hospital, Belfast BT9 7AB, UK.

    Desmosomes are highly organized intercellular adhesive junctions that are particularly prominent in epidermis and other tissues experiencing mechanical stress. Desmoplakin, a constitutive component of the desmosomal plaque, is the most abundant protein present in such junctions and plays a critical role in linking the intermediate filament network to the plasma membrane in these tissues. Here we report the first mutation in the gene encoding desmoplakin. The identified mutation, resulting in a null allele and haploinsufficiency, was observed in genomic DNA from a kindred with the dominantly inherited skin disorder, striate palmoplantar keratoderma. Affected individuals had a linear pattern of skin thickening on the fingers and palms and circumscribed areas of skin thickening on the soles. Affected skin demonstrated loosening of intercellular connections, disruption of desmosome-keratin intermediate filament interactions and a proportion of rudimentary desmosomal structures. The disorder mapped to chromosome 6p21 with a maximum lod score of 10.67. The mutation was a heterozygous C-->T transition in exon 4 of the desmoplakin gene and predicted a premature termination codon in the N-terminal region of the peptide. This is the first reported mutation of desmo-plakin and also the first inherited skin disorder in which haploinsufficiency of a structural component has been implicated. It identifies dosage of desmoplakin as critical in maintaining epidermal integrity.

    Funded by: NIAMS NIH HHS: R01 AR43380; Wellcome Trust

    Human molecular genetics 1999;8;1;143-8

  • VE-cadherin and desmoplakin are assembled into dermal microvascular endothelial intercellular junctions: a pivotal role for plakoglobin in the recruitment of desmoplakin to intercellular junctions.

    Kowalczyk AP, Navarro P, Dejana E, Bornslaeger EA, Green KJ, Kopp DS and Borgwardt JE

    Departments of Dermatology, Pathology, and The Robert H. Lurie Cancer Center, Northwestern University Medical School, Chicago, IL, USA. akowalc@emory.edu.

    Vascular endothelial cells assemble adhesive intercellular junctions comprising a unique cadherin, VE-cadherin, which is coupled to the actin cytoskeleton through cytoplasmic interactions with plakoglobin, beta-catenin and alpha -catenin. However, the potential linkage between VE-cadherin and the vimentin intermediate filament cytoskeleton is not well characterized. Recent evidence indicates that lymphatic and vascular endothelial cells express desmoplakin, a cytoplasmic desmosomal protein that attaches intermediate filaments to the plasma membrane in epithelial cells. In the present study, desmoplakin was localized to intercellular junctions in human dermal microvascular endothelial cells. To determine if VE-cadherin could associate with desmoplakin, VE-cadherin, plakoglobin, and a desmoplakin amino-terminal polypeptide (DP-NTP) were co-expressed in L-cell fibroblasts. In the presence of VE-cadherin, both plakoglobin and DP-NTP were recruited to cell-cell borders. Interestingly, beta-catenin could not substitute for plakoglobin in the recruitment of DP-NTP to cell borders, and DP-NTP bound to plakoglobin but not beta-catenin in the yeast two-hybrid system. In addition, DP-NTP colocalized at cell-cell borders with alpha-catenin in the L-cell lines, and endogenous desmoplakin and alpha-catenin colocalized in cultured dermal microvascular endothelial cells. This is in striking contrast to epithelial cells, where desmoplakin and alpha -+catenin are restricted to desmosomes and adherens junctions, respectively. These results suggest that endothelial cells assemble unique junctional complexes that couple VE-cadherin to both the actin and intermediate filament cytoskeleton.

    Funded by: NIAMS NIH HHS: KO1AR02039, R01AR44380

    Journal of cell science 1998;111 ( Pt 20);3045-57

  • A novel E2F binding protein with Myc-type HLH motif stimulates E2F-dependent transcription by forming a heterodimer.

    Suzuki M, Okuyama S, Okamoto S, Shirasuna K, Nakajima T, Hachiya T, Nojima H, Sekiya S and Oda K

    Department of Biological Science and Technology, Science University of Tokyo, Chiba, Japan.

    The human embryonal carcinoma cells NEC14 can be induced to differentiate morphologically by the addition of 10(-2) M N, N'-hexamethylene-bis-acetamide and cease to grow in several days. Transcription factors of the E2F/DP family have been shown to be closely related to the regulation of cell proliferation. To analyse cellular proteins which interact with E2F in NEC14 cells, cDNA clones encoding E2F binding proteins were isolated from a lambdaZAP II NEC14 cell library with the 32P-labeled GST (Glutathione S-transferase)-E2F-1 fusion protein as a probe. One of the clones encodes E2FBP1 which has the helix-loop-helix (HLH) motif, but lacks the basic domain and the zipper structure usually found at N- and C-terminal sides to the HLH motif, respectively. The arrangement of amino acids in the helix 1 and helix 2 regions is quite similar to those of Mxi and Mad, but different from those of E2F-1 and DP-1. Western blot analysis of the immunoprecipitates prepared with anti-E2FBP1 antibody showed that E2FBP1 associates with both E2F-1 and DP-1 in vivo. E2FBP1 alone has no DNA binding activity, but bind to the E2F site through heterodimerization with E2F-1 but not with DP-1. Although E2FBP1 lacks the transactivation domain, it stimulates E2F site-dependent transcription in cooperation with E2F-1.

    Oncogene 1998;17;7;853-65

  • Ceramides are bound to structural proteins of the human foreskin epidermal cornified cell envelope.

    Marekov LN and Steinert PM

    Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892-2752, USA.

    An important component of barrier function in human epidermis is contributed by ceramides that are bound by ester linkages to undefined proteins of the cornified cell envelope (CE). In this paper, we have examined the protein targets for the ceramide attachment. By partial saponification of isolated foreskin epidermal CEs followed by limited proteolysis, we have recovered several lipopeptides. Biochemical and mass spectroscopic characterization revealed that all contained near stoichiometric amounts of ceramides of masses ranging from about 690 to 890 atomic mass units, of which six quantitatively major species were common. The array of ceramides was similar to that obtained from pig skin, the composition of which is known, thereby providing strong indirect data for their fatty acid and sphingosine compositions. The recovered peptides accounted for about 20% of the total foreskin CE ceramides. By amino acid sequencing, about 35% of the peptides were derived from ancestral glutamine-glutamate-rich regions of involucrin, an important CE structural protein. Another 18% derived from rod domain sequences of periplakin and envoplakin, which are also known or suspected CE proteins. Other peptides were too short for unequivocal identification. Together, these data indicate that involucrin, envoplakin, periplakin, and possibly other structural proteins serve as substrates for the attachment of ceramides by ester linkages to the CE for barrier function in human epidermis.

    The Journal of biological chemistry 1998;273;28;17763-70

  • Defining the interactions between intermediate filaments and desmosomes.

    Smith EA and Fuchs E

    Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637, USA.

    Desmoplakin (DP), plakoglobin (PG), and plakophilin 1 (PP1) are desmosomal components lacking a transmembrane domain, thus making them candidate linker proteins for connecting intermediate filaments and desmosomes. Using deletion and site-directed mutagenesis, we show that remarkably, removal of approximately 1% of DP's sequence obliterates its ability to associate with desmosomes. Conversely, when linked to a foreign protein, as few as 86 NH2-terminal DP residues are sufficient to target to desmosomes efficiently. In in vitro overlay assays, the DP head specifically associates with itself and with desmocollin 1a (Dsc1a). In similar overlay assays, PP1 binds to DP and Dsc1a, and to a lesser extent, desmoglein 1 (Dsg1), while PG binds to Dsg1 and more weakly to Dsc1a and DP. Interestingly, like DP, PG and PP1 associate with epidermal keratins, although PG is considerably weaker in its ability to do so. As judged by overlay assays, the amino terminal head domain of type II keratins appears to have a special importance in establishing these connections. Taken together, our findings provide new insights into the complexities of the links between desmosomes and intermediate filaments (IFs). Our results suggest a model whereby at desmosome sites within dividing epidermal cells, DP and PG anchor to desmosomal cadherins and to each other, forming an ordered array of nontransmembrane proteins that then bind to keratin IFs. As epidermal cells differentiate, PP1 is added as a molecular reinforcement to the plaque, enhancing anchorage to IFs and accounting at least partially for the increase in numbers and stability of desmosomes in suprabasal cells.

    The Journal of cell biology 1998;141;5;1229-41

  • Fine mapping of 39 ESTs on human chromosome 6p23-p25.

    Olavesen MG, Bentley E, Mason RV, Stephens RJ and Ragoussis J

    Division of Medical Molecular Genetics, UMDS, Guy's Hospital, London, United Kingdom.

    Loci conferring susceptibility to schizophrenia, coeliac disease, and orofacial clefting have been assigned to the 6p23-p25 region of human chromosome 6. To facilitate the identification of candidate genes we have sublocalized and ordered 39 ESTs assigned to this interval by radiation hybrid mapping. This was achieved by generating PAC contigs containing the ESTs, genetic markers, and random STSs. For full integration into previously published data a single YAC contig spanning 6p23-p25 was used to unambiguously order the PAC contigs and ESTs along the chromosome. The majority of the ESTs (31/39) were positioned in the 6p23-p24 interval at the proximal half of the map, and of these 8 are located within a single PAC clone. The order of known genes in this region is cen-CD83-ZNF40-EDN1-(GCNT2, CAPZB)-TFAP2-BMP6-DSP-tel.

    Genomics 1997;46;2;303-6

  • The amino-terminal domain of desmoplakin binds to plakoglobin and clusters desmosomal cadherin-plakoglobin complexes.

    Kowalczyk AP, Bornslaeger EA, Borgwardt JE, Palka HL, Dhaliwal AS, Corcoran CM, Denning MF and Green KJ

    Department of Dermatology, Northwestern University Medical School, Chicago, Illinois 60611, USA.

    The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell-cell adhesion. Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin. Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome. However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified. To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components. When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution. However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP. In addition, plakoglobin and DP-NTP were recruited to cell-cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome. In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin. Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1. These results identify a role for desmoplakin in organizing the desmosomal cadherin-plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.

    Funded by: NIAMS NIH HHS: R01 AR041836, R01 AR043380, R01AR41836, R01AR43380

    The Journal of cell biology 1997;139;3;773-84

  • Two-hybrid analysis reveals fundamental differences in direct interactions between desmoplakin and cell type-specific intermediate filaments.

    Meng JJ, Bornslaeger EA, Green KJ, Steinert PM and Ip W

    Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521, USA.

    Desmosomes are cell junctions that act as sites of strong intercellular adhesion and also serve to anchor the intermediate filament (IF) cytoskeleton to the plasma membrane of a variety of cell types. Previous studies demonstrated that the COOH terminus of the desmosomal plaque protein, desmoplakin (DP), is required for the association of DP with IF networks in cultured cells and that this domain interacts directly with type II epidermal keratin polypeptides in vitro. However, these studies left open the question of how desmosomes might anchor other IF types known to associate with these junctions. In this report we used yeast two-hybrid and in vitro dot blot assays to further examine the requirements for direct interactions between desmoplakin and various IF types. Our results confirm the ability of the DP COOH terminus (DPCT) to interact with at least two regions of the head domain of the type II epidermal keratin K1 and also demonstrate that DPCT can interact with the type III IF family members, vimentin and desmin, as well as simple epithelial keratins. Unlike the situation for type II epidermal keratins, the interaction between DPCT and simple epithelial keratins appears to depend on heterodimerization of the type I and II keratin polypeptides, since both are required to detect an interaction. Furthermore, although the interaction between DPCT and K1 requires the keratin head domain, deletion of this domain from the simple epithelial keratins does not compromise interaction with DPCT. The interaction between DPCT and type III or simple epithelial keratins also appeared to be less robust than that between DPCT and K1. In the case of K8/K18, however, the interaction as assessed by yeast two-hybrid assays increased 9-fold when a serine located in a protein kinase A consensus phosphorylation site 23 residues from the end of DP was altered to a glycine. Taken together, these data indicate that DP interacts directly with different IF types in specific ways.

    Funded by: NIAMS NIH HHS: R01 AR35973, R01 AR43380

    The Journal of biological chemistry 1997;272;34;21495-503

  • Direct evidence that involucrin is a major early isopeptide cross-linked component of the keratinocyte cornified cell envelope.

    Steinert PM and Marekov LN

    Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892-2752, USA. pemast@helix.nih.gov

    Involucrin was the first protein to be identified as a likely constituent of the insoluble cornified cell envelope (CE) of stratified squamous epithelia. However, to date, direct isolation from CEs of involucrin cross-linked by way of the transglutaminase-induced isopeptide bond has not been reported. We have treated human foreskin CEs with methanol/KOH (saponification) to hydrolyze off much of the lipids. By immunogold electron microscopy, this exposed large amounts of involucrin epitopes as well as of desmoplakin, a desmosomal structural protein. About 20% of the total CE protein could be solubilized by proteolytic digestion after saponification, of which involucrin was the most abundant. Subsequent amino acid sequencing revealed many peptides involving involucrin cross-linked either to itself or to a variety of other known CE protein components, including cystatin alpha, desmoplakin, elafin, keratins, members of the small proline-rich superfamily, loricrin, and unknown proteins related to the desmoplakin family. Specific glutamines or lysines of involucrin were used to cross-link the different proteins, such as glutamines 495 and 496 to desmoplakin, glutamine 288 to keratins, and lysines 468, 485, and 508 and glutamines 465 and 489 for interchain involucrin cross-links. Many identical peptides were obtained from immature CEs isolated from the inner living cell layers of foreskin epidermis. The multiple cross-linked partners of involucrin provide experimental confirmation that involucrin is an important early scaffold protein in the CE. Further, these data suggest that there is significant redundancy in the structural organization of the CE.

    The Journal of biological chemistry 1997;272;3;2021-30

  • Phosphorylation of the desmoplakin COOH terminus negatively regulates its interaction with keratin intermediate filament networks.

    Stappenbeck TS, Lamb JA, Corcoran CM and Green KJ

    Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611.

    Desmoplakins (DPs) are the most abundant proteins in the innermost portion of the desmosomal plaque and have been proposed to play a role in the attachment of intermediate filaments (IF) to cell-cell contact sites. Our previous results suggest that the globular end domains of DP perform dual functions: first, to target DP to the desmosome via the NH2 terminus and second, to attach IF to the desmosomal plaque via the COOH terminus. When ectopically expressed in most cultured cells, the COOH terminus plus the rod domain (DP. delta N.SerC23) exhibits striking coalignment with keratin IF networks. However, in certain cell types (e.g. PtK2) or in cells treated with forskolin to activate protein kinase A, DP. delta N.SerC23 exhibits a diffuse cytoplasmic distribution. A variant molecule (DP. delta N.GlyC23) in which a serine located 23 amino acids from the COOH terminus is altered to a glycine, thereby disrupting a protein kinase A consensus phosphorylation site, co-localizes with keratin IF networks regardless of cell type or forskolin treatment. Analysis of the phosphopeptide maps of these DP variants and endogenous DP is consistent with the phosphorylation of the serine 23 residues from the COOH terminus. These results suggest that phosphorylation of a specific residue in the DP COOH terminus may negatively regulate its interaction with keratin IF networks.

    The Journal of biological chemistry 1994;269;47;29351-4

  • Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins.

    Kouklis PD, Hutton E and Fuchs E

    Howard Hughes Medical Institute, Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637.

    In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution-binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.

    Funded by: NIAMS NIH HHS: AR27883

    The Journal of cell biology 1994;127;4;1049-60

  • Molecular structure of the human desmoplakin I and II amino terminus.

    Virata ML, Wagner RM, Parry DA and Green KJ

    Department of Pathology, Northwestern University Medical School, Chicago, IL 60611.

    Desmoplakins (DPs) I and II are closely related proteins found in the innermost region of the desmosomal plaque, which serves as a cell surface attachment site for cytoplasmic intermediate filaments. Overlapping cDNA clones comprising 9.2 kilobases of DP-I, predicted to encode a full-length 310-kDa polypeptide (2677 amino acid residues), have now been identified. Here we report the predicted protein sequence and structural analysis of the N terminus of DP, extending our previous study of the rod and carboxyl domains. The N terminus contains groups of heptad repeats that are predicted to form at least two major alpha-helical-rich bundles. Unlike the rod and carboxyl domains, the N terminus did not display a periodic distribution of charged residues. Northern blot mapping and genomic sequence analysis were also undertaken to examine the organization of the DP mRNAs. A 1-kilobase intron was located at the 3' boundary of a DP-I-specific region; however, instead of an intron at the 5' junction, a possible splice donor site was observed within a potential coding sequence, suggesting alternative RNA splicing from an internal donor site.

    Funded by: NICHD NIH HHS: HD24430

    Proceedings of the National Academy of Sciences of the United States of America 1992;89;2;544-8

  • Undulin is a novel member of the fibronectin-tenascin family of extracellular matrix glycoproteins.

    Just M, Herbst H, Hummel M, Dürkop H, Tripier D, Stein H and Schuppan D

    Institut für Pathologie, Klinikum Steglitz, Freie Universität Berlin, Federal Republic of Germany.

    We characterized cDNA clones specific for the extracellular matrix glycoprotein undulin. Two sets of cDNA clones were isolated from a human placental lambda gt11 expression library and from a rhabdomyosarcoma cell line encoding two partially identical carboxyl-terminal polypeptides of 843 (Un1) and 443 (Un2) amino acids suggesting differential splicing of a single gene transcript. Northern blot analysis of human rhabdomyosarcoma cell poly (A) RNA with cDNA specific for Un1 identified transcripts of approximately 4.2, 6.5, and 8.5 kilobases, whereas a probe specific for Un2 detected a single mRNA of approximately 5 kilobases. Since a monoclonal antibody that is reactive with a sequence encoded by Un1 and not by Un2 detects the bands considered characteristic for undulin in Western blots, the mRNAs related to Un1 may code for the major part of the undulin molecule. The protein sequences deduced from Un1 and Un2 reveal an amino-terminal differentially spliced von Willebrand factor A domain, characteristic of proteins that interact with interstitial collagens, which is linked to fibronectin-like type III homology units by a unique sequence of 57 amino acids. Whereas Un2 encodes two complete and one incomplete type III homologies followed by a unique acidic carboxyl-terminal domain of 118 amino acids, Un1 codes for seven complete and one truncated type III homologies, followed by a short proline-rich carboxyl-terminal segment of 23 amino acids. Considering the 298 amino acids occurring in identical segments, the 989 different amino acid positions deduced from clones Un1 and Un2 represent an estimated 40% of the overall undulin sequence. In the context of 1) rotary shadowing electron microscopy data showing undulin as a structure composed of nodules that are interconnected by flexible rods of varying size, 2) the presence of three major bands of Mr 270,000, 190,000, and 180,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with 3) common antigenic epitopes and similar peptide maps (Schuppan, D., Cantaluppi, M.C., Becker, J., Veit, A., Bunte, T., Troyer, D., Schuppan, F., Schmid, M., Ackermann, R., and Hahn, E.G. (1990) J. Biol. Chem. 265, 8823-8832), our finding of differentially spliced type III homology units, as found in tenascin and fibronectin, suggests that undulin is another member of the fibronectin-tenascin family of extracellular matrix glycoproteins. Furthermore, as in fibronectin and tenascin, undulin bears an additional subset of interactive domains tailored to specific structural and functional roles in development and differentiation.

    The Journal of biological chemistry 1991;266;26;17326-32

  • Chromosomal assignment of the human genes coding for the major proteins of the desmosome junction, desmoglein DGI (DSG), desmocollins DGII/III (DSC), desmoplakins DPI/II (DSP), and plakoglobin DPIII (JUP).

    Arnemann J, Spurr NK, Wheeler GN, Parker AE and Buxton RS

    Laboratory of Eukaryotic Molecular Genetics, National Institute for Medical Research, London, United Kingdom.

    We have established PCR assays for the genes coding for the major proteins of the desmosome type of cell junction, the desmosomal cadherins DGI (desmoglein) and DGII/III (desmocollins), and the plaque proteins DPI/II (desmoplakin) and DPIII (plakoglobin) and used them to test human-mouse and human-rat somatic cell hybrids with different contents of human chromosomes. From these data we were able to assign DGI to chromosome 18 (DSG), DGII/III to chromosome 9p (DSC), DPI/II to chromosome 6p21-ter(DSP), and DPIII to chromosome 7 (JUP).

    Genomics 1991;10;3;640-5

  • Paraneoplastic pemphigus. An autoimmune mucocutaneous disease associated with neoplasia.

    Anhalt GJ, Kim SC, Stanley JR, Korman NJ, Jabs DA, Kory M, Izumi H, Ratrie H, Mutasim D, Ariss-Abdo L et al.

    Department of Dermatology, Johns Hopkins University, School of Medicine, Baltimore, MD 21205.

    We describe five patients with underlying neoplasms in whom painful mucosal ulcerations and polymorphous skin lesions developed, usually with progression to blistering eruptions on the trunk and extremities. Histologic examination showed vacuolization of epidermal basal cells, keratinocyte necrosis, and acantholysis. Immunofluorescence testing revealed atypical pemphigus-like autoantibodies in perilesional epithelium and serum from all five patients. We studied the antigenic specificities of the autoantibodies by indirect immunofluorescence and immunoprecipitation, using extracts of 14C-labeled human keratinocytes. IgG purified from the serum of one patient was passively transferred to four neonatal mice to test for pathogenicity.

    Results: Immunofluorescence testing showed that the autoantibodies bound to the surface of tissues containing desmosomes, including complex and simple epithelia, and myocardium. An identical and unique complex of four polypeptides with molecular weights of 250, 230, 210, and 190 was immunoprecipitated by all serum samples. The 250-kd polypeptide comigrated with desmoplakin I (a protein found in the desmosomes of all epithelia), and the 230-kd antigen comigrated with the antigen of bullous pemphigoid. Cutaneous blisters, a positive Nikolsky's sign, and epidermal and esophageal acantholysis developed in all mice into which the autoantibody was injected. Electron microscopy showed epidermal acantholysis similar to lesions of experimentally induced pemphigus vulgaris.

    Conclusion: These five patients with cancer had a novel acantholytic mucocutaneous disease characterized by autoantibodies that were pathogenic after passive transfer. The autoantibodies from these patients reacted with an antigen complex composed of desmoplakin I and the 230-kd antigen of bullous pemphigoid and two as yet unidentified epithelial antigens. We suggest the term "paraneoplastic pemphigus" for this disease.

    Funded by: NIAMS NIH HHS: KO4-AR-01686, R01-AR-32490

    The New England journal of medicine 1990;323;25;1729-35

  • Structure of the human desmoplakins. Implications for function in the desmosomal plaque.

    Green KJ, Parry DA, Steinert PM, Virata ML, Wagner RM, Angst BD and Nilles LA

    The Journal of biological chemistry 1990;265;19;11406-7

  • Structure of the human desmoplakins. Implications for function in the desmosomal plaque.

    Green KJ, Parry DA, Steinert PM, Virata ML, Wagner RM, Angst BD and Nilles LA

    Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611.

    Desmoplakins (DPs) I and II are two major related proteins located in the innermost portion of the desmosomal plaque where it is thought they may play a role in attaching intermediate filaments (IF) to the cell surface. We have isolated and sequenced human cDNA clones encoding two major DP domains and a portion of a third. These clones can be divided into two classes that we believe to represent DPI and DPII cDNAs; our evidence suggests that the DPII message is derived at least in part from the processing of a larger transcript encoded by a single gene. Computer-aided analysis of the DPI-predicted amino acid sequence indicates that the central domain, which contains the heptad repeat characteristic of many alpha-fibrous proteins, will participate in the formation of a coiled coil dimer approximately 130 nm in length. The periodicity of acidic and basic residues in the rod suggests that DPI will aggregate with itself or similar molecules into higher order filamentous structures. The carboxyl terminus contains three regions with significant homology, each of which comprises almost five repeats of a 38-residue motif. It is likely that these regions each fold into a compact globular conformation stabilized by intrachain ionic interactions. Comparison of the predicted amino acid sequence of a cDNA encoding a portion of the 230-kDa bullous pemphigoid antigen (Stanley, J. R., Tanaka, T., Mueller, S., Klaus-Kovtun, V., and Roop, D. (1988) J. Clin. Invest. 82, 1864-1870) with DP revealed the presence of a 38-residue repeat with striking similarity to that of the DPs. Significantly, the periodicity in acidic and basic residues of these domains is the same as that found in the 1B rod domain of IF proteins. This suggests the possibility that the DPs might interact with IF via their common periodicity of charged residues.

    Funded by: NICHD NIH HHS: HD24430

    The Journal of biological chemistry 1990;265;5;2603-12

Gene lists (4)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000014 G2C Homo sapiens Human ARC Human orthologues of mouse AMPA receptor complex adapted from Collins et al (2006) 9
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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