G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
bridging integrator 1
G00000676 (Mus musculus)

Databases (8)

ENSG00000136717 (Ensembl human gene)
274 (Entrez Gene)
1081 (G2Cdb plasticity & disease)
BIN1 (GeneCards)
601248 (OMIM)
Marker Symbol
HGNC:1052 (HGNC)
Protein Expression
1945 (human protein atlas)
Protein Sequence
O00499 (UniProt)

Synonyms (2)

  • AMPH2
  • SH3P9

Literature (75)

Pubmed - other

  • Phenotype of a patient with recessive centronuclear myopathy and a novel BIN1 mutation.

    Claeys KG, Maisonobe T, Böhm J, Laporte J, Hezode M, Romero NB, Brochier G, Bitoun M, Carlier RY and Stojkovic T

    Groupe Hospitalier Pitié-Salpêtrière, Institut de Myologie, Unité de Morphologie Neuromusculaire (Pavillon Risler), 47-83, Boulevard de l'Hôpital, 75651 Paris Cedex 13, France. k.claeys@institut-myologie.org

    Neurology 2010;74;6;519-21

  • Assessment of a polymorphism of SDK1 with hypertension in Japanese Individuals.

    Oguri M, Kato K, Yokoi K, Yoshida T, Watanabe S, Metoki N, Yoshida H, Satoh K, Aoyagi Y, Nozawa Y and Yamada Y

    Department of Cardiology, Japanese Red Cross Nagoya First Hospital, Nagoya, Japan.

    Background: Hypertension is a major risk factor for cardiovascular disease. Although genetic studies have suggested that several genetic variants increase the risk for hypertension, the genes that underlie genetic susceptibility to this condition remain to be identified definitively. The purpose of the present study was to identify genetic variants that confer susceptibility to hypertension in Japanese individuals.

    Methods: A total of 5,734 Japanese individuals from two independent populations were examined: subject panel A comprised 2,066 hypertensive individuals and 824 controls; and subject panel B comprised 834 hypertensive individuals and 2,010 controls. The 150 polymorphisms examined in the present study were selected by genome-wide association studies of myocardial infarction and ischemic stroke with the use of the GeneChip Human Mapping 500K Array Set (Affymetrix).

    Results: The chi(2)-test revealed that 10 polymorphisms were significantly (P < 0.05) related to the prevalence of hypertension in subject panel A. To validate the relations, these polymorphisms were examined in subject panel B. The A-->G polymorphism (rs645106) of SDK1 and the C-->G polymorphism (rs12078839) of RABGAP1L were significantly associated with hypertension in subject panel B. Multivariable logistic regression analysis with adjustment for covariates, as well as a stepwise forward selection procedure revealed that the A-->G polymorphism of SDK1 was significantly associated with hypertension in both subject panels A and B, with the G allele protecting against this condition.

    Conclusions: SDK1 may be a susceptibility gene for hypertension in Japanese individuals, although the functional relevance of the identified polymorphism was not determined.

    American journal of hypertension 2010;23;1;70-7

  • The c-MYC-interacting proapoptotic tumor suppressor BIN1 is a transcriptional target for E2F1 in response to DNA damage.

    Cassimere EK, Pyndiah S and Sakamuro D

    Division of Cancer Biology, Department of Pathology, School of Medicine and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA, USA.

    The E2F1 transcription factor, which was originally identified as a cell-cycle initiator, mediates apoptosis in response to DNA damage. As E2F1-induced apoptosis is an attractive mechanism for cancer therapy, it is critical to fully elucidate its effector pathways. Here, we show that the c-MYC-interacting proapoptotic tumor suppressor, BIN1, is transcriptionally activated by E2F1 and mediates E2F1-induced apoptosis in response to DNA damage. Acting through the DNA-binding and transactivation domains, ectopically expressed E2F1 activated the human BIN1 promoter, which contains canonical E2F-recognition sites. Conversely, depletion of E2F1 by small interfering RNA or germline deletion led to BIN1 deficiency. DNA-damaging agents (which included etoposide) increased BIN1 levels, unless E2F1 was deficient. Moreover, endogenous E2F1 protein interacted directly with the BIN1 gene promoter in chromatin, particularly after etoposide treatment. Notably, suppression of BIN1 expression using an antisense (AS) technique attenuated the cell death mediated by E2F1 and etoposide. Although the p53 tumor suppressor, its sibling protein p73, and caspases are well-known E2F1 effectors for DNA damage-induced apoptosis, AS-BIN1 did not compromise their apoptotic functions. Our results collectively suggest that BIN1 is a novel transcriptional target of E2F1 that triggers a unique mode of cell death in response to DNA damage.

    Funded by: NCI NIH HHS: F31 CA110205

    Cell death and differentiation 2009;16;12;1641-53

  • AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling.

    Pant S, Sharma M, Patel K, Caplan S, Carr CM and Grant BD

    Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA.

    RME-1/EHD1 (receptor mediated endocytosis/Eps15 homology-domain containing 1) family proteins are key residents of the recycling endosome, which are required for endosome-to-plasma membrane transport in Caenorhabditis elegans and mammals. Recent studies suggest similarities between the RME-1/EHD proteins and the Dynamin GTPase superfamily of mechanochemical pinchases, which promote membrane fission. Here we show that endogenous C. elegans AMPH-1, the only C. elegans member of the Amphiphysin/BIN1 family of BAR (Bin1-Amphiphysin-Rvs161p/167p)-domain-containing proteins, colocalizes with RME-1 on recycling endosomes in vivo, that amph-1-deletion mutants are defective in recycling endosome morphology and function, and that binding of AMPH-1 Asn-Pro-Phe(Asp/Glu) sequences to the RME-1 EH-domain promotes the recycling of transmembrane cargo. We also show a requirement for human BIN1 (also known as Amphiphysin 2) in EHD1-regulated endocytic recycling. In vitro, we find that purified recombinant AMPH-1-RME-1 complexes produce short, coated membrane tubules that are qualitatively distinct from those produced by either protein alone. Our results indicate that AMPH-1 and RME-1 cooperatively regulate endocytic recycling, probably through functions required for the production of cargo carriers that exit the recycling endosome for the cell surface.

    Funded by: NCRR NIH HHS: R24 RR022234, R24RR22234; NIGMS NIH HHS: GM074876, GM67237, R01 GM067237, R01 GM067237-07, R01 GM074876

    Nature cell biology 2009;11;12;1399-410

  • Bin1 is linked to metastatic potential and chemosensitivity in neuroblastoma.

    Zhong X, Hoelz DJ, Kumar HR, Sandoval JA, Rescorla FJ, Hickey RJ and Malkas LH

    Department of Surgery, Section of Pediatric Surgery, Riley Children's Hospital, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA. xzhong@iupui.edu

    Background: Neuroblastoma (NB) is the most common extracranial solid tumor in children. At the time of diagnosis, the tumor has metastasized in as many as 7 of 10 cases, and survival in high-risk patients remains poor. Accurate classification of high-risk patients is very important since this determines treatment plan, and although a consensus risk classification system has been established for NB, it contains few specific molecular markers that account for aggressive nature and metastatic potential of the tumor. Bin1 expression is reduced in breast, NB, and other cancer types and the reduction correlates with high-risk clinical features. Here we hypothesize that Bin1 has an inhibitory role in metastasis, and therefore decrease in its expression may be a marker of high-risk NB.

    Procedure: Initially, breast cancer and NB cell lines derived from metastasis were examined for Bin1 expression. Then, a stable Bin1-overexpressing NB cell line was created and evaluated for in vitro metastatic behaviors using anoikis, invasion, and migration assays, and chemoresponsiveness using MTT assay.

    Results: Reduced Bin1 was detected in all cancer cell lines examined, and forced Bin1 overexpression increased NB cell anoikis and enhanced the cell killing by doxorubicin. However, Bin1 overexpression did not significantly affect cell invasion, motility, or proliferation.

    Conclusions: Bin1 appears to function as a metastasis suppressor and chemosensitizer in NB, and resistance to anoikis may be an important metastatic mechanism. Thus, Bin1 expression status could serve as a marker for metastatic potential and chemosensitivity thereby allowing for more accurate classifications of high-risk NB patients.

    Pediatric blood & cancer 2009;53;3;332-7

  • The membrane-tubulating potential of amphiphysin 2/BIN1 is dependent on the microtubule-binding cytoplasmic linker protein 170 (CLIP-170).

    Meunier B, Quaranta M, Daviet L, Hatzoglou A and Leprince C

    Analysis of Signal Transduction Group, INSERM U830, Institut Curie, Paris, France.

    Amphiphysins are BIN-amphiphysin-RVS (BAR) domain-containing proteins that influence membrane curvature in sites such as T-tubules in muscular cells, endocytic pits in neuronal as well as non-neuronal cells, and possibly cytoplasmic endosomes. This effect on lipid membranes is fulfilled by diverse amphiphysin 2/BIN1 isoforms, generated by alternative splicing and showing distinct structural and functional properties. In this study, our goal was to characterize the functional role of a ubiquitously expressed amphiphysin 2/BIN1 by the characterization of new molecular partners. We performed a two-hybrid screen with an isoform of amphiphysin 2/BIN1 expressed in HeLa cells. We identified CLIP-170 as an amphiphysin 2/BIN1-interacting molecule. CLIP-170 is a plus-end tracking protein involved in microtubule (MT) stability and recruitment of dynactin. The binding between amphiphysin 2/BIN1 and CLIP-170 is dependent on the N-terminal part of amphiphysin 2 (mostly the BAR domain) and an internal coiled-coil region of CLIP-170. This partnership was confirmed by GST pull-down assay and by co-immunoprecipitation in HeLa cells that express endogenous amphiphysin 2 (mostly isoforms 6, 9 and 10). When overexpressed in HeLa cells, amphiphysin 2/BIN1 leads to the formation of intracellular tubules which can closely align with MTs. After MT depolymerization by nocodazole, amphiphysin 2-stained tubules disappear, and reappear after nocodazole washout. Furthermore, depletion of CLIP-170 by RNAi induced a decrease in the proportion of cells with amphiphysin 2-stained tubules and an increase in the proportion of cells with no tubules. This result suggests the existence of a mechanistic link between the two types of tubules, which is likely to involve the +TIP protein, CLIP-170. Amphiphysin 2/BIN1 may be an anchoring point on membranes for CLIP-170, and consequently for MT. Then, the pushing force of polymerizing MT could help amphiphysin 2/BIN1 in its tubulation potential. We propose that amphiphysin 2/BIN1 participates in the tubulation of traffic intermediates and intracellular organelles first via its intrinsic tubulating potential and second via its ability to bind CLIP-170 and MT.

    European journal of cell biology 2009;88;2;91-102

  • Structure and dynamics of helix-0 of the N-BAR domain in lipid micelles and bilayers.

    Löw C, Weininger U, Lee H, Schweimer K, Neundorf I, Beck-Sickinger AG, Pastor RW and Balbach J

    Institut für Physik, Biophysik, Martin-Luther-Universität Halle-Wittenberg, D-06120 Halle (Saale), Germany.

    Bin/Amphiphysin/Rvs-homology (BAR) domains generate and sense membrane curvature by binding the negatively charged membrane to their positively charged concave surfaces. N-BAR domains contain an N-terminal extension (helix-0) predicted to form an amphipathic helix upon membrane binding. We determined the NMR structure and nano-to-picosecond dynamics of helix-0 of the human Bin1/Amphiphysin II BAR domain in sodium dodecyl sulfate and dodecylphosphocholine micelles. Molecular dynamics simulations of this 34-amino acid peptide revealed electrostatic and hydrophobic interactions with the detergent molecules that induce helical structure formation from residues 8-10 toward the C-terminus. The orientation in the micelles was experimentally confirmed by backbone amide proton exchange. The simulation and the experiment indicated that the N-terminal region is disordered, and the peptide curves to adopted the micelle shape. Deletion of helix-0 reduced tubulation of liposomes by the BAR domain, whereas the helix-0 peptide itself was fusogenic. These findings support models for membrane curving by BAR domains in which helix-0 increases the binding affinity to the membrane and enhances curvature generation.

    Biophysical journal 2008;95;9;4315-23

  • Adenovirus E1A oncoprotein liberates c-Myc activity to promote cell proliferation through abating Bin1 expression via an Rb/E2F1-dependent mechanism.

    Kinney EL, Tanida S, Rodrigue AA, Johnson JK, Tompkins VS and Sakamuro D

    Division of Cancer Biology, Department of Pathology, School of Medicine and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.

    Adenovirus E1A oncogene transforms primary rodent fibroblasts in cooperation with activated Ras. Conversely, the c-Myc oncoprotein-binding tumor suppressor, Bin1, inhibits Ras/E1A-mediated cell transformation. Since E1A does not directly bind to and inhibit Bin1, the primary mechanism by which E1A counteracts Bin1 to liberate oncogenic c-Myc activity is poorly understood. Here we show that wild-type E1A, but not an Rb binding-defective E1A mutant, suppresses endogenous Bin1 expression in cultured rodent fibroblasts. Similarly, other anti-Rb agents, such as human papillomavirus E7, mitogenic stimuli, and small interfering RNA (siRNA) for Rb, consistently decrease Bin1 promoter activity. In contrast, serum starvation, which activates Rb, enhances endogenous Bin1 levels. These findings suggest that Bin1 may be a novel component of Rb-mediated G1 arrest. Consistent with this premise, chromatin immunoprecipitation assays demonstrate that Rb protein directly interacts with the Bin1 promoter only upon removal of serum. Furthermore, ectopically expressed E2F1, which is primarily inhibited by Rb under serum-starved condition, represses Bin1 promoter activity in a manner that is dependent on the DNA-binding and transactivation domains of E2F1. Lastly, depletion of endogenous Bin1 per se is biologically meaningful since antisense or siRNA of Bin1 transfection releases endogenous c-Myc transcriptional activity and, concomitantly, accelerates cell proliferation. Our results suggest that Bin1 gene suppression caused by oncogenic E1A via Rb inactivation is an essential step in cell cycle progression promoted by c-Myc, and subsequently, E1A transformation. We propose a novel G1 arrest signaling mechanism by which Rb indirectly curbs oncogenic c-Myc activity via sustaining Bin1 expression.

    Journal of cellular physiology 2008;216;3;621-31

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • [Mutations in amphiphysin 2 (BIN1) cause autosomal recessive centronuclear myopathy].

    Toussaint A, Nicot AS, Mandel JL and Laporte J

    Medecine sciences : M/S 2007;23;12;1080-2

  • Mutations in amphiphysin 2 (BIN1) disrupt interaction with dynamin 2 and cause autosomal recessive centronuclear myopathy.

    Nicot AS, Toussaint A, Tosch V, Kretz C, Wallgren-Pettersson C, Iwarsson E, Kingston H, Garnier JM, Biancalana V, Oldfors A, Mandel JL and Laporte J

    Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Department of Molecular Pathology, F-67400 Illkirch, France.

    Centronuclear myopathies are characterized by muscle weakness and abnormal centralization of nuclei in muscle fibers not secondary to regeneration. The severe neonatal X-linked form (myotubular myopathy) is due to mutations in the phosphoinositide phosphatase myotubularin (MTM1), whereas mutations in dynamin 2 (DNM2) have been found in some autosomal dominant cases. By direct sequencing of functional candidate genes, we identified homozygous mutations in amphiphysin 2 (BIN1) in three families with autosomal recessive inheritance. Two missense mutations affecting the BAR (Bin1/amphiphysin/RVS167) domain disrupt its membrane tubulation properties in transfected cells, and a partial truncation of the C-terminal SH3 domain abrogates the interaction with DNM2 and its recruitment to the membrane tubules. Our results suggest that mutations in BIN1 cause centronuclear myopathy by interfering with remodeling of T tubules and/or endocytic membranes, and that the functional interaction between BIN1 and DNM2 is necessary for normal muscle function and positioning of nuclei.

    Nature genetics 2007;39;9;1134-9

  • Bin1 ablation increases susceptibility to cancer during aging, particularly lung cancer.

    Chang MY, Boulden J, Katz JB, Wang L, Meyer TJ, Soler AP, Muller AJ and Prendergast GC

    Lankenau Institute for Medical Research, Lankenau Hospital, Wynnewood, Pennsylvania 19096, USA.

    Age is the major risk factor for cancer, but few genetic pathways that modify cancer incidence during aging have been described. Bin1 is a prototypic member of the BAR adapter gene family that functions in vesicle dynamics and nuclear processes. Bin1 limits oncogenesis and is often attenuated in human cancers, but its role in cancer suppression has yet to be evaluated fully in vivo. In the mouse, homozygous deletion of Bin1 causes developmental lethality, so to assess this role, we examined cancer incidence in mosaic null mice generated by a modified Cre-lox technology. During study of these animals, one notable phenotype was an extended period of female fecundity during aging, with mosaic null animals retaining reproductive capability until the age of 17.3 +/- 1.1 months. Through 1 year of age, cancer incidence was unaffected by Bin1 ablation; however, by 18 to 20 months of age, approximately 50% of mosaic mice presented with lung adenocarcinoma and approximately 10% with hepatocarcinoma. Aging mosaic mice also displayed a higher incidence of inflammation and/or premalignant lesions, especially in the heart and prostate. In mice where colon tumors were initiated by a ras-activating carcinogen, Bin1 ablation facilitated progression to more aggressive invasive status. In cases of human lung and colon cancers, immunohistochemical analyses evidenced frequent attenuation of Bin1 expression, paralleling observations in other solid tumors. Taken together, our findings highlight an important role for Bin1 as a negative modifier of inflammation and cancer susceptibility during aging.

    Funded by: NCI NIH HHS: CA100123, CA10954, CA82222, R01 CA100123

    Cancer research 2007;67;16;7605-12

  • Bin1 interacts with and restrains the DNA end-binding protein complex Ku.

    Ramalingam A, Farmer GE, Stamato TD and Prendergast GC

    Lankenau Institute for Medical Research, Wynnewood, Pennsylvania 19096, USA.

    The Bin1 gene encodes a BAR adapter protein that suppresses cancer by poorly defined mechanisms. In an effort to gain insights, we identified cellular proteins that form biochemical complexes with Bin1 protein. Here we report that Bin1 physically binds to Ku, a DNA end-binding protein that functions in telomere maintenance, apoptosis, and DNA repair. Both Ku70 and Ku80 were purified from human and murine cell extracts using the Bin1 BAR domain as an affinity matrix. A BAR domain mutation that destroys antioncogenic activity completely abolished Ku binding, supporting functional relevance. To further evaluate meaning, we investigated interactions between the Bin1 homolog hob1+ and the Ku homologs pku70+ and pku80+ in fission yeast. Notably, deleting pku70+ or pku80+ relieved the survival defect displayed by hob1delta cells after treatment with the DNA damaging agent phleomycin, suggesting that hob1+ may restrain Ku. Consistent with this notion, telomere length was altered in hob1delta cells. The potential relevance of Bin1-Ku interaction to cancer are discussed in light of these findings.

    Funded by: NCI NIH HHS: CA82222, R01 CA100123, R01 CA109542

    Cell cycle (Georgetown, Tex.) 2007;6;15;1914-8

  • Bin1 homolog hob1 supports a Rad6-Set1 pathway of transcriptional repression in fission yeast.

    Ramalingam A and Prendergast GC

    Lankenau Institute for Medical Research, Wynnewood, Pennsylvania 19096, USA.

    Bin1 encodes a mammalian BAR adapter protein with a nuclear anti-oncogenic function that is poorly understood. To gain functional insights, we investigated the role of the fission yeast homolog hob1+ in growth arrest and survival of cells treated with phleomycin, a DNA damaging drug. Unlike wild-type cells, hob1delta cells treated with phleomycin displayed a defective growth arrest phenotype, elongating abnormally without septation or cytokinesis and eventually losing viability. Genetic investigations suggested that the survival defect in hob1delta cells reflected a deficiency in a Rad6 pathway involving histone methyltransferase Set1 that leads to transcriptional repression. In support of this connection, transcription of telomeric and centromeric heterochromatin that is normally silenced by a Rad6/Set1-dependent mechanism was aberrantly activated in hob1delta cells. Taken together, these findings suggest that hob1+ may support a mechanism of transcriptional repression possibly relevant to the role of Bin1 in cancer suppression.

    Funded by: NCI NIH HHS: R01 CA100123, R01 CA109542, R01 CA82222

    Cell cycle (Georgetown, Tex.) 2007;6;13;1655-62

  • Systematic identification of SH3 domain-mediated human protein-protein interactions by peptide array target screening.

    Wu C, Ma MH, Brown KR, Geisler M, Li L, Tzeng E, Jia CY, Jurisica I and Li SS

    Department of Biochemistry and the Siebens-Drake Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

    Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCgamma1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCgamma1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCgamma1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.

    Proteomics 2007;7;11;1775-85

  • Bin1 attenuation in breast cancer is correlated to nodal metastasis and reduced survival.

    Ghaneie A, Zemba-Palko V, Itoh H, Itoh K, Sakamuro D, Nakamura S, Soler AP and Prendergast GC

    Lankenau Cancer Center and Departments of Hematology/Oncology, Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096, USA.

    Clinical outcomes in breast cancer are likely influenced by modifier genes that affect tumor dormancy versus progression. The Bin1 gene encodes a nucleocytosolic adapter protein that suppresses neoplastic cell transformation and that is often attenuated in human breast carcinoma. Recent mouse genetic studies indicate that Bin1 loss cooperates with ras activation to drive progression of mammary carcinoma, establishing Bin1 as a negative modifier of tumor progression in breast cancer. In this study, we investigated whether immunohistochemical losses of nuclear Bin1 proteins in cases of human breast cancer were correlated to progression status. In American and Japanese groups of low or middle grade breast cancers, losses were associated with reduced survival and increased nodal metastasis, respectively. Taken together with recent findings from mouse genetic studies, these findings encourage further evaluation of the potential utility of Bin1 as a clinical prognostic marker in breast cancer.

    Funded by: NCI NIH HHS: CA 100123, CA 109542, CA 82222, R01 CA100123

    Cancer biology & therapy 2007;6;2;192-4

  • Bin1 ablation in mammary gland delays tissue remodeling and drives cancer progression.

    Chang MY, Boulden J, Sutanto-Ward E, Duhadaway JB, Soler AP, Muller AJ and Prendergast GC

    Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096, USA.

    Genes that modify oncogenesis may influence dormancy versus progression in cancer, thereby affecting clinical outcomes. The Bin1 gene encodes a nucleocytosolic adapter protein that interacts with and suppresses the cell transforming activity of Myc. Bin1 is often attenuated in breast cancer but its ability to negatively modify oncogenesis or progression in this context has not been gauged directly. In this study, we investigated the effects of mammary gland-specific deletion of Bin1 on initiation and progression of breast cancer in mice. Bin1 loss delayed the outgrowth and involution of the glandular ductal network during pregnancy but had no effect on tumor susceptibility. In contrast, in mice where tumors were initiated by the ras-activating carcinogen 7,12-dimethylbenz(a)anthracene, Bin1 loss strongly accentuated the formation of poorly differentiated tumors characterized by increased proliferation, survival, and motility. This effect was specific as Bin1 loss did not accentuate progression of tumors initiated by an overexpressed mouse mammary tumor virus-c-myc transgene, which on its own produced poorly differentiated and aggressive tumors. These findings suggest that Bin1 loss cooperates with ras activation to drive progression, establishing a role for Bin1 as a negative modifier of oncogenicity and progression in breast cancer.

    Funded by: NCI NIH HHS: CA 100123, R01 CA100123

    Cancer research 2007;67;1;100-7

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • The crystal structure of the BAR domain from human Bin1/amphiphysin II and its implications for molecular recognition.

    Casal E, Federici L, Zhang W, Fernandez-Recio J, Priego EM, Miguel RN, DuHadaway JB, Prendergast GC, Luisi BF and Laue ED

    Department of Biochemistry, University of Cambridge, UK.

    BAR domains are found in proteins that bind and remodel membranes and participate in cytoskeletal and nuclear processes. Here, we report the crystal structure of the BAR domain from the human Bin1 protein at 2.0 A resolution. Both the quaternary and tertiary architectures of the homodimeric Bin1BAR domain are built upon "knobs-into-holes" packing of side chains, like those found in conventional left-handed coiled-coils, and this packing governs the curvature of a putative membrane-engaging concave face. Our calculations indicate that the Bin1BAR domain contains two potential sites for protein-protein interactions on the convex face of the dimer. Comparative analysis of structural features reveals that at least three architectural subtypes of the BAR domain are encoded in the human genome, represented by the Arfaptin, Bin1/Amphiphysin, and IRSp53 BAR domains. We discuss how these principal groups may differ in their potential to form regulatory heterotypic interactions.

    Funded by: NCI NIH HHS: R01 CA100123, R01 CA100123-03; Wellcome Trust

    Biochemistry 2006;45;43;12917-28

  • The SH3 binding motif of HCV [corrected] NS5A protein interacts with Bin1 and is important for apoptosis and infectivity.

    Nanda SK, Herion D and Liang TJ

    Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

    HCV nonstructural protein 5A (NS5A) has been implicated in regulating cell growth and interferon response. The NS5A protein contains proline-rich regions that are highly conserved among HCV genotypes and match Src homology 3 (SH3)-binding motifs (PxxP) found in various cellular signaling molecules.

    Methods: We screened for HCV NS5A interacting proteins by using the yeast 2-hybrid system and studied the functional consequence of this interaction.

    Results: Several independent clones containing SH3 domains were isolated along with Bin1, a tumor suppressor with pro-apoptotic properties, being the most frequently identified clone. The protein-protein interaction between NS5A and Bin1 was confirmed by in vitro binding, in vivo co-immunoprecipitation, and confocal microscopy. Deletion and mutation analyses indicated that the SH3 binding motif of HCV NS5A and SH3 domain of Bin1 are essential for interaction. Human hepatoma (HepG2) cells lacking expression of Bin1 undergo apoptosis upon infection with adeno-Bin1. Bin1-induced apoptosis was inhibited in HepG2 cells expressing wild-type NS5A but not NS5A mutant with mutations in the SH3 binding motif. Infectious HCV genome containing mutations in the SH3 binding motif was not infectious in chimpanzees.

    Conclusions: Our results indicate that this interaction is implicated in productive HCV infection and may contribute to the pathogenesis of hepatocellular carcinoma. In addition, the NS5A PxxP motif may represent a novel target for antiviral development.

    Funded by: Intramural NIH HHS

    Gastroenterology 2006;130;3;794-809

  • Biological diagnosis for neuroblastoma using the combination of highly sensitive analysis of prognostic factors.

    Tajiri T, Tanaka S, Higashi M, Kinoshita Y, Takahashi Y, Tatsuta K and Suita S

    Department of Pediatric Surgery, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan. taji@pedsurg.med.kyusyu-u.ac.jp

    To select the optimal treatment according to the degree of malignancy of neuroblastoma, it is essential to accurately and rapidly identify any genetic abnormalities associated with the prognosis. This study aims to assess the correlation between the combination of prognostic factors and the biologic findings of neuroblastoma using a highly sensitive analysis of prognostic factors.

    Methods: In 44 neuroblastoma primary samples, we determined the gene dosages of MYCN and Survivin (as the target of 17q gain) and the expression levels of MYCN, Survivin, and BIN1 using highly sensitive analysis (the quantitative polymerase chain reaction method); furthermore, we assessed the correlation between the combination of their prognostic factors and the biology of neuroblastoma.

    Results: The gene dosage of MYCN or Survivin was significantly associated with all known prognostic factors. The expression level of MYCN or Survivin was not significantly associated with any prognostic factors, whereas the expression level of BIN1 was significantly associated with 5 of 6 prognostic factors. Regarding the combination of MYCN amplification and 17q gain (the gene dosage of Survivin), and the low expression of BIN1, the rates of advanced stages (stage III or IV) were 100% for the cases with 3 factors, 63% for the cases with 2 factors, 42% for the cases with 1 factor, and 0% for the cases with null factor. Furthermore, the survival rates were 20% for the cases with 3 factors, 50% for the cases with 2 factors, 100% for the cases with 1 factor, and 100% for the cases with null factor.

    Conclusion: The combination of gene dosages of MYCN and Survivin and the expression level of BIN1 using the quantitative polymerase chain reaction method was significantly correlated with the clinical stage and the patients' outcome. This combination of biologic factors may enhance the accuracy to the conventional criteria, but this would have to be shown in a much larger study that is adequately powered to detect such an advantage.

    Journal of pediatric surgery 2006;41;3;560-6

  • Identification of VCP/p97, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners using membrane-based human proteome arrays.

    Grelle G, Kostka S, Otto A, Kersten B, Genser KF, Müller EC, Wälter S, Böddrich A, Stelzl U, Hänig C, Volkmer-Engert R, Landgraf C, Alberti S, Höhfeld J, Strödicke M and Wanker EE

    Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Strasse 10, D-13125 Berlin-Buch, Germany.

    Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts. Many novel interaction pairs such as valosin-containing protein/autocrine motility factor receptor, CHIP/caytaxin, or amphiphysin II/DLP4 were identified and subsequently confirmed by pull-down, two-hybrid and co-immunoprecipitation experiments. In addition, assays were performed to validate the interactions functionally. CHIP e.g. was found to efficiently polyubiquitinate caytaxin in vitro, suggesting that it might influence caytaxin degradation in vivo. Using peptide arrays, we also identified the binding motifs in the proteins DLP4, XRCC4, and fructose-1,6-bisphosphatase, which are crucial for the association with the Src homology 3 domain of amphiphysin II. Together these studies indicate that our human proteome array technology permits the identification of protein-protein interactions that are functionally involved in neurodegenerative disease processes, the degradation of protein substrates, and the transport of membrane vesicles.

    Molecular & cellular proteomics : MCP 2006;5;2;234-44

  • The LIFEdb database in 2006.

    Mehrle A, Rosenfelder H, Schupp I, del Val C, Arlt D, Hahne F, Bechtel S, Simpson J, Hofmann O, Hide W, Glatting KH, Huber W, Pepperkok R, Poustka A and Wiemann S

    Division Molecular Genome Analysis, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany. a.mehrle@dkfz.de

    LIFEdb (http://www.LIFEdb.de) integrates data from large-scale functional genomics assays and manual cDNA annotation with bioinformatics gene expression and protein analysis. New features of LIFEdb include (i) an updated user interface with enhanced query capabilities, (ii) a configurable output table and the option to download search results in XML, (iii) the integration of data from cell-based screening assays addressing the influence of protein-overexpression on cell proliferation and (iv) the display of the relative expression ('Electronic Northern') of the genes under investigation using curated gene expression ontology information. LIFEdb enables researchers to systematically select and characterize genes and proteins of interest, and presents data and information via its user-friendly web-based interface.

    Nucleic acids research 2006;34;Database issue;D415-8

  • Ataxin-2 and huntingtin interact with endophilin-A complexes to function in plastin-associated pathways.

    Ralser M, Nonhoff U, Albrecht M, Lengauer T, Wanker EE, Lehrach H and Krobitsch S

    Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany.

    Spinocerebellar ataxia type 2 is an inherited neurodegenerative disorder that is caused by an expanded trinucleotide repeat in the SCA2 gene, encoding a polyglutamine stretch in the gene product ataxin-2. Although evidence has been provided that ataxin-2 is involved in RNA metabolism, the physiological function of ataxin-2 remains unclear. Here, we demonstrate that ataxin-2 interacts with two members of the endophilin family, endophilin-A1 and endophilin-A3. To elucidate the physiological implications of these interactions, we exploited yeast as a model system and discovered that expression of ataxin-2 as well as both endophilin proteins is toxic for yeast lacking the SAC6 gene product fimbrin, a protein involved in actin filament organization and endocytotic processes. Intriguingly, expression of huntingtin, another polyglutamine protein interacting with endophilin-A3, was also toxic in Deltasac6 yeast. These effects can be suppressed by simultaneous expression of one of the two human fimbrin orthologs, L- or T-plastin. Moreover, we have discovered that ataxin-2 associates with L- and T-plastin and that overexpression of ataxin-2 leads to accumulation of T-plastin in mammalian cells. Thus, our findings suggest an interplay between ataxin-2, endophilin proteins and huntingtin in plastin-associated cellular pathways.

    Human molecular genetics 2005;14;19;2893-909

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • A structure-based model of the c-Myc/Bin1 protein interaction shows alternative splicing of Bin1 and c-Myc phosphorylation are key binding determinants.

    Pineda-Lucena A, Ho CS, Mao DY, Sheng Y, Laister RC, Muhandiram R, Lu Y, Seet BT, Katz S, Szyperski T, Penn LZ and Arrowsmith CH

    Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ont., Canada M5G 2M9.

    The N terminus of the c-Myc oncoprotein interacts with Bin1, a ubiquitously expressed nucleocytoplasmic protein with features of a tumor suppressor. The c-Myc/Bin1 interaction is dependent on the highly conserved Myc Box 1 (MB1) sequence of c-Myc. The c-Myc/Bin1 interaction has potential regulatory significance as c-Myc-mediated transformation and apoptosis can be modulated by the expression of Bin1. Multiple splicing of the Bin1 transcript results in ubiquitous, tissue-specific and tumor-specific populations of Bin1 proteins in vivo. We report on the structural features of the interaction between c-Myc and Bin1, and describe two mechanisms by which the binding of different Bin1 isoforms to c-Myc may be regulated in cells. Our findings identify a consensus class II SH3-binding motif in c-Myc and the C-terminal SH3 domain of Bin1 as the primary structure determinants of their interaction. We present biochemical and structural evidence that tumor-specific isoforms of Bin1 are precluded from interaction with c-Myc through an intramolecular polyproline-SH3 domain interaction that inhibits the Bin1 SH3 domain from binding to c-Myc. Furthermore, c-Myc/Bin1 interaction can be inhibited by phosphorylation of c-Myc at Ser62, a functionally important residue found within the c-Myc SH3-binding motif. Our data provide a structure-based model of the c-Myc/Bin1 interaction and suggest a mode of regulation that may be important for c-Myc function as a regulator of gene transcription.

    Journal of molecular biology 2005;351;1;182-94

  • Regulation of Bin1 SH3 domain binding by phosphoinositides.

    Kojima C, Hashimoto A, Yabuta I, Hirose M, Hashimoto S, Kanaho Y, Sumimoto H, Ikegami T and Sabe H

    Department of Molecular Biology, Osaka Bioscience Institute, Suita, Japan.

    Bin1/M-amphiphysin-II is an amphiphysin-II isoform highly expressed in transverse tubules of adult striated muscle and is implicated in their biogenesis. Bin1 contains a basic unique amino-acid sequence, Exon10, which interacts with certain phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), to localize to membranes. Here we found that Exon10 also binds to the src homology 3 (SH3) domain of Bin1 itself, and hence blocks the binding of the SH3 domain to its canonical PxxP ligands, including dynamin. This blockage was released by addition of PI(4,5)P(2) in vitro or in cells overexpressing phosphatidylinositol 4-phosphate 5-kinase. The Exon10-binding interface of the Bin1 SH3 domain largely overlapped with its PxxP-binding interface. We also show that the PLCdelta pleckstrin homology domain, another PI(4,5)P(2)-binding module, cannot substitute for Exon10 in Bin1 function in transverse tubule formation, and suggest the importance of the dual biochemical properties of Exon10 in myogenesis. Our results exemplify a novel mechanism of SH3 domain regulation, and suggest that the SH3-mediated protein-protein interactions of Bin1 are regulated by Exon10 so that it may only occur when Bin1 localizes to certain submembrane areas.

    The EMBO journal 2004;23;22;4413-22

  • From ORFeome to biology: a functional genomics pipeline.

    Wiemann S, Arlt D, Huber W, Wellenreuther R, Schleeger S, Mehrle A, Bechtel S, Sauermann M, Korf U, Pepperkok R, Sültmann H and Poustka A

    Molecular Genome Analysis, German Cancer Research Center, 69120 Heidelberg, Germany. s.wiemann@dkfz.de

    As several model genomes have been sequenced, the elucidation of protein function is the next challenge toward the understanding of biological processes in health and disease. We have generated a human ORFeome resource and established a functional genomics and proteomics analysis pipeline to address the major topics in the post-genome-sequencing era: the identification of human genes and splice forms, and the determination of protein localization, activity, and interaction. Combined with the understanding of when and where gene products are expressed in normal and diseased conditions, we create information that is essential for understanding the interplay of genes and proteins in the complex biological network. We have implemented bioinformatics tools and databases that are suitable to store, analyze, and integrate the different types of data from high-throughput experiments and to include further annotation that is based on external information. All information is presented in a Web database (http://www.dkfz.de/LIFEdb). It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy.

    Genome research 2004;14;10B;2136-44

  • 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

  • Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation.

    Stoletov KV and Terman BI

    Cardiology Division, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

    We had previously shown that Rap1 mediates certain of the signaling pathways involved in VEGF-induced endothelial cell migration, although the downstream Rap1 effectors are not known. Towards the goal of identifying those effectors, we utilized a commercially available antibody array filter to identify proteins that either directly interact with Rap1 or interact indirectly through a multi-protein complex. The protocol identified 10 possible Rap1-interacting proteins, including the Bmx non-receptor tyrosine kinase. The conclusion that VEGF treatment leads to a Rap1/Bmx complex was confirmed by an experiment in which cell lysates from VEGF and control cells were immunoprecipitated with Bmx antibodies and Western blotting was done using anti-Rap1 antibodies. VEGF treatment led to the recruitment of Bmx to the CAS scaffolding protein, and inhibition of the Bmx kinase blocked VEGF-induced cell migration. Formation of a Rap1/Bmx complex was not observed in cells transfected with an expression vector for a dominant-negative Rap1, indicating that Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation.

    Funded by: NCI NIH HHS: CA086289; NHLBI NIH HHS: HL67019

    Biochemical and biophysical research communications 2004;320;1;70-5

  • Myc-interacting protein 1 target gene profile: a link to microtubules, extracellular signal-regulated kinase, and cell growth.

    Ziegelbauer J, Wei J and Tjian R

    Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.

    To study the role of the transcription factor Myc-interacting protein 1 (MIZ-1) in activating various target genes after induction with the microtubule disrupting agent T113242, we have used small interfering RNA duplexes (siRNAs) to knockdown the expression of MIZ-1. As expected, depletion of MIZ-1 resulted in the inhibition of T113242-dependent activation of the low-density lipoprotein receptor (LDLR) gene in hepatocytes. Cells transfected with MIZ-1 siRNAs also exhibited growth arrest. In addition, inhibition of the extracellular signal-regulated kinase (ERK) pathway inhibited T113242-induced nuclear accumulation of MIZ-1 and activation of LDLR. Gene expression microarray analysis under various induction conditions identified other T113242-activated genes affected by a decrease in MIZ-1 and inhibition of the ERK pathway. We also found that the accumulation of MIZ-1 in the nucleus is influenced by cell-cell contact and/or growth. Taken together, our studies suggest that MIZ-1 regulates a specific set of genes that includes LDLR and that the ERK pathway plays a role in the activation of target promoters by MIZ-1.

    Funded by: NCI NIH HHS: CA25417, R37 CA025417

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;2;458-63

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • RIN3: a novel Rab5 GEF interacting with amphiphysin II involved in the early endocytic pathway.

    Kajiho H, Saito K, Tsujita K, Kontani K, Araki Y, Kurosu H and Katada T

    Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.

    The small GTPase Rab5, which cycles between active (GTP-bound) and inactive (GDP-bound) states, plays essential roles in membrane budding and trafficking in the early endocytic pathway. However, the molecular mechanisms underlying the Rab5-regulated processes are not fully understood other than the targeting event to early endosomes. Here, we report a novel Rab5-binding protein, RIN3, that contains many functional domains shared with other RIN members and additional Pro-rich domains. RIN3 displays the same biochemical properties as RIN2, the stimulator and stabilizer of GTP-Rab5. In addition, RIN3 exhibits its unique intracellular localization. RIN3 expressed in HeLa cells localized to cytoplasmic vesicles and the RIN3-positive vesicles contained Rab5 but not the early endosomal marker EEA1. Transferrin appeared to be transported partly through the RIN3-positive vesicles to early endosomes. RIN3 was also capable of interacting via its Pro-rich domain with amphiphysin II, which contains SH3 domain and participates in receptor-mediated endocytosis. Interestingly, cytoplasmic amphiphysin II was translocated into the RIN3- and Rab5-positive vesicles when co-expressed with RIN3. These results indicate that RIN3 biochemically characterized as the stimulator and stabilizer for GTP-Rab5 plays an important role in the transport pathway from plasma membrane to early endosomes.

    Journal of cell science 2003;116;Pt 20;4159-68

  • Targeted disruption of the murine Bin1/Amphiphysin II gene does not disable endocytosis but results in embryonic cardiomyopathy with aberrant myofibril formation.

    Muller AJ, Baker JF, DuHadaway JB, Ge K, Farmer G, Donover PS, Meade R, Reid C, Grzanna R, Roach AH, Shah N, Soler AP and Prendergast GC

    DuPont Pharmaceuticals Company, Wilmington, Delaware, USA.

    The mammalian Bin1/Amphiphysin II gene encodes an assortment of alternatively spliced adapter proteins that exhibit markedly divergent expression and subcellular localization profiles. Bin1 proteins have been implicated in a variety of different cellular processes, including endocytosis, actin cytoskeletal organization, transcription, and stress responses. To gain insight into the physiological functions of the Bin1 gene, we have disrupted it by homologous recombination in the mouse. Bin1 loss had no discernible impact on either endocytosis or phagocytosis in mouse embryo-derived fibroblasts and macrophages, respectively. Similarly, actin cytoskeletal organization, proliferation, and apoptosis in embryo fibroblasts were all unaffected by Bin1 loss. In vivo, however, Bin1 loss resulted in perinatal lethality. Bin1 has been reported to affect muscle cell differentiation and T-tubule formation. No striking histological abnormalities were evident in skeletal muscle of Bin1 null embryos, but severe ventricular cardiomyopathy was observed in these embryos. Ultrastructurally, myofibrils in ventricular cardiomyocytes of Bin1 null embryos were severely disorganized. These results define a developmentally critical role for the Bin1 gene in cardiac muscle development.

    Molecular and cellular biology 2003;23;12;4295-306

  • Sorting nexin 4 and amphiphysin 2, a new partnership between endocytosis and intracellular trafficking.

    Leprince C, Le Scolan E, Meunier B, Fraisier V, Brandon N, De Gunzburg J and Camonis J

    INSERM U528, Institut Curie Section de Recherche, 26 rue d'Ulm, 75248 Paris Cedex 05, France. Corinne.Leprince@curie.fr

    Endocytosis is a regulated physiological process by which membrane receptors and their extracellular ligands are internalized. After internalization, they enter the endosomal trafficking pathway for sorting and processing. Amphiphysins consist of a family of proteins conserved throughout evolution that are crucial elements of the endocytosis machinery in mammalian cells. They act as adaptors for a series of proteins important for the endocytic process, such as dynamin. In order to improve our knowledge of amphiphysin function, we performed a two-hybrid screen with the N-terminal part of murine amphiphysin 2 (residues 1-304). One of the interacting clones corresponded to sorting nexin 4 (SNX4), a member of the SNX family of proteins which are suspected to regulate vesicular trafficking. This interaction was confirmed in vivo by co-immunoprecipitation. Immunofluorescence analysis revealed that amphiphysin 2 might bind reticulo-vesicular structures present throughout the cell body and be associated with SNX4 on these structures. In an endocytosis assay, overexpressed C-terminal or full-length SNX4 was able to inhibit transferrin receptor endocytosis as efficiently as the SH3 domain of amphiphysin 2. At lower levels of expression, SNX4 colocalized with transferrin-containing vesicles, some of which were also positive for amphiphysin 2. These results indicate that SNX4 may be part of the endocytic machinery or, alternatively, that SNX4 may associate with key elements of endocytosis such as amphiphysin 2 and sequester them when overexpressed. The presence of amphiphysin 2 on intracellular vesicles and its interplay with SNX4, which is likely to take part in intracellular trafficking, suggest that amphiphysin 2 is not only a regulator of the early steps of endocytosis. It could also play a role at the surface of the endocytic vesicle that has just been formed and of the future endosomes, in order to regulate intracellular trafficking.

    Journal of cell science 2003;116;Pt 10;1937-48

  • Identification and characterization of amphiphysin II as a novel cellular interaction partner of the hepatitis C virus NS5A protein.

    Zech B, Kurtenbach A, Krieger N, Strand D, Blencke S, Morbitzer M, Salassidis K, Cotten M, Wissing J, Obert S, Bartenschlager R, Herget T and Daub H

    Axxima Pharmaceuticals AG, Am Klopferspitz 19, 82152 Martinsried, Germany.

    The hepatitis C virus (HCV) NS5A protein is highly phosphorylated by cellular protein kinases. To study how NS5A might be integrated in cellular kinase signalling, we isolated phosphoproteins from HuH-7 hepatoma cells that specifically interacted with recombinant NS5A protein. Subsequent mass spectrometry identified the adaptor protein amphiphysin II as a novel interaction partner of NS5A. Mutational analysis revealed that complex formation is primarily mediated by a proline-rich region in the C-terminal part of NS5A, which interacts with the amphiphysin II Src homology 3 domain. Importantly, we could further demonstrate specific co-precipitation and cellular co-localization of endogenous amphiphysin II with NS5A in HuH-7 cells carrying a persistently replicating subgenomic HCV replicon. Although the NS5A-amphiphysin II interaction appeared to be dispensable for replication of these HCV RNAs in cell culture, our results indicate that NS5A-amphiphysin II complex formation might be of physiological relevance for the HCV life cycle.

    The Journal of general virology 2003;84;Pt 3;555-60

  • Immunohistochemical analysis of Bin1/Amphiphysin II in human tissues: diverse sites of nuclear expression and losses in prostate cancer.

    DuHadaway JB, Lynch FJ, Brisbay S, Bueso-Ramos C, Troncoso P, McDonnell T and Prendergast GC

    Lankenau Institute for Medical Research, Wynnewood, Pennsylvania 19096, USA.

    The Bin1/Amphiphysin II gene encodes at least seven alternately spliced adapter proteins that have been implicated in membrane dynamics and nuclear processes. Nuclear localized Bin1 polypeptides have tumor suppressor and proapoptotic activities, suggesting that Bin1 may suppress cancer in tissues where nuclear expression may occur. One question is the extent to which human tissues express nuclear Bin1 isoforms. A secondary issue has been the need for a specific antibody that can detect all the splice isoforms expressed by the human, mouse, and rat Bin1 genes. Using a novel mouse monoclonal antibody with these characteristics, we performed an immunohistochemical analysis of Bin1 expression in a panel of normal human tissues. We also compared the expression profile of Bin1 in normal or malignant tissues derived from human prostate, where Bin1 is a candidate tumor suppressor gene. In brain, a distinct nuclear staining pattern overlapped with a cytosolic staining pattern present in certain layers of the cerebral cortex and cerebellum. Bone marrow cells displayed mainly nuclear localization whereas peripheral lymphoid cells exhibited mainly cytosolic localization. In several epithelial tissues, nuclear or nucleocytosolic staining patterns were displayed by basal cells in skin, breast, or prostate, whereas cytosolic or plasma membrane-associated staining patterns were noted in gastrointestinal cells. Interestingly, a striking gradient of expression was observed in gastrointestinal epithelia, particularly in the large intestine, with the strongest staining displayed by cells destined to undergo apoptosis at the villus tip. In prostate, Bin1 staining was frequently absent in cases of primary prostate adenocarcinoma. This study used a novel reagent to document the extent of expression of nuclear Bin1 isoforms, which exhibit cancer suppression and proapoptotic activity in human cells.

    Journal of cellular biochemistry 2003;88;3;635-42

  • Characterization of Endophilin B1b, a brain-specific membrane-associated lysophosphatidic acid acyl transferase with properties distinct from endophilin A1.

    Modregger J, Schmidt AA, Ritter B, Huttner WB and Plomann M

    Center for Biochemistry II, Medical Faculty, Joseph-Stelzmann-Strasse 52, University of Cologne, Germany.

    We have characterized mammalian endophilin B1, a novel member of the endophilins and a representative of their B subgroup. The endophilins B show the same domain organization as the endophilins A, which contain an N-terminal domain responsible for lipid binding and lysophosphatidic acid acyl transferase activity, a central coiled-coil domain for oligomerization, a less conserved linker region, and a C-terminal Src homology 3 (SH3) domain. The endophilin B1 gene gives rise to at least three splice variants, endophilin B1a, which shows a widespread tissue distribution, and endophilins B1b and B1c, which appear to be brain-specific. Endophilin B1, like endophilins A, binds to palmitoyl-CoA, exhibits lysophosphatidic acid acyl transferase activity, and interacts with dynamin, amphiphysins 1 and 2, and huntingtin. However, in contrast to endophilins A, endophilin B1 does not bind to synaptojanin 1 and synapsin 1, and overexpression of its SH3 domain does not inhibit transferrin endocytosis. Consistent with this, immunofluorescence analysis of endophilin B1b transfected into fibroblasts shows an intracellular reticular staining, which in part overlaps with that of endogenous dynamin. Upon subcellular fractionation of brain and transfected fibroblasts, endophilin B1 is largely recovered in association with membranes. Together, our results suggest that the action of the endophilins is not confined to the formation of endocytic vesicles from the plasma membrane, with endophilin B1 being associated with, and presumably exerting a functional role at, intracellular membranes.

    The Journal of biological chemistry 2003;278;6;4160-7

  • Specific interactions of neuronal focal adhesion kinase isoforms with Src kinases and amphiphysin.

    Messina S, Onofri F, Bongiorno-Borbone L, Giovedì S, Valtorta F, Girault JA and Benfenati F

    Department of Experimental Medicine, Section of Human Physiology, University of Genova, Italy.

    Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that activates Src family kinases via SH2- and SH3-mediated interactions. Specific FAK isoforms (FAK+), responsive to depolarization and neurotransmitters, are enriched in neurons. We analyzed the interactions of endogenous FAK+ and recombinant FAK+ isoforms containing amino acid insertions (boxes 6,7,28) with an array of SH3 domains and the c-Src SH2/SH3 domain tandem. Endogenous FAK+ bound specifically to the SH3 domains of c-Src (but not n-Src), Fyn, Yes, phosphtidylinositol-3 kinase, amphiphysin II, amphiphysin I, phospholipase Cgamma and NH2-terminal Grb2. The inclusion of boxes 6,7 was associated with a significant decrease in the binding of FAK+ to the c-Src and Fyn SH3 domains, and a significant increase in the binding to the Src SH2 domain, as a consequence of the higher phosphorylation of Tyr-397. The novel interaction with the amphiphysin SH3 domain, involving the COOH-terminal proline-rich region of FAK, was confirmed by coimmunoprecipitation of the two proteins and a closely similar response to stimuli affecting the actin cytoskeleton. Moreover, an impairment of endocytosis was observed in synaptosomes after internalization of a proline-rich peptide corresponding to the site of interaction. The data account for the different subcellular distribution of FAK and Src kinases and the specific regulation of the transduction pathways linked to FAK activation in the brain and implicate FAK in the regulation of membrane trafficking in nerve terminals.

    Funded by: Telethon: 1131

    Journal of neurochemistry 2003;84;2;253-65

  • Amphiphysin 2 (Bin1) and T-tubule biogenesis in muscle.

    Lee E, Marcucci M, Daniell L, Pypaert M, Weisz OA, Ochoa GC, Farsad K, Wenk MR and De Camilli P

    Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06510, USA.

    In striated muscle, the plasma membrane forms tubular invaginations (transverse tubules or T-tubules) that function in depolarization-contraction coupling. Caveolin-3 and amphiphysin were implicated in their biogenesis. Amphiphysin isoforms have a putative role in membrane deformation at endocytic sites. An isoform of amphiphysin 2 concentrated at T-tubules induced tubular plasma membrane invaginations when expressed in nonmuscle cells. This property required exon 10, a phosphoinositide-binding module. In developing myotubes, amphiphysin 2 and caveolin-3 segregated in tubular and vesicular portions of the T-tubule system, respectively. These findings support a role of the bilayer-deforming properties of amphiphysin at T-tubules and, more generally, a physiological role of amphiphysin in membrane deformation.

    Funded by: NCI NIH HHS: CA46128; NINDS NIH HHS: NS36251

    Science (New York, N.Y.) 2002;297;5584;1193-6

  • Amphiphysin IIb-1, a novel splicing variant of amphiphysin II, regulates p73beta function through protein-protein interactions.

    Kim KC, Kim TS, Kang KH and Choi KH

    Laboratory of Molecular Biology, Department of Biology, College of Natural Sciences, Chung-Ang University, Heuksuk Dong, Dongjak Ku, Seoul 156-756, Korea.

    p73 is a nuclear protein that is similar in structure and function to p53. Notably, the C-terminal region of p73 has a regulatory function, through interactions with a positive or negative regulator. In this study, we use the yeast two-hybrid technique to identify a novel p73beta binding protein, designated amphiphysin IIb-1. Amphiphysin IIb-1 is one of the splicing variants of amphiphysin II, and has a shorter protein product than amphiphysin IIb, which has been previously reported. We confirmed that amphiphysin IIb-1 binds full-length p73beta, both in vitro and in vivo. This association is mediated via the SH3 domain of amphiphysin IIb-1 and C-terminal amino acids 321-376 of p73beta. Double immunofluorescence patterns revealed that p73beta is relocalized to the cytoplasm in the presence of amphiphysin IIb-1. Overexpression of amphiphysin IIb-1 was found to significantly inhibit the transcriptional activity of p73beta in a dose-dependent manner. In addition, the cell death function of p73beta was inhibited by amphiphysin IIb-1. These findings offer a new insight into the regulation mechanism of p73beta, and suggest that amphiphysin IIb-1 modulates p73beta function by direct binding.

    Oncogene 2001;20;46;6689-99

  • Endophilin regulates JNK activation through its interaction with the germinal center kinase-like kinase.

    Ramjaun AR, Angers A, Legendre-Guillemin V, Tong XK and McPherson PS

    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.

    The endophilin family of proteins function in clathrin-mediated endocytosis. Here, we have identified and cloned the rat germinal center kinase-like kinase (rGLK), a member of the GCK (germinal center kinase) family of c-Jun N-terminal kinase (JNK) activating enzymes, as a novel endophilin I-binding partner. The interaction occurs both in vitro and in cells and is mediated by the Src homology 3 domain of endophilin I and a region of rGLK containing the endophilin consensus-binding sequence PPRPPPPR. Overlay analysis of rat brain extracts demonstrates that endophilin I is a major Src homology 3 domain-binding partner for rGLK. Overexpression of full-length endophilin I activates rGLK-mediated JNK activation, whereas N- and C-terminal fragments of endophilin I block JNK activation. Thus, endophilin I appears to have a novel function in JNK activation.

    The Journal of biological chemistry 2001;276;31;28913-9

  • Pattern of expression of c-Myc, Max and Bin1 in human anagen hair follicles.

    Barajon I, Rumio C, Donetti E, Imberti A, Brivio M and Castano P

    Institute of Human Anatomy, University of Milan, Via Mangiagalli 31, 20133 Milan.

    Background: We recently reported the presence of c-Myc immunoreactivity in two distinct regions of the inner root sheath (IRS) of human anagen hair follicles; they corresponded to the regions where keratinocytes of Henle's and Huxley's layers enter the terminal differentiation phase that will lead to their exfoliation in the pilary canal. These regions were denoted lower (LR) ring and upper ring (UR).

    Objectives: To extend these observations to other genes connected to c-Myc and specifically to Max and Bin1. Max is the best known heterodimeric partner of c-Myc, interacting with its C-terminal domain, and Bin1 is an adaptor protein interacting with its N-terminal domain.

    Methods: Human anagen hair follicles were processed for c-Myc, Max and Bin1 immunohistochemistry and immunofluorescence. The presence of different isoforms of Bin1 was evaluated by Western blot analysis.

    Results: Analysis of sections cut in several planes, including tangential, demonstrated the presence of a third ring of c-Myc-positive cells (intermediate ring; IR) in the cuticle of the IRS corresponding to the region where this thin layer undergoes keratinization. Max immunoreactivity was observed in the three layers of the IRS starting in the lower bulbar region and ending in each of them at the level of the corresponding c-Myc-positive ring. Bin1 immunoreactivity was clearly distinguished only in Huxley's layer and in the cuticle, starting in some cells below the UR and terminating at the level of the latter. The companion layer of the outer root sheath was also labelled up to the infundibular region. Max and Bin1 immunostaining were less consistently observed in other skin adnexae and in the epidermis.

    Conclusions: The results indicate that the asynchronous differentiation along the axis of the hair follicle of the different layers of the IRS and of the companion layer involves the expression of different genes that are interrelated in the so-called 'Myc network'. The specific localization of c-Myc in the IRS only at the level of the discrete and limited regions of the three rings appears to be the hallmark of the switch from differentiation to terminal differentiation/cell deletion.

    The British journal of dermatology 2001;144;6;1193-203

  • Bin1 mediates apoptosis by c-Myc in transformed primary cells.

    DuHadaway JB, Sakamuro D, Ewert DL and Prendergast GC

    The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.

    The Bin1 gene encodes a c-Myc-interacting adapter protein with tumor suppressor and cell death properties. In this study, we offer evidence that Bin1 participates in a mechanism through which c-Myc activates programmed cell death in transformed primary chick or rat cells. Antisense or dominant inhibitory Bin1 genes did not affect the ability of c-Myc to drive proliferation or transformation, but they did reduce the susceptibility of cells to c-Myc-induced apoptosis. Protein-protein interaction was implicated, suggesting that Bin1 mediates a death or death sensitization signal from c-Myc. Our findings offer direct support for the "dual signal" model of Myc apoptotic function, based on interactions with a binding protein. Loss of Bin1 in human tumors may promote malignant progression in part by helping to stanch the death penalty associated with c-Myc activation.

    Funded by: NCI NIH HHS: CA10815

    Cancer research 2001;61;7;3151-6

  • Toward a catalog of human genes and proteins: sequencing and analysis of 500 novel complete protein coding human cDNAs.

    Wiemann S, Weil B, Wellenreuther R, Gassenhuber J, Glassl S, Ansorge W, Böcher M, Blöcker H, Bauersachs S, Blum H, Lauber J, Düsterhöft A, Beyer A, Köhrer K, Strack N, Mewes HW, Ottenwälder B, Obermaier B, Tampe J, Heubner D, Wambutt R, Korn B, Klein M and Poustka A

    Molecular Genome Analysis, German Cancer Research Center, 69120 Heidelberg, Germany. s.wiemann@dkfz.de

    With the complete human genomic sequence being unraveled, the focus will shift to gene identification and to the functional analysis of gene products. The generation of a set of cDNAs, both sequences and physical clones, which contains the complete and noninterrupted protein coding regions of all human genes will provide the indispensable tools for the systematic and comprehensive analysis of protein function to eventually understand the molecular basis of man. Here we report the sequencing and analysis of 500 novel human cDNAs containing the complete protein coding frame. Assignment to functional categories was possible for 52% (259) of the encoded proteins, the remaining fraction having no similarities with known proteins. By aligning the cDNA sequences with the sequences of the finished chromosomes 21 and 22 we identified a number of genes that either had been completely missed in the analysis of the genomic sequences or had been wrongly predicted. Three of these genes appear to be present in several copies. We conclude that full-length cDNA sequencing continues to be crucial also for the accurate identification of genes. The set of 500 novel cDNAs, and another 1000 full-coding cDNAs of known transcripts we have identified, adds up to cDNA representations covering 2%--5 % of all human genes. We thus substantially contribute to the generation of a gene catalog, consisting of both full-coding cDNA sequences and clones, which should be made freely available and will become an invaluable tool for detailed functional studies.

    Genome research 2001;11;3;422-35

  • DNA cloning using in vitro site-specific recombination.

    Hartley JL, Temple GF and Brasch MA

    Life Technologies, Inc., Rockville, Maryland 20850, USA. jhartley@lifetech.com

    As a result of numerous genome sequencing projects, large numbers of candidate open reading frames are being identified, many of which have no known function. Analysis of these genes typically involves the transfer of DNA segments into a variety of vector backgrounds for protein expression and functional analysis. We describe a method called recombinational cloning that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. Numerous DNA segments can be transferred in parallel into many different vector backgrounds, providing an approach to high-throughput, in-depth functional analysis of genes and rapid optimization of protein expression. The resulting subclones maintain orientation and reading frame register, allowing amino- and carboxy-terminal translation fusions to be generated. In this paper, we outline the concepts of this approach and provide several examples that highlight some of its potential.

    Genome research 2000;10;11;1788-95

  • The c-Myc-interacting adaptor protein Bin1 activates a caspase-independent cell death program.

    Elliott K, Ge K, Du W and Prendergast GC

    The Wistar Institute, Philadelphia, PA, USA.

    Cell death processes are progressively inactivated during malignant development, in part by loss of tumor suppressors that can promote cell death. The Bin1 gene encodes a nucleocytosolic adaptor protein with tumor suppressor properties, initially identified through its ability to interact with and inhibit malignant transformation by c-Myc and other oncogenes. Bin1 is frequently missing or functionally inactivated in breast and prostate cancers and in melanoma. In this study, we show that Bin1 engages a caspase-independent cell death process similar to type II apoptosis, characterized by cell shrinkage, substratum detachment, vacuolated cytoplasm, and DNA degradation. Cell death induction was relieved by mutation of the BAR domain, a putative effector domain, or by a missplicing event that occurs in melanoma and inactivates suppressor activity. Cells in all phases of the cell cycle were susceptible to death and p53 and Rb were dispensable. Notably, Bin1 did not activate caspases and the broad spectrum caspase inhibitor ZVAD.fmk did not block cell death. Consistent with the lack of caspase involvement, dying cells lacked nucleosomal DNA cleavage and nuclear lamina degradation. Moreover, neither Bcl-2 or dominant inhibition of the Fas pathway had any effect. In previous work, we showed that Bin1 could not suppress cell transformation by SV40 large T antigen. Consistent with this finding, we observed that T antigen suppressed the death program engaged by Bin1. This observation was interesting in light of emerging evidence that T antigen has roles in cell immortalization and human cell transformation beyond Rb and p53 inactivation. In support of a link to c-Myc-induced death processes, AEBSF, a serine protease inhibitor that inhibits apoptosis by c-Myc, potently suppressed DNA degradation by Bin1. Our findings suggest that the tumor suppressor activity of Bin1 reflects engagement of a unique cell death program. We propose that loss of Bin1 may promote malignancy by blunting death penalties associated with oncogene activation.

    Oncogene 2000;19;41;4669-84

  • Specificity of the binding of synapsin I to Src homology 3 domains.

    Onofri F, Giovedi S, Kao HT, Valtorta F, Bongiorno Borbone L, De Camilli P, Greengard P and Benfenati F

    Department of Experimental Medicine, Section of Physiology, University of Genova, Via Benedetto XV 3, I-16132 Genova, Italy.

    Synapsins are synaptic vesicle-associated phosphoproteins involved in synapse formation and regulation of neurotransmitter release. Recently, synapsin I has been found to bind the Src homology 3 (SH3) domains of Grb2 and c-Src. In this work we have analyzed the interactions between synapsins and an array of SH3 domains belonging to proteins involved in signal transduction, cytoskeleton assembly, or endocytosis. The binding of synapsin I was specific for a subset of SH3 domains. The highest binding was observed with SH3 domains of c-Src, phospholipase C-gamma, p85 subunit of phosphatidylinositol 3-kinase, full-length and NH(2)-terminal Grb2, whereas binding was moderate with the SH3 domains of amphiphysins I/II, Crk, alpha-spectrin, and NADPH oxidase factor p47(phox) and negligible with the SH3 domains of p21(ras) GTPase-activating protein and COOH-terminal Grb2. Distinct sites in the proline-rich COOH-terminal region of synapsin I were found to be involved in binding to the various SH3 domains. Synapsin II also interacted with SH3 domains with a partly distinct binding pattern. Phosphorylation of synapsin I in the COOH-terminal region by Ca(2+)/calmodulin-dependent protein kinase II or mitogen-activated protein kinase modulated the binding to the SH3 domains of amphiphysins I/II, Crk, and alpha-spectrin without affecting the high affinity interactions. The SH3-mediated interaction of synapsin I with amphiphysins affected the ability of synapsin I to interact with actin and synaptic vesicles, and pools of synapsin I and amphiphysin I were shown to associate in isolated nerve terminals. The ability to bind multiple SH3 domains further implicates the synapsins in signal transduction and protein-protein interactions at the nerve terminal level.

    Funded by: NIA NIH HHS: AG15072; NIMH NIH HHS: MH39327; Telethon: 1131

    The Journal of biological chemistry 2000;275;38;29857-67

  • Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing.

    Simpson JC, Wellenreuther R, Poustka A, Pepperkok R and Wiemann S

    Department of Cell Biology and Biophysics, EMBL Heidelberg, Germany.

    As a first step towards a more comprehensive functional characterization of cDNAs than bioinformatic analysis, which can only make functional predictions for about half of the cDNAs sequenced, we have developed and tested a strategy that allows their systematic and fast subcellular localization. We have used a novel cloning technology to rapidly generate N- and C-terminal green fluorescent protein fusions of cDNAs to examine the intracellular localizations of > 100 expressed fusion proteins in living cells. The entire analysis is suitable for automation, which will be important for scaling up throughput. For > 80% of these new proteins a clear intracellular localization to known structures or organelles could be determined. For the cDNAs where bioinformatic analyses were able to predict possible identities, the localization was able to support these predictions in 75% of cases. For those cDNAs where no homologies could be predicted, the localization data represent the first information.

    EMBO reports 2000;1;3;287-92

  • Bin2, a functionally nonredundant member of the BAR adaptor gene family.

    Ge K and Prendergast GC

    The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.

    BAR family proteins are a unique class of adaptor proteins characterized by a common N-terminal fold of undetermined function termed the BAR domain. This set of adaptors, which includes the mammalian proteins amphiphysin and Bin1 and the yeast proteins Rvs167p and Rvs161p, has been implicated in diverse cellular processes, including synaptic vesicle endocytosis, actin regulation, differentiation, cell survival, and tumorigenesis. Here we report the identification and characterization of Bin2, a novel protein that contains a BAR domain but that is otherwise structurally dissimilar to other members of the BAR adaptor family. The Bin2 gene is located at chromosome 4q22.1 and is expressed predominantly in hematopoietic cells. Bin2 is upregulated during differentiation of granulocytes, suggesting that it functions in that lineage. Bin2 formed a stable complex in cells with Bin1, but not with amphiphysin, in a BAR domain-dependent manner. This finding indicates that BAR domains have specific preferences for interaction. However, Bin2 did not influence endocytosis in the same manner as brain-specific splice isoforms of Bin1, nor did it exhibit the tumor suppressor properties inherent to ubiquitous splice isoforms of Bin1. Thus, Bin2 appears to encode a nonredundant function in the BAR adaptor gene family.

    Genomics 2000;67;2;210-20

  • Inhibition of phospholipase D by amphiphysins.

    Lee C, Kim SR, Chung JK, Frohman MA, Kilimann MW and Rhee SG

    Laboratory of Cell Signaling, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-0320, USA.

    Two distinct proteins inhibiting phospholipase D (PLD) activity in rat brain cytosol were previously purified and identified as synaptojanin and AP180, which are specific to nerve terminals and associate with the clathrin coat. Two additional PLD-inhibitory proteins have now been purified and identified as the amphiphysins I and II, which forms a heterodimer that also associates with the clathrin coat. Bacterially expressed recombinant amphiphysins inhibited both PLD1 and PLD2 isozymes in vitro with a potency similar to that of brain amphiphysin (median inhibitory concentration of approximately 15 nm). Expressions of either amphiphysin in COS-7 cells reduced activity of endogenous PLD as well as exogenously expressed PLD1 and PLD2. Coprecipitation experiments suggested that the inhibitory effect of amphiphysins results from their direct interaction with PLDs. The NH(2) terminus of amphiphysin I was critical for both inhibition of and binding to PLD. Phosphatidic acid formed by signal-induced PLD is thought to be required for the assembly of clathrin-coated vesicles during endocytosis. Thus, the inhibition of PLD by amphiphysins, synaptojanin, and AP180 might play an important role in synaptic vesicle trafficking.

    The Journal of biological chemistry 2000;275;25;18751-8

  • Loss of heterozygosity and tumor suppressor activity of Bin1 in prostate carcinoma.

    Ge K, Minhas F, Duhadaway J, Mao NC, Wilson D, Buccafusca R, Sakamuro D, Nelson P, Malkowicz SB, Tomaszewski J and Prendergast GC

    The Wistar Institute, Philadelphia, Pennsylvania, USA.

    The genetic events underlying the development of prostate cancer are poorly defined. c-Myc is often activated in tumors that have progressed to metastatic status, so events that promote this process may be important. Bin1 is a nucleocytoplasmic adaptor protein with features of a tumor suppressor that was identified through its ability to interact with and inhibit malignant transformation by c-Myc. We investigated a role for Bin1 loss or inactivation in prostate cancer because the human Bin1 gene is located at chromosome 2q14 within a region that is frequently deleted in metastatic prostate cancer but where no tumor suppressor candidate has been located. A novel polymorphic microsatellite marker located within intron 5 of the human Bin1 gene was used to demonstrate loss of heterozygosity and coding alteration in 40% of informative cases of prostate neoplasia examined. RNA and immunohistochemical analyses indicated that Bin1 was expressed in most primary tumors, even at slightly elevated levels relative to benign tissues, but that it was frequently missing or inactivated by aberrant splicing in metastatic tumors and androgen-independent tumor cell lines. Ectopic expression of Bin1 suppressed the growth of prostate cancer lines in vitro. Our findings support the candidacy of Bin1 as the chromosome 2q prostate tumor suppressor gene.

    International journal of cancer 2000;86;2;155-61

  • Losses of the tumor suppressor BIN1 in breast carcinoma are frequent and reflect deficits in programmed cell death capacity.

    Ge K, Duhadaway J, Sakamuro D, Wechsler-Reya R, Reynolds C and Prendergast GC

    The Wistar Institute, Philadelphia, PA, USA.

    Oncogenic activation of MYC occurs often in breast carcinoma and is associated with poor prognosis. Loss or inactivation of mechanisms that restrain MYC may therefore be involved in tumor progression. In this study, we show that the MYC-interacting adaptor protein BIN1 is frequently missing in malignant breast cells and that this loss is functionally significant. BIN1 was expressed in normal and benign cells and tissues but was undetectable in 6/6 estrogen receptor-positive or estrogen receptor-negative carcinoma cell lines examined. Similarly, complete or partial losses of BIN1 were documented in 30/50 (60%) cases of malignant breast tissue analyzed by immuno-histochemistry or RT-PCR. Abnormalities in the organization of the BIN1 gene were apparent in only a minority of these cases, suggesting that most losses were due to epigenetic causes. Nevertheless, they were functionally significant because ectopic BIN1 induced programmed cell death in malignant cells lacking endogenous BIN1 but had no effect on the viability of benign cells. We propose that loss of BIN1 may contribute to breast cancer progression by eliminating a mechanism that restrains the ability of activated MYC to drive cell division inappropriately.

    Funded by: NCI NIH HHS: CA10851

    International journal of cancer 2000;85;3;376-83

  • Mechanism for elimination of a tumor suppressor: aberrant splicing of a brain-specific exon causes loss of function of Bin1 in melanoma.

    Ge K, DuHadaway J, Du W, Herlyn M, Rodeck U and Prendergast GC

    The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA.

    Loss of tumor suppressors that restrain important oncoproteins such as c-Myc may contribute to malignant progression. Bin1 is an adapter protein with features of a tumor suppressor that was identified through its interaction with and inhibition of the oncogenic properties of c-Myc. In this study, we analyzed the patterns of Bin1 expression in normal melanocytes and melanoma cells at different stages of tumor progression. Evidence is provided that Bin1 function is abrogated in melanoma cells by a mechanism based on aberrant splicing of a tissue-specific exon. Specifically, most melanoma cells inappropriately expressed exon 12A, which is spliced alternately into Bin1 isoforms found in brain but not into isoforms found in melanocytes and many other nonneuronal cells. Exon 12A sequences abolished the ability of Bin1 to inhibit malignant transformation by c-Myc or adenovirus E1A. Similarly, these sequences abolished the ability of Bin1 to induce programmed cell death in melanoma cells that endogenously expressed exon 12A. Our findings suggest that aberrant splicing of Bin1 may contribute to melanoma progression, and they define a mechanism by which the activity of a tumor suppressor can be eliminated in cells.

    Funded by: NCI NIH HHS: CA10851; NIDDK NIH HHS: P30 DK050306, P30 DK50306

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;17;9689-94

  • Crystal structure of the alpha appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly.

    Traub LM, Downs MA, Westrich JL and Fremont DH

    Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.

    AP-2 adaptors regulate clathrin-bud formation at the cell surface by recruiting clathrin trimers to the plasma membrane and by selecting certain membrane proteins for inclusion within the developing clathrin-coat structure. These functions are performed by discrete subunits of the adaptor heterotetramer. The carboxyl-terminal appendage of the AP-2 alpha subunit appears to regulate the translocation of several endocytic accessory proteins to the bud site. We have determined the crystal structure of the alpha appendage at 1.4-A resolution by multiwavelength anomalous diffraction phasing. It is composed of two distinct structural modules, a beta-sandwich domain and a mixed alpha-beta platform domain. Structure-based mutagenesis shows that alterations to the molecular surface of a highly conserved region on the platform domain differentially affect associations of the appendage with amphiphysin, eps15, epsin, and AP180, revealing a common protein-binding interface.

    Funded by: NIDDK NIH HHS: DK53249, R01 DK053249

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;16;8907-12

  • The N terminus of amphiphysin II mediates dimerization and plasma membrane targeting.

    Ramjaun AR, Philie J, de Heuvel E and McPherson PS

    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.

    Amphiphysin I and II are nerve terminal-enriched proteins containing SH3 domains that interact with dynamin and synaptojanin. The amphiphysins may function in synaptic vesicle endocytosis by targeting synaptojanin and dynamin to emerging endocytic buds through SH3 domain-independent interactions with clathrin and AP2. We have recently identified and cloned several amphiphysin II splice variants that differentially incorporate clathrin-binding domains. To determine whether these domains function in membrane targeting, we used immunofluorescence to examine the potential localization of amphiphysin II variants to clathrin-coated pits on plasma membranes purified from transfected COS-7 cells. Full-length amphiphysin II targets to the plasma membrane where it partially co-localizes with clathrin. However, splice variants and deletion constructs lacking clathrin-binding domains still target to the plasma membrane, and removal of clathrin from the membrane does not affect amphiphysin II distribution. Surprisingly, plasma membrane targeting was dependent on the presence of a 31-amino acid alternatively spliced sequence at the N terminus of amphiphysin II, a result confirmed using subcellular fractionation. In binding assays, the 31-amino acid sequence was also found to facilitate amphiphysin dimerization mediated through the N terminus. Taken together, these data support a role for the N terminus of amphiphysin II in membrane targeting during endocytosis.

    The Journal of biological chemistry 1999;274;28;19785-91

  • Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains.

    Wixler V, Laplantine E, Geerts D, Sonnenberg A, Petersohn D, Eckes B, Paulsson M and Aumailley M

    Institute for Biochemistry II, Medical Faculty of the University of Cologne, Germany.

    The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit. In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified. Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of alpha subunits. The conserved membrane proximal region of the alpha3A chain was identified as the binding site for all four proteins. They, therefore, may be involved in the regulation of general functions of integrins.

    FEBS letters 1999;445;2-3;351-5

  • The murine Bin1 gene functions early in myogenesis and defines a new region of synteny between mouse chromosome 18 and human chromosome 2.

    Mao NC, Steingrimsson E, DuHadaway J, Wasserman W, Ruiz JC, Copeland NG, Jenkins NA and Prendergast GC

    The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania, 19104, USA.

    We cloned and functionally characterized the murine Bin1 gene as a first step to investigate its physiological roles in differentiation, apoptosis, and tumorigenesis. The exon-intron organization of the >/=55-kb gene is similar to that of the human gene. Consistent with a role for Bin1 in apoptosis, the promoter included a functional consensus motif for activation by NF-kappaB, an important regulator of cell death. A muscle regulatory module defined in the human promoter that includes a consensus recognition site for myoD family proteins was not conserved in the mouse promoter. However, Bin1 is upregulated in embryonic development by E10.5 in myotomes, the progenitors of skeletal muscle, supporting a role in myogenesis and suggesting that the mouse and human genes may be controlled somewhat differently during development. In C2C12 myoblasts antisense Bin1 prevents induction of the cell cycle kinase inhibitor p21WAF1, suggesting that it acts at an early time during the muscle differentiation program. Interspecific mouse backcross mapping located the Bin1 locus between Mep1b and Apc on chromosome 18. Since the human gene was mapped previously to chromosome 2q14, the location of Bin1 defines a previously unrecognized region of synteny between human chromosome 2 and mouse chromosome 18.

    Genomics 1999;56;1;51-8

  • Multiple amphiphysin II splice variants display differential clathrin binding: identification of two distinct clathrin-binding sites.

    Ramjaun AR and McPherson PS

    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Quebec, Canada.

    Amphiphysin I and II are nerve terminal-enriched proteins that display src homology 3 domain-mediated interactions with dynamin and synaptojanin. It has been demonstrated that the amphiphysins also bind to clathrin, and we have proposed that this interaction may help to target synaptojanin and dynamin to sites of synaptic vesicle endocytosis. To understand better this potential functional role, we have begun to characterize clathrin-amphiphysin interactions. Using PCR from adult human cortex cDNA, we have cloned a number of amphiphysin II splice variants. In in vitro binding assays, the amphiphysin II splice variants display differential clathrin binding and define a 44-amino acid region mediating the interaction. Amphiphysin II truncation and deletion mutants identify two distinct clathrin-binding domains within this region: one with the sequence LLDLDFDP, the second with the sequence PWDLW. Both domains are conserved in amphiphysin I, and saturation binding analysis demonstrates that both sites bind clathrin with approximately equal affinity. The elucidation of clathrin as a splice-specific binding partner for amphiphysin II begins to address the potential functional role(s) for the multiple amphiphysin II splice variants and further supports an important function for clathrin-amphiphysin interactions in protein targeting during endocytosis.

    Journal of neurochemistry 1998;70;6;2369-76

  • A role for the putative tumor suppressor Bin1 in muscle cell differentiation.

    Wechsler-Reya RJ, Elliott KJ and Prendergast GC

    The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.

    Bin1 is a Myc-interacting protein with features of a tumor suppressor. The high level of Bin1 expression in skeletal muscle prompted us to investigate its role in muscle differentiation. Significant levels of Bin1 were observed in undifferentiated C2C12 myoblasts, a murine in vitro model system. Induction of differentiation by growth factor withdrawal led to an upregulation of Bin1 mRNA and to the generation of higher-molecular-weight forms of Bin1 protein by alternate splicing. While Bin1 in undifferentiated cells was localized exclusively in the nucleus, differentiation-associated isoforms of Bin1 were found in the cytoplasm as well. To examine the function of Bin1 during differentiation, we generated stable cell lines that express exogenous human Bin1 cDNA in the sense or antisense orientation. Cells overexpressing Bin1 grew more slowly than control cells and differentiated more rapidly when deprived of growth factors. In contrast, C2C12 cells expressing antisense Bin1 showed an impaired ability to undergo differentiation. Taken together, the results indicated that Bin1 expression, structure, and localization are tightly regulated during muscle differentiation and suggested that Bin1 plays a functional role in the differentiation process.

    Funded by: NICHD NIH HHS: N01-HD-7-3263

    Molecular and cellular biology 1998;18;1;566-75

  • Structural analysis of the human BIN1 gene. Evidence for tissue-specific transcriptional regulation and alternate RNA splicing.

    Wechsler-Reya R, Sakamuro D, Zhang J, Duhadaway J and Prendergast GC

    The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.

    BIN1 is a putative tumor suppressor that was identified through its interaction with the MYC oncoprotein. To begin to identify elements of BIN1 whose alteration may contribute to malignancy, we cloned and characterized the human BIN1 gene and promoter. Nineteen exons were identified in a region of >54 kilobases, six of which were alternately spliced in a cell type-specific manner. One alternately spliced exon encodes part of the MYC-binding domain, suggesting that splicing controls the MYC-binding capacity of BIN1 polypeptides. Four other alternately spliced exons encode amphiphysin-related sequences that were included in brain-specific BIN1 species, also termed amphiphysin isoforms or amphiphysin II. The 5'-flanking region of BIN1 is GC-rich and lacks a TATA box but directs transcriptional initiation from a single site. A approximately 0. 9-kilobase fragment from this region was sufficient for basal transcription and transactivation by MyoD, which may account for the high levels of BIN1 observed in skeletal muscle. This study lays the foundation for genetic and epigenetic investigations into the role of BIN1 in normal and neoplastic cell regulation.

    The Journal of biological chemistry 1997;272;50;31453-8

  • The amphiphysin-like protein 1 (ALP1) interacts functionally with the cABL tyrosine kinase and may play a role in cytoskeletal regulation.

    Kadlec L and Pendergast AM

    Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.

    cABL is a protooncogene, activated in a subset of human leukemias, whose protein product is a nonreceptor tyrosine kinase of unknown function. cABL has a complex structure that includes several domains and motifs found in proteins implicated in signal transduction pathways. An approach to elucidate cABL function is to identify proteins that interact directly with cABL and that may serve as regulators or effectors of its activity. To this end, a protein-interaction screen of a phage expression library was undertaken to identify proteins that interact with specific domains of cABL. An SH3-domain-containing protein has been identified that interacts with sequences in the cABL carboxyl terminus. The cDNA encoding ALP1 (amphiphysin-like protein 1) was isolated from a 16-day mouse embryo. ALP1 has high homology to BIN1, a recently cloned myc-interacting protein, and also shows significant homology to amphiphysin, a neuronal protein cloned from human and chicken. The amino terminus has homology to two yeast proteins, Rvs167 and Rvs161, which are involved in cell entry into stationary phase and cytoskeletal organization. ALP1 binds cABL in vitro and in vivo. Expression of ALP1 results in morphological transformation of NIH 3T3 fibroblasts in a cABL-dependent manner. The properties of ALP1 suggest that it may be involved in possible cytoskeletal functions of the cABL kinase. Additionally, these results provide further evidence for the importance of the cABL carboxyl terminus and its binding proteins in the regulation of cABL function.

    Funded by: NCI NIH HHS: CA70940, R01 CA070940

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;23;12390-5

  • Synaptojanin forms two separate complexes in the nerve terminal. Interactions with endophilin and amphiphysin.

    Micheva KD, Kay BK and McPherson PS

    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, 3801 Rue University, Montreal, Quebec H3A 2B4, Canada.

    Endophilin is a recently discovered src homology 3 domain-containing protein that is a major in vitro binding partner for synaptojanin. To further characterize endophilin, we generated an antipeptide antibody. Endophilin is enriched in the brain, and immunofluorescence analysis reveals a high concentration of the protein in synaptic terminals, where it colocalizes with synaptojanin. In vitro binding assays demonstrate that endophilin binds through its src homology 3 domain to synaptojanin, and immunoprecipitation analysis with the antiendophilin antibody reveals that endophilin is stably associated with synaptojanin in the nerve terminal. Immunoprecipitation with an antibody against amphiphysin I and II, which interact through their src homology 3 domains with dynamin and synaptojanin at sites distinct from those for endophilin, reveals a second stable complex, which includes dynamin and synaptojanin but excludes endophilin. These data demonstrate that synaptojanin is present in two separate complexes in the nerve terminal and support an important role for endophilin in the regulation of synaptojanin function.

    The Journal of biological chemistry 1997;272;43;27239-45

  • Amphiphysin heterodimers: potential role in clathrin-mediated endocytosis.

    Wigge P, Köhler K, Vallis Y, Doyle CA, Owen D, Hunt SP and McMahon HT

    Neurobiology Division, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.

    Amphiphysin (Amph) is a src homology 3 domain-containing protein that has been implicated in synaptic vesicle endocytosis as a result of its interaction with dynamin. In a screen for novel members of the amphiphysin family, we identified Amph2, an isoform 49% identical to the previously characterized Amph1 protein. The subcellular distribution of this isoform parallels Amph1, both being enriched in nerve terminals. Like Amph1, a role in endocytosis at the nerve terminal is supported by the rapid dephosphorylation of Amph2 on depolarization. Importantly, the two isoforms can be coimmunoprecipitated from the brain as an equimolar complex, suggesting that the two isoforms act in concert. As determined by cross-linking of brain extracts, the Amph1-Amph2 complex is a 220- to 250-kDa heterodimer. COS cells transfected with either Amph1 or Amph2 show greatly reduced transferrin uptake, but coexpression of the two proteins rescues this defect, supporting a role for the heterodimer in clathrin-mediated endocytosis. Although the src homology 3 domains of both isoforms interact with dynamin, the heterodimer can associate with multiple dynamin molecules in vitro and activates dynamin's GTPase activity. We propose that it is an amphiphysin heterodimer that drives the recruitment of dynamin to clathrin-coated pits in endocytosing nerve terminals.

    Molecular biology of the cell 1997;8;10;2003-15

  • SH3 domain-dependent interactions of endophilin with amphiphysin.

    Micheva KD, Ramjaun AR, Kay BK and McPherson PS

    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Que., Canada.

    Amphiphysin I and II are nerve terminal-enriched proteins thought to function in synaptic vesicle endocytosis. In addition to a C-terminal SH3 domain, the proteins contain a highly conserved putative SH3 binding site and numerous consensus phosphorylation sites. We now demonstrate that amphiphysin I but not amphiphysin II is a phosphoprotein which undergoes dephosphorylation during nerve terminal depolarization. Further, both amphiphysin I and II interact with the SH3 domain of endophilin, a synaptically enriched protein implicated in synaptic vesicle endocytosis. The interaction is direct and mediated through a 43 amino acid region of amphiphysin containing the putative SH3 binding site. These data further support a role for amphiphysin I, II and endophilin in synaptic vesicle endocytosis.

    FEBS letters 1997;414;2;308-12

  • Clathrin interacts specifically with amphiphysin and is displaced by dynamin.

    McMahon HT, Wigge P and Smith C

    Neurobiology Division, MRC-LMB, Cambridge, UK. hmm@mrc-lmb.cam.ac.uk

    Amphiphysin is an SH3 domain protein that has been implicated in synaptic vesicle endocytosis. We have recently cloned a second amphiphysin isoform, Amph2 (sequence submitted to GenBank, Y13380). Proteins capable of forming a complex with amphiphysin were isolated from rat brain by using recombinant GST-Amph2 for binding experiments. As well as interacting with dynamin I, the full-length protein bound to a weaker 180-kDa band. Immunoblotting demonstrated this protein to be clathrin. To address whether this is a direct interaction, the clathrin binding to amphiphysin was reconstituted in vitro with purified proteins. The N-terminal domain of Amph2 is sufficient for clathrin binding. Dynamin, which interacts with the SH3 domain of Amph2, displaces clathrin from the N-terminus. We propose a model that may explain how clathrin and dynamin are recruited to non-overlapping sites of the coated pit.

    FEBS letters 1997;413;2;319-22

  • cDNA cloning of a novel amphiphysin isoform and tissue-specific expression of its multiple splice variants.

    Tsutsui K, Maeda Y, Tsutsui K, Seki S and Tokunaga A

    Department of Molecular Biology, Institute of Cellular and Molecular Biology, Okayama University Medical School, Shikata-cho, Japan.

    Amphiphysin, an SH3-domain containing protein concentrated in nerve terminals, is believed to be involved in the synaptic vesicle recycling. We have cloned cDNAs of a novel isoform of amphiphysin (amphiphysin II) by exploiting sequence information for homologous ESTs deposited in databases. At least 9 different subtypes of the isoform with 50-60% amino acid identity to the human amphiphysin were identified by a conventional library screening and PCR amplification of cDNA libraries. Each subtype probably represents a splice variant of a single gene transcript. Analysis of mRNA expression in various tissues by RT-PCR showed that the isoform is ubiquitously distributed. The expression spectrum of the isoform subtypes, however, is significantly different in several tissues examined, suggesting that they are involved in the regulation of endocytic processes that are unique to each cell type.

    Biochemical and biophysical research communications 1997;236;1;178-83

  • Identification and characterization of a nerve terminal-enriched amphiphysin isoform.

    Ramjaun AR, Micheva KD, Bouchelet I and McPherson PS

    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Québec H3A 2B4, Canada.

    Amphiphysin is a nerve terminal-enriched protein thought to function in synaptic vesicle endocytosis, in part through Src homology 3 (SH3) domain-mediated interactions with dynamin and synaptojanin. Here, we report the characterization of a novel amphiphysin isoform (termed amphiphysin II) that was identified through a homology search of the data base of expressed sequence tags. Antibodies specific to amphiphysin II recognize a 90-kDa protein on Western blot that is brain-specific and highly enriched in nerve terminals. Like amphiphysin (now referred to as amphiphysin I), amphiphysin II binds to dynamin and synaptojanin through its SH3 domain. Further, both proteins bind directly to clathrin in an SH3 domain-independent manner. Taken together, these data suggest that amphiphysin II may participate with amphiphysin I in the regulation of synaptic vesicle endocytosis.

    The Journal of biological chemistry 1997;272;26;16700-6

  • Amphiphysin II (SH3P9; BIN1), a member of the amphiphysin/Rvs family, is concentrated in the cortical cytomatrix of axon initial segments and nodes of ranvier in brain and around T tubules in skeletal muscle.

    Butler MH, David C, Ochoa GC, Freyberg Z, Daniell L, Grabs D, Cremona O and De Camilli P

    Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

    Amphiphysin (amphiphysin I), a dominant autoantigen in paraneoplastic Stiff-man syndrome, is a neuronal protein highly concentrated in nerve terminals, where it has a putative role in endocytosis. The yeast homologue of amphiphysin, Rvs167, has pleiotropic functions, including a role in endocytosis and in actin dynamics, suggesting that amphiphysin may also be implicated in the function of the presynaptic actin cytoskeleton. We report here the characterization of a second mammalian amphiphysin gene, amphiphysin II (SH3P9; BIN1), which encodes products primarily expressed in skeletal muscle and brain, as differentially spliced isoforms. In skeletal muscle, amphiphysin II is concentrated around T tubules, while in brain it is concentrated in the cytomatrix beneath the plasmamembrane of axon initial segments and nodes of Ranvier. In both these locations, amphiphysin II is colocalized with splice variants of ankyrin3 (ankyrinG), a component of the actin cytomatrix. In the same regions, the presence of clathrin has been reported. These findings support the hypothesis that, even in mammalian cells, amphiphysin/Rvs family members have a role both in endocytosis and in actin function and suggest that distinct amphiphysin isoforms contribute to define distinct domains of the cortical cytoplasm. Since amphiphysin II (BIN1) was reported to interact with Myc, it may also be implicated in a signaling pathway linking the cortical cytoplasm to nuclear function.

    Funded by: NCI NIH HHS: CA46128, P01 CA046128

    The Journal of cell biology 1997;137;6;1355-67

  • A new member of the amphiphysin family connecting endocytosis and signal transduction pathways.

    Leprince C, Romero F, Cussac D, Vayssiere B, Berger R, Tavitian A and Camonis JH

    INSERM U248, Institut Curie, 26 rue d'Ulm, 75231 Paris Cedex 05, France. leprince@curie.fr

    Src homology 3 (SH3) domains are conserved modules which participate in protein interaction by recognizing proline-rich motifs on target molecules. To identify new SH3-containing proteins, we performed a two-hybrid screen with a proline-rich region of human SOS-1. One of the specific SOS-1 interacting clones that were isolated from a mouse brain cDNA library defines a new protein that was named amphiphysin 2 because of its homology to the previously reported amphiphysin. Amphiphysin 2 is expressed in a number of mouse tissues through multiple RNA transcripts. Here, we report the amino acid sequence of a brain form of amphiphysin 2 (BRAMP2) encoded by a 2. 5-kilobase mRNA. BRAMP2 associates in vitro with elements of the endocytosis machinery such as alpha-adaptin and dynamin. On a biosensor surface, the BRAMP2/dynamin interaction appeared to be direct and partly dependent on a proline-rich sequence of dynamin. Association with dynamin was also observed in PC12 cells after cell stimulation with nerve growth factor, suggesting that amphiphysin 2 may be connected to receptor-dependent signaling pathways. This hypothesis is strengthened by the ability of BRAMP2 to interact with the p21(ras) exchange factor SOS, in vitro, as a possible point of interconnection between the endocytic and signaling pathways.

    The Journal of biological chemistry 1997;272;24;15101-5

  • Large-scale concatenation cDNA sequencing.

    Yu W, Andersson B, Worley KC, Muzny DM, Ding Y, Liu W, Ricafrente JY, Wentland MA, Lennon G and Gibbs RA

    A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (> 20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (> or = 98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.

    Funded by: NHGRI NIH HHS: 1F32 HG00169-01, F32 HG000169, F33 HG000210, P30 HG00210-05, R01 HG00823, U54 HG003273

    Genome research 1997;7;4;353-8

  • BIN1 is a novel MYC-interacting protein with features of a tumour suppressor.

    Sakamuro D, Elliott KJ, Wechsler-Reya R and Prendergast GC

    Wistar Institute, Philadelphia, Pennsylvania 19104, USA.

    BIN1 is a novel protein that interacts with the functionally critical Myc box regions at the N terminus of the MYC oncoprotein. BIN1 is structurally related to amphiphysin, a breast cancer-associated autoimmune antigen, and RVS167, a negative regulator of the yeast cell cycle, suggesting roles in malignancy and cell cycle control. Consistent with this likelihood, BIN1 inhibited malignant cell transformation by MYC. Although BIN1 is expressed in many normal cells, its levels were greatly reduced or undetectable in 14/27 carcinoma cell lines and 3/6 primary breast tumours. Deficits were functionally significant because ectopic expression of BIN1 inhibited the growth of tumour cells lacking endogenous message. We conclude that BIN1 is an MYC-interacting protein with features of a tumour suppressor.

    Funded by: NCI NIH HHS: CA09171, CA10815, CA66179

    Nature genetics 1996;14;1;69-77

  • The Bin1 gene localizes to human chromosome 2q14 by PCR analysis of somatic cell hybrids and fluorescence in situ hybridization.

    Negorev D, Riethman H, Wechsler-Reya R, Sakamuro D, Prendergast GC and Simon D

    Medical College of Pennsylvania, Philadelphia, USA.

    Funded by: NCI NIH HHS: CA10815, CA58525; NHGRI NIH HHS: HG600954

    Genomics 1996;33;2;329-31

  • A "double adaptor" method for improved shotgun library construction.

    Andersson B, Wentland MA, Ricafrente JY, Liu W and Gibbs RA

    Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.

    The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.

    Funded by: NHGRI NIH HHS: R01 HG00823

    Analytical biochemistry 1996;236;1;107-13

Gene lists (8)

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
L00000011 G2C Homo sapiens Human clathrin Human orthologues of mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000012 G2C Homo sapiens Human Synaptosome Human orthologues of mouse synaptosome adapted from Collins et al (2006) 152
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© 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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