G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
dynamin 2
G00000340 (Mus musculus)

Databases (7)

ENSG00000079805 (Ensembl human gene)
1785 (Entrez Gene)
694 (G2Cdb plasticity & disease)
DNM2 (GeneCards)
602378 (OMIM)
Marker Symbol
HGNC:2974 (HGNC)
Protein Sequence
P50570 (UniProt)

Synonyms (5)

  • CMTDI1
  • DYN2

Literature (72)

Pubmed - other

  • Dynamin 2 and c-Abl are novel regulators of hyperoxia-mediated NADPH oxidase activation and reactive oxygen species production in caveolin-enriched microdomains of the endothelium.

    Singleton PA, Pendyala S, Gorshkova IA, Mambetsariev N, Moitra J, Garcia JG and Natarajan V

    Department of Medicine, Pritzker School of Medicine, The University of Chicago, Chicago, Illinois 60637, USA. psinglet@medicine.bsd.uchicago.edu

    Reactive oxygen species (ROS) generation, particularly by the endothelial NADPH oxidase family of proteins, plays a major role in the pathophysiology associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. We examined potential regulators of ROS production and discovered that hyperoxia treatment of human pulmonary artery endothelial cells induced recruitment of the vesicular regulator, dynamin 2, the non-receptor tyrosine kinase, c-Abl, and the NADPH oxidase subunit, p47(phox), to caveolin-enriched microdomains (CEMs). Silencing caveolin-1 (which blocks CEM formation) and/or c-Abl expression with small interference RNA inhibited hyperoxia-mediated tyrosine phosphorylation and association of dynamin 2 with p47(phox) and ROS production. In addition, treatment of human pulmonary artery endothelial cells with dynamin 2 small interfering RNA or the dynamin GTPase inhibitor, Dynasore, attenuated hyperoxia-mediated ROS production and p47(phox) recruitment to CEMs. Using purified recombinant proteins, we observed that c-Abl tyrosine-phosphorylated dynamin 2, and this phosphorylation increased p47(phox)/dynamin 2 association (change in the dissociation constant (K(d)) from 85.8 to 6.9 nm). Furthermore, exposure of mice to hyperoxia increased ROS production, c-Abl activation, dynamin 2 association with p47(phox), and pulmonary leak, events that were attenuated in the caveolin-1 knock-out mouse confirming a role for CEMs in ROS generation. These results suggest that hyperoxia induces c-Abl-mediated dynamin 2 phosphorylation required for recruitment of p47(phox) to CEMs and subsequent ROS production in lung endothelium.

    Funded by: NHLBI NIH HHS: P01 HL058064, P01-HL 58064, R01 HL 085553, R01 HL085553, R01 HL095723, R01-HL 095723

    The Journal of biological chemistry 2009;284;50;34964-75

  • Dynamin 2 mutations associated with human diseases impair clathrin-mediated receptor endocytosis.

    Bitoun M, Durieux AC, Prudhon B, Bevilacqua JA, Herledan A, Sakanyan V, Urtizberea A, Cartier L, Romero NB and Guicheney P

    Institut National de la Santé et de la Recherche Médicale, U582, Institut de Myologie, Paris, France. m.bitoun@institut-myologie.org

    Dynamin 2 (DNM2) is a large GTPase involved in the release of nascent vesicles during endocytosis and intracellular membrane trafficking. Distinct DNM2 mutations, affecting the middle domain (MD) and the Pleckstrin homology domain (PH), have been identified in autosomal dominant centronuclear myopathy (CNM) and in the intermediate and axonal forms of the Charcot-Marie-Tooth peripheral neuropathy (CMT). We report here the first CNM mutation (c.1948G>A, p.E650 K) in the DNM2 GTPase effector domain (GED), leading to a slowly progressive moderate myopathy. COS7 cells transfected with DNM2 constructs harboring a disease-associated mutation in MD, PH, or GED show a reduced uptake of transferrin and low-density lipoprotein (LDL) complex, two markers of clathrin-mediated receptor endocytosis. A decrease in clathrin-mediated endocytosis was also identified in skin fibroblasts from one CNM patient. We studied the impact of DNM2 mutant overexpression on epidermal growth factor (EGF)-induced extracellular signal-regulated kinase 1 (ERK1) and ERK2 activation, known to be an endocytosis- and DNM2-dependent process. Activation of ERK1/2 was impaired for all the transfected mutants in COS7 cells, but not in CNM fibroblasts. Our results indicate that impairment of clathrin-mediated endocytosis may play a role in the pathophysiological mechanisms leading to DNM2-related diseases, but the tissue-specific impact of DNM2 mutations in both diseases remains unclear.

    Human mutation 2009;30;10;1419-27

  • Phenotypic spectrum of dynamin 2 mutations in Charcot-Marie-Tooth neuropathy.

    Claeys KG, Züchner S, Kennerson M, Berciano J, Garcia A, Verhoeven K, Storey E, Merory JR, Bienfait HM, Lammens M, Nelis E, Baets J, De Vriendt E, Berneman ZN, De Veuster I, Vance JM, Nicholson G, Timmerman V and De Jonghe P

    Neurogenetics Group, VIB Department of Molecular Genetics, University of Antwerp, Antwerpen, Belgium.

    Dominant intermediate Charcot-Marie-Tooth neuropathy type B is caused by mutations in dynamin 2. We studied the clinical, haematological, electrophysiological and sural nerve biopsy findings in 34 patients belonging to six unrelated dominant intermediate Charcot-Marie-Tooth neuropathy type B families in whom a dynamin 2 mutation had been identified: Gly358Arg (Spain); Asp551_Glu553del; Lys550fs (North America); Lys558del (Belgium); Lys558Glu (Australia, the Netherlands) and Thr855_Ile856del (Belgium). The Gly358Arg and Thr855_Ile856del mutations were novel, and in contrast to the other Charcot-Marie-Tooth-related mutations in dynamin 2, which are all located in the pleckstrin homology domain, they were situated in the middle domain and proline-rich domain of dynamin 2, respectively. We report the first disease-causing mutation in the proline-rich domain of dynamin 2. Patients with a dynamin 2 mutation presented with a classical Charcot-Marie-Tooth phenotype, which was mild to moderately severe since only 3% of the patients were wheelchair-bound. The mean age at onset was 16 years with a large variability ranging from 2 to 50 years. Interestingly, in the Australian and Belgian families, which carry two different mutations affecting the same amino acid (Lys558), Charcot-Marie-Tooth cosegregated with neutropaenia. In addition, early onset cataracts were observed in one of the Charcot-Marie-Tooth families. Our electrophysiological data indicate intermediate or axonal motor median nerve conduction velocities (NCV) ranging from 26 m/s to normal values in four families, and less pronounced reduction of motor median NCV (41-46 m/s) with normal amplitudes in two families. Sural nerve biopsy in a Dutch patient with Lys558Glu mutation showed diffuse loss of large myelinated fibres, presence of many clusters of regenerating myelinated axons and fibres with focal myelin thickenings--findings very similar to those previously reported in the Australian family. We conclude that dynamin 2 mutations should be screened in the autosomal dominant Charcot-Marie-Tooth neuropathy families with intermediate or axonal NCV, and in patients with a classical mild to moderately severe Charcot-Marie-Tooth phenotype, especially when Charcot-Marie-Tooth is associated with neutropaenia or cataracts.

    Funded by: NINDS NIH HHS: 5R01NS052767

    Brain : a journal of neurology 2009;132;Pt 7;1741-52

  • Dynamic instability of microtubules requires dynamin 2 and is impaired in a Charcot-Marie-Tooth mutant.

    Tanabe K and Takei K

    Department of Neuroscience, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558, Japan.

    Dynamin is a fission protein that participates in endocytic vesicle formation. Although dynamin was originally identified as a microtubule-binding protein, the physiological relevance of this function was unclear. Recently, mutations in the ubiquitously expressed dynamin 2 (dyn2) protein were found in patients with Charcot-Marie-Tooth (CMT) disease, which is an inherited peripheral neuropathy. In this study, we show that one of these mutations, 551Delta3, induces prominent decoration of microtubules with the mutant dyn2. Dyn2 was required for proper dynamic instability of microtubules, and this was impaired in cells expressing the 551Delta3 mutant, which showed a remarkable increase in microtubule acetylation, a marker of stable microtubules. Depletion of endogenous dyn2 with a small interfering RNA also resulted in the accumulation of stable microtubules. Furthermore, the formation of mature Golgi complexes, which depends on microtubule-dependent membrane transport, was impaired in both dyn2 knockdown cells and cells expressing the 551Delta3 mutant. Collectively, our results suggest that dyn2 regulates dynamic instability of microtubules, which is essential for organelle motility, and that this function may be impaired in CMT disease.

    The Journal of cell biology 2009;185;6;939-48

  • New function of the proline rich domain in dynamin-2 to negatively regulate its interaction with microtubules in mammalian cells.

    Hamao K, Morita M and Hosoya H

    Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, 739-8526, Japan.

    Microtubule reorganization is necessary for many cellular functions such as cell migration, cell polarity and cell division. Dynamin was originally identified as a microtubule-binding protein. Previous limited digestion experiment revealed that C-terminal 100-amino acids proline rich domain (PRD) of dynamin is responsible for microtubule binding in vitro. However, as obvious localization of dynamin along microtubules is only observed at the spindle midzone during mitosis but not in interphase cells, it remains unclear how dynamin interacts with microtubules in vivo. Here, we report that GFP-dynamin-2-(1-786), a truncated mutant lacking a C-terminal portion of the PRD, localized along microtubules in interphase HeLa cells. GFP-dynamin-2-wild type (WT) and GFP-dynamin-2-(1-745), a construct that was further truncated to remove the entire PRD, localized in discrete punctate structures but not along microtubules. These data suggest that the N-terminal (residues 746-786) but not the entire PRD is necessary for the interaction of dynamin-2 with microtubules in the cell and that the C-terminus of PRD (787-870) negatively regulate this interaction. Microtubules in cells expressing GFP-dynamin-2-(1-786) were stabilized against exposure to cold. These results provide a first evidence for a regulated interaction of dynamin-2 with microtubules in cultured mammalian cells.

    Experimental cell research 2009;315;7;1336-45

  • FBP17 Mediates a Common Molecular Step in the Formation of Podosomes and Phagocytic Cups in Macrophages.

    Tsuboi S, Takada H, Hara T, Mochizuki N, Funyu T, Saitoh H, Terayama Y, Yamaya K, Ohyama C, Nonoyama S and Ochs HD

    Infectious and Inflammatory Disease Center, Burnham Institute for Medical Research, La Jolla, California 92037, USA. tsuboi@oyokyo.jp

    Macrophages act to protect the body against inflammation and infection by engaging in chemotaxis and phagocytosis. In chemotaxis, macrophages use an actin-based membrane structure, the podosome, to migrate to inflamed tissues. In phagocytosis, macrophages form another type of actin-based membrane structure, the phagocytic cup, to ingest foreign materials such as bacteria. The formation of these membrane structures is severely affected in macrophages from patients with Wiskott-Aldrich syndrome (WAS), an X chromosome-linked immunodeficiency disorder. WAS patients lack WAS protein (WASP), suggesting that WASP is required for the formation of podosomes and phagocytic cups. Here we have demonstrated that formin-binding protein 17 (FBP17) recruits WASP, WASP-interacting protein (WIP), and dynamin-2 to the plasma membrane and that this recruitment is necessary for the formation of podosomes and phagocytic cups. The N-terminal EFC (extended FER-CIP4 homology)/F-BAR (FER-CIP4 homology and Bin-amphiphysin-Rvs) domain of FBP17 was previously shown to have membrane binding and deformation activities. Our results suggest that FBP17 facilitates membrane deformation and actin polymerization to occur simultaneously at the same membrane sites, which mediates a common molecular step in the formation of podosomes and phagocytic cups. These results provide a potential mechanism underlying the recurrent infections in WAS patients.

    Funded by: NICHD NIH HHS: R01HD042752

    The Journal of biological chemistry 2009;284;13;8548-56

  • Decrease of dynamin 2 levels in late-onset Alzheimer's disease alters Abeta metabolism.

    Kamagata E, Kudo T, Kimura R, Tanimukai H, Morihara T, Sadik MG, Kamino K and Takeda M

    Department of Geriatric Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, Japan.

    Late-onset Alzheimer's disease (LOAD) is significantly associated with a single nucleotide polymorphism located in the dynamin (DNM) 2 gene, especially in non-carriers of the apolipoprotein E-epsilon4 allele. In this study we used real-time PCR to show that DNM2 mRNA is significantly reduced in the cortex of AD brains and in the peripheral blood of dementia patients. Neuroblastoma cells transfected with a dominant negative DNM2 had increased amyloid beta protein (Abeta) secretion and most of the amyloid precursor protein (APP) in these cells was localized to the plasma membrane. In addition, these cells were rich in flotillin, which is a component of lipid rafts. These data suggest that DNM2 expression is reduced in LOAD, which results in the accumulation of APP in lipid raft-rich plasma membranes. Consequently, Abeta secretion may increase in LOAD neurons.

    Biochemical and biophysical research communications 2009;379;3;691-5

  • A new centronuclear myopathy phenotype due to a novel dynamin 2 mutation.

    Bitoun M, Bevilacqua JA, Eymard B, Prudhon B, Fardeau M, Guicheney P and Romero NB

    INSERM U582, Institut de Myologie, Groupe Hospitalier Pitié-Salpêtrière, 75013, Paris, France. m.bitoun@institut-myologie.org

    Neurology 2009;72;1;93-5

  • Dynamin 2 regulates granule exocytosis during NK cell-mediated cytotoxicity.

    Arneson LN, Segovis CM, Gomez TS, Schoon RA, Dick CJ, Lou Z, Billadeau DD and Leibson PJ

    Department of Immunology, College of Medicine, Mayo Clinic, Rochester, MN 55905, USA.

    NK cells are innate immune cells that can eliminate their targets through granule release. In this study, we describe a specialized role for the large GTPase Dynamin 2 (Dyn2) in the regulation of these secretory events leading to cell-mediated cytotoxicity. By modulating the expression of Dyn2 using small interfering RNA or by inhibiting its activity using a pharmacological agent, we determined that Dyn2 does not regulate conjugate formation, proximal signaling, or granule polarization. In contrast, during cell-mediated killing, Dyn2 localizes with lytic granules and polarizes to the NK cell-target interface where it regulates the final fusion of lytic granules with the plasma membrane. These findings identify a novel role for Dyn2 in the exocytic events required for effective NK cell-mediated cytotoxicity.

    Funded by: NCI NIH HHS: CA47752, R01 CA047752, R01 CA047752-19A1; NIAID NIH HHS: AI065474, R01 AI065474, R01 AI065474-04

    Journal of immunology (Baltimore, Md. : 1950) 2008;181;10;6995-7001

  • A canine DNM1 mutation is highly associated with the syndrome of exercise-induced collapse.

    Patterson EE, Minor KM, Tchernatynskaia AV, Taylor SM, Shelton GD, Ekenstedt KJ and Mickelson JR

    Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St Paul, Minnesota 55108, USA. patte037@umn.edu

    Labrador retrievers are the most common dog breed in the world, with over 200,000 new kennel club registrations per year. The syndrome of exercise-induced collapse (EIC) in this breed is manifested by muscle weakness, incoordination and life-threatening collapse after intense exercise. Using a genome-wide microsatellite marker scan for linkage in pedigrees, we mapped the EIC locus to canine chromosome 9. We then used SNP association and haplotype analysis to fine map the locus, and identified a mutation in the dynamin 1 gene (DNM1) that causes an R256L substitution in a highly conserved region of the protein. This first documented mammalian DNM1 mutation is present at a high frequency in the breed and is a compelling candidate causal mutation for EIC, as the dynamin 1 protein has an essential role in neurotransmission and synaptic vesicle endocytosis.

    Nature genetics 2008;40;10;1235-9

  • Magnetic resonance imaging findings of leg musculature in Charcot-Marie-Tooth disease type 2 due to dynamin 2 mutation.

    Gallardo E, Claeys KG, Nelis E, García A, Canga A, Combarros O, Timmerman V, De Jonghe P and Berciano J

    Services of Radiology, Clinical Neurophysiology and Neurology, University Hospital Marqués de Valdecilla, (CIBERNED and IFIMAV), Santander, Spain.

    The purpose of the study was to prospectively assess magnetic resonance (MR) imaging findings of lower limb musculature in an axonal Charcot-Marie-Tooth disease (CMT2) pedigree due to mutation in the dynamin 2 gene (DNM2). The series comprises a proband patient aged 55 years and her two affected daughters aged 32 and 23. MR imaging study included T1- and fat suppressed T2-weighted spin-echo sequences. MR imaging study showed extensive fatty infiltration of all calf muscle compartments with relative preservation of the deep posterior one. Fatty muscle infiltration increased distally in 19 out of 66 (23%) visualized calf muscles in the three patients, but this percentage increased to 64% in the youngest and least severe patient. Muscle edema without contrast enhancement was present in 23% of calf muscles. There was massive fatty atrophy of foot musculature. We conclude that MR imaging study accurately depicts lower limb muscle involvement in CMT2 caused by DNM2 mutation.

    Journal of neurology 2008;255;7;986-92

  • A novel mutation in the dynamin 2 gene in a Charcot-Marie-Tooth type 2 patient: clinical and pathological findings.

    Bitoun M, Stojkovic T, Prudhon B, Maurage CA, Latour P, Vermersch P and Guicheney P

    INSERM, U582, Institut de Myologie, Paris (F-75013), France. m.bitoun@institut-mykologie.org

    Mutations in dynamin 2 (DNM2) have been associated with autosomal dominant centronuclear myopathy, dominant intermediate Charcot-Marie-Tooth (CMT) type B and CMT2. Here, we report a novel DNM2 mutation in the Pleckstrin homology domain of DNM2 (p.K559del) in a patient with an axonal length-dependent sensorimotor polyneuropathy predominantly affecting the lower limbs. Neuropathy is associated with congenital cataracts, ophthalmoparesis, ptosis and neutropenia. There was no evidence of a skeletal myopathy on EMG or muscle biopsy. We suggest that this constellation of clinical features can help the diagnosis and selection of patients for direct DNM2 genetic analysis.

    Neuromuscular disorders : NMD 2008;18;4;334-8

  • Dynamin 2 gene is a novel susceptibility gene for late-onset Alzheimer disease in non-APOE-epsilon4 carriers.

    Aidaralieva NJ, Kamino K, Kimura R, Yamamoto M, Morihara T, Kazui H, Hashimoto R, Tanaka T, Kudo T, Kida T, Okuda J, Uema T, Yamagata H, Miki T, Akatsu H, Kosaka K and Takeda M

    Department of Psychiatry, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.

    Alzheimer disease (AD) is characterized by progressive cognitive decline caused by synaptic dysfunction and neurodegeneration in the brain, and late-onset AD (LOAD), genetically classified as a polygenetic disease, is the major form of dementia in the elderly. It has been shown that beta amyloid, deposited in the AD brain, interacts with dynamin 1 and that the dynamin 2 (DNM2) gene homologous to the dynamin 1 gene is encoded at chromosome 19p13.2 where a susceptibility locus has been detected by linkage analysis. To test the genetic association of LOAD with the DNM2 gene, we performed a case-control study of 429 patients with LOAD and 438 sex- and age-matched control subjects in a Japanese population. We found a significant association of LOAD with single nucleotide polymorphism markers of the DNM2 gene, especially in non-carriers of the apolipoprotein E-epsilon4 allele. Even though subjects with the genotype homozygous for the risk allele at rs892086 showed no mutation in exons of the DNM2 gene, expression of DNM2 mRNA in the hippocampus was decreased in the patients compared to non-demented controls. We propose that the DNM2 gene is a novel susceptibility gene for LOAD.

    Journal of human genetics 2008;53;4;296-302

  • Dynamin 2 mediates fluid-phase micropinocytosis in epithelial cells.

    Cao H, Chen J, Awoniyi M, Henley JR and McNiven MA

    Mayo Clinic, Department of Biochemistry and Molecular Biology and the Miles and Shirley Fiterman Center for Digestive Diseases, Rochester, MN 55905, USA.

    It is well-known that dynamin 2 (Dyn2) participates in clathrin- and caveolae-mediated endocytosis; however, the role of Dyn2 in coat-independent endocytic processes remains controversial. Here we demonstrate a role for specific spliced variants of Dyn2 in the micropinocytosis of fluid in epithelial cells, independent of coat-mediated endocytic pathways. A general inhibition of Dyn2 was first performed using either microinjection of anti-dynamin antibodies or Dyn2-siRNA treatment. Both of these methods resulted in reduced uptake of transferrin, a marker for clathrin-mediated endocytosis, and, under unstimulated conditions, reduced the uptake of the fluid-phase markers dextran and horseradish peroxidase (HRP). By contrast, cells treated similarly but stimulated with serum or EGF internalized substantial amounts of dextran or HRP, indicating that Dyn2 is not required for stimulated fluid uptake via macropinocytosis. We next tested whether a specific spliced variant might selectively affect fluid-phase endocytosis. Mutation of specific Dyn2 spliced variants resulted in a differential attenuation of transferrin and dextran internalization. Furthermore, the reduction in fluid uptake in Dyn2-siRNA-treated cells was only rescued upon re-expression of select spliced variants. These findings suggest that Dyn2 function is required for the coat-independent internalization of fluid through endocytic pathways distinct from macropinocytosis and, in addition, implicate different Dyn2 spliced variants in specific endocytic functions.

    Funded by: NIDDK NIH HHS: DK44650

    Journal of cell science 2007;120;Pt 23;4167-77

  • Dynamin 2 mutations cause sporadic centronuclear myopathy with neonatal onset.

    Bitoun M, Bevilacqua JA, Prudhon B, Maugenre S, Taratuto AL, Monges S, Lubieniecki F, Cances C, Uro-Coste E, Mayer M, Fardeau M, Romero NB and Guicheney P

    Institut National de la Sante et de la Recherche Médicale, U582, Institut de Myologie, Paris, France.

    We report four heterozygous dynamin 2 (DNM2) mutations in five centronuclear myopathy patients aged 1 to 15 years. They all presented with neonatal hypotonia with weak suckling. Thereafter, their phenotype progressively improved. All patients demonstrated muscle weakness prominent in the lower limbs, and most of them also presented with facial weakness, open mouth, arched palate, ptosis, and ophthalmoparesis. Electrophysiology showed only myopathic changes, and muscle biopsies showed central nuclei and type 1 fiber hypotrophy and predominance. Our results expand the phenotypic spectrum of dynamin 2-related centronuclear myopathy from the classic mild form to the more severe neonatal phenotype.

    Annals of neurology 2007;62;6;666-70

  • Subtle central and peripheral nervous system abnormalities in a family with centronuclear myopathy and a novel dynamin 2 gene mutation.

    Echaniz-Laguna A, Nicot AS, Carré S, Franques J, Tranchant C, Dondaine N, Biancalana V, Mandel JL and Laporte J

    Département de Neurologie, Hôpital Civil de Strasbourg, 1 Place de l'Hôpital, BP426, 67091 Strasbourg, France. Echaniz-Laguna@medecine.u-strasbg.fr

    Mutations in dynamin 2 (DNM2), an ubiquitously-expressed large GTPase, cause autosomal dominant centronuclear myopathy (DNM2-CNM) and AD Charcot-Marie-Tooth disease type 2B (DNM2-CMT2B). We report a series of 5 patients from the same family who all presented with dominant centronuclear myopathy, mild cognitive impairment, mild axonal peripheral nerve involvement, and the novel E368Q mutation in the DNM2 gene. This study suggests that the phenotypes of dynamin 2 related centronuclear myopathy and Charcot-Marie-Tooth disease overlap and that DNM2 mutations may alter cerebral function. This report extends the clinical knowledge of DNM2-centronuclear myopathy and shows that the role of DNM2 mutations in the central nervous system should be further studied.

    Neuromuscular disorders : NMD 2007;17;11-12;955-9

  • CD44 regulates hepatocyte growth factor-mediated vascular integrity. Role of c-Met, Tiam1/Rac1, dynamin 2, and cortactin.

    Singleton PA, Salgia R, Moreno-Vinasco L, Moitra J, Sammani S, Mirzapoiazova T and Garcia JG

    Department of Medicine, Pritzker School of Medicine, University of Chicago, Chicago, Illinois 60637, USA.

    The preservation of vascular endothelial cell (EC) barrier integrity is critical to normal vessel homeostasis, with barrier dysfunction being a feature of inflammation, tumor angiogenesis, atherosclerosis, and acute lung injury. Therefore, agents that preserve or restore vascular integrity have important therapeutic implications. In this study, we explored the regulation of hepatocyte growth factor (HGF)-mediated enhancement of EC barrier function via CD44 isoforms. We observed that HGF promoted c-Met association with CD44v10 and recruitment of c-Met into caveolin-enriched microdomains (CEM) containing CD44s (standard form). Treatment of EC with CD44v10-blocking antibodies inhibited HGF-mediated c-Met phosphorylation and c-Met recruitment to CEM. Silencing CD44 expression (small interfering RNA) attenuated HGF-induced recruitment of c-Met, Tiam1 (a Rac1 exchange factor), cortactin (an actin cytoskeletal regulator), and dynamin 2 (a vesicular regulator) to CEM as well as HGF-induced trans-EC electrical resistance. In addition, silencing Tiam1 or dynamin 2 reduced HGF-induced Rac1 activation, cortactin recruitment to CEM, and EC barrier regulation. We observed that both HGF- and high molecular weight hyaluronan (CD44 ligand)-mediated protection from lipopolysaccharide-induced pulmonary vascular hyperpermeability was significantly reduced in CD44 knock-out mice, thus validating these in vitro findings in an in vivo murine model of inflammatory lung injury. Taken together, these results suggest that CD44 is an important regulator of HGF/c-Met-mediated in vitro and in vivo barrier enhancement, a process with essential involvement of Tiam1, Rac1, dynamin 2, and cortactin.

    Funded by: NHLBI NIH HHS: F32 HL68472, HL58064

    The Journal of biological chemistry 2007;282;42;30643-57

  • Myoferlin regulates vascular endothelial growth factor receptor-2 stability and function.

    Bernatchez PN, Acevedo L, Fernandez-Hernando C, Murata T, Chalouni C, Kim J, Erdjument-Bromage H, Shah V, Gratton JP, McNally EM, Tempst P and Sessa WC

    Department of Pharmacology and Vascular Biology & Transplantation Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536, USA.

    Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling.

    Funded by: NHLBI NIH HHS: HL64793, N01-HV-28186, P01 HL 70295, R01 HL 57665, R01 HL61371

    The Journal of biological chemistry 2007;282;42;30745-53

  • Dynamin 2 regulates riboflavin endocytosis in human placental trophoblasts.

    Foraker AB, Ray A, Da Silva TC, Bareford LM, Hillgren KM, Schmittgen TD and Swaan PW

    Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201, USA.

    Riboflavin is thoroughly established to be indispensable in a multitude of cellular oxidation-reduction reactions through its conversion to coenzyme forms flavin mononucleotide and flavin adenine dinucleotide. Despite its physiological importance, little is known about specific mechanisms or proteins involved in regulating its cellular entry in humans. Studies involving biochemical modulators and immunological inhibition assays have indirectly revealed that riboflavin internalization and trafficking occurs at least in part through a clathrin-dependent receptor-mediated endocytic process. Here, using a two-tiered strategy involving RNA interference and the overexpression of dominant-negative constructs, we directly show the involvement of this endocytic mechanism through the requirement of the pluripotent endocytic vesicle scission enzyme, dynamin 2 GTPase, in human placental trophoblasts. Similar to the endocytic control ligand, transferrin, riboflavin is shown to exhibit 50% dependence on the functional expression of dynamin 2 for its active cellular entry. Furthermore, this reduced vitamin uptake correlates with >2-fold higher riboflavin association at the cell surface. In addition, fluorescent ligand endocytosis assays showing colocalization between rhodamine-riboflavin and the immunostained caveolar coat protein, caveolin 1, suggest that the active absorption of this important nutrient involves multiple and distinct endocytosis pathways.

    Funded by: NIDDK NIH HHS: DK56631

    Molecular pharmacology 2007;72;3;553-62

  • Human papillomavirus type 31 uses a caveolin 1- and dynamin 2-mediated entry pathway for infection of human keratinocytes.

    Smith JL, Campos SK and Ozbun MA

    Department of Molecular Genetics and Microbiology, The University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.

    Papillomaviruses are species-specific and epitheliotropic DNA viruses that cause tumors in their natural hosts. Certain infections with genital human papillomavirus (HPV) types are causally related to cervical cancer development. Most papillomaviruses are thought to infect cells via a clathrin-dependent pathway, yet no studies have determined the entry route in permissive host epithelial cells. Employing fluorescently labeled and native virions, we tested the effects of dominant-negative and biochemical inhibitors of cellular endocytosis pathways. Infections of human keratinocytes, a natural host cell type for HPVs, were assessed visually and by infectious entry assays. We found that HPV type 31 (HPV31) entry and initiation of early infection events require both caveolin 1 and dynamin 2 and occur independently of clathrin-mediated endocytosis. Treatment with chlorpromazine and filipin had opposing effects on HPV31 and HPV16 infection. HPV31 entry was remarkably slow, with a half-time of approximately 14 h, whereas the entry half-time of HPV16 was 4 h. Consistent with a caveola-mediated entry pathway for HPV31, the virions associated with detergent-resistant lipid rafts. During a 16-h microscopic tracking of HPV31 and HPV16 virions, no colocalization of the two viral types was observed. These data suggest that HPV31 and HPV16 virions use distinct routes for host epithelial cell entry.

    Funded by: NCI NIH HHS: CA 085747, F32 CA 123842, F32 CA123842, R01 CA085747; NIAID NIH HHS: T32 AI 07538, T32 AI007538

    Journal of virology 2007;81;18;9922-31

  • Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.

    Jeronimo C, Forget D, Bouchard A, Li Q, Chua G, Poitras C, Thérien C, Bergeron D, Bourassa S, Greenblatt J, Chabot B, Poirier GG, Hughes TR, Blanchette M, Price DH and Coulombe B

    Laboratory of Gene Transcription and Proteomics Discovery Platform, Institut de Recherches Cliniques de Montréal, Montréal, QC, Canada.

    We have performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Thirty-two tagged polypeptides yielded a network of 805 high-confidence interactions. Remarkably, the network is significantly enriched in proteins that regulate the formation of protein complexes, including a number of previously uncharacterized proteins for which we have inferred functions. The RNA polymerase II (RNAP II)-associated proteins (RPAPs) are physically and functionally associated with RNAP II, forming an interface between the enzyme and chaperone/scaffolding proteins. BCDIN3 is the 7SK snRNA methylphosphate capping enzyme (MePCE) present in an snRNP complex containing both RNA processing and transcription factors, including the elongation factor P-TEFb. Our results define a high-density protein interaction network for the mammalian transcription machinery and uncover multiple regulatory factors that target the transcription machinery.

    Funded by: Canadian Institutes of Health Research: 14309-3, 82851-1

    Molecular cell 2007;27;2;262-74

  • Two novel mutations in dynamin-2 cause axonal Charcot-Marie-Tooth disease.

    Fabrizi GM, Ferrarini M, Cavallaro T, Cabrini I, Cerini R, Bertolasi L and Rizzuto N

    Section of Clinical Neurology, Department of Neurological and Visual Sciences, University of Verona, Verona, Italy. gianmaria.fabrizi@univr.it

    Background: Recently, mutations affecting different domains of dynamin-2 (DNM2) were associated alternatively with autosomal dominant centronuclear myopathy or dominant intermediate (demyelinating and axonal) Charcot-Marie-Tooth disease (CMT) type B.

    Objective: To assess the etiologic role of DNM2 in CMT.

    Methods: We performed a mutational screening of DNM2 exons 13 through 16 encoding the pleckstrin homology domain in a large series of CMT patients with a broad range of nerve conduction velocities and without mutations in more common genes.

    Results: We identified two novel DNM2 mutations that cosegregated with purely axonal CMT in two pedigrees without clinical evidence of primary myopathy.

    Conclusion: Patients with axonal Charcot-Marie-Tooth disease type 2 neuropathy without mutations in more common genes should undergo investigation for DNM2 pleckstrin homology.

    Funded by: Telethon: GUP04009

    Neurology 2007;69;3;291-5

  • 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

  • Dynamin 2 is required for the enhancement of HIV-1 infectivity by Nef.

    Pizzato M, Helander A, Popova E, Calistri A, Zamborlini A, Palù G and Göttlinger HG

    Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115, USA. m.pizzato@imperial.ac.uk

    Nef is a virulence factor of HIV-1 and other primate lentiviruses that is crucial for rapid progression to AIDS. In cell culture, Nef increases the infectivity of HIV-1 progeny virions by an unknown mechanism. We now show that dynamin 2 (Dyn2), a key regulator of vesicular trafficking, is a binding partner of Nef that is required for its ability to increase viral infectivity. Dominant-negative Dyn2 or the depletion of Dyn2 by small interfering RNA potently inhibited the effect of Nef on HIV-1 infectivity. Furthermore, in Dyn2-depleted cells, this function of Nef could be rescued by ectopically expressed Dyn2 but not by Dyn1, a closely related isoform that does not bind Nef. The infectivity enhancement by Nef also depended on clathrin, because it was diminished in clathrin-depleted cells and profoundly inhibited in cells expressing the clathrin-binding domain of AP180, which blocks clathrin-coated pit formation but not clathrin-independent endocytosis. Together, these findings imply that the infectivity enhancement activity of Nef depends on Dyn2- and clathrin-mediated membrane invagination events.

    Funded by: NIAID NIH HHS: AI29873, AI54261, R01 AI029873, R37 AI029873

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;16;6812-7

  • Two mechanistically distinct forms of endocytosis in adrenal chromaffin cells: Differential effects of SH3 domains and amphiphysin antagonism.

    Elhamdani A, Azizi F, Solomaha E, Palfrey HC and Artalejo CR

    Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA. aelhamda@med.wayne.edu

    We previously identified two forms of endocytosis using capacitance measurements in chromaffin cells: rapid endocytosis (RE), dynamin-1 dependent but clathrin-independent and slow endocytosis (SE), dynamin-2 and clathrin-dependent. Various recombinant SH3 domains that interact with the proline-rich domain of dynamin were introduced into single cells via the patch pipette. GST-SH3 domains of amphiphysin-1, intersectin-IC, and endophilin-I inhibited SE but had no effect on RE. Grb2-SH3 (N-terminal) or a mutant of amphiphysin-1-SH3 was inactive on either process. These data confirm that dynamin-1 dependent RE is independent of clathrin and show that amphiphysin is exclusively associated with clathrin and dynamin-2-dependent SE.

    Funded by: NIDDK NIH HHS: DK-58921; NIGMS NIH HHS: GM-56396; NIMH NIH HHS: MH-47181

    FEBS letters 2006;580;13;3263-9

  • Differential regulation of interleukin 5-stimulated signaling pathways by dynamin.

    Gorska MM, Cen O, Liang Q, Stafford SJ and Alam R

    Division of Allergy and Immunology, Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.

    Through the yeast two-hybrid screen we have identified dynamin-2 as a molecule that interacts with the alpha subunit of the interleukin (IL) 5 receptor. Dynamin-2 is a GTPase that is critical for endocytosis. We have shown that dynamin-2 interacts with the IL-5 receptor-associated tyrosine kinases, Lyn and JAK2, in eosinophils. Tyrosine phosphorylation of dynamin is markedly enhanced upon IL-5 stimulation. The inhibition of tyrosine kinases results in complete abolition of ligand-induced receptor endocytosis. Inhibition of dynamin by a dominant-negative mutant or by small interfering RNA results in enhancement of IL-5-stimulated ERK1/2 signaling and cell proliferation. In contrast, the absence of a functional dynamin does not affect STAT5 or AKT phosphorylation or cell survival. Thus, we have identified specific functions for dynamin in the IL-5 signaling pathway and demonstrated its role in receptor endocytosis and termination of the ERK1/2 signaling pathway.

    Funded by: NIAID NIH HHS: AI059719, AI68088, R01 AI50179

    The Journal of biological chemistry 2006;281;20;14429-39

  • Complexes of syndapin II with dynamin II promote vesicle formation at the trans-Golgi network.

    Kessels MM, Dong J, Leibig W, Westermann P and Qualmann B

    Department of Neurochemistry and Molecular Biology, AG Membrane Trafficking and Cytoskeleton, Leibniz Institute for Neurobiology, 39118 Magdeburg, Germany.

    The role of dynamin and so-called accessory proteins in endocytosis is well established. However, molecular details of the function(s) of dynamin II at the Golgi are largely unclear. We demonstrate that the ubiquitously expressed syndapin II isoform interacts with the proline-rich domain (PRD) of dynamin II through its Src-homology 3 (SH3) domain. Co-immunoprecipitation of endogenous syndapin II and dynamin II, and successful reconstitutions of such complexes at membranes in COS-7 cells, show the in vivo relevance of the interaction. Syndapin II can associate with Golgi membranes and this association increases upon Golgi exit block. Brefeldin A treatment clearly shows that the observed perinuclear localization of syndapin II co-localizing with syntaxin 6 reflects the Golgi complex and that it requires functional integrity of the Golgi. Syndapins are crucial for Golgi vesicle formation because anti-syndapin antibodies, used either in in vitro reconstitutions or in living cells, inhibited this process. Both types of assays additionally revealed the essential role of syndapin II SH3 interactions with the dynamin II PRD in vesicle formation. An excess of the syndapin SH3 domain strongly inhibited budding from Golgi membranes in vitro. Likewise, overexpression of the syndapin SH3 domain or of a dynamin II variant incapable of associating with syndapin II (dynamin IIDeltaPRD) impaired trafficking of vesicular stomatitis virus glycoprotein (VSVG)-GFP in vivo. By contrast, full-length syndapin II-l had no negative effect, and instead promoted VSVG-GFP export from the Golgi. Importantly, a cytosolic fraction containing endogenous syndapin-dynamin complexes was sufficient to promote vesicle formation from Golgi membranes in a syndapin-dependent manner. Thus, syndapin-dynamin complexes are crucial and sufficient to promote vesicle formation from the trans-Golgi network.

    Journal of cell science 2006;119;Pt 8;1504-16

  • TTP specifically regulates the internalization of the transferrin receptor.

    Tosoni D, Puri C, Confalonieri S, Salcini AE, De Camilli P, Tacchetti C and Di Fiore PP

    IFOM, Istituto FIRC di Oncologia Molecolare, Via Adamello 16, 20139 Milan, Italy.

    Different plasma membrane receptors are internalized through saturable/noncompetitive pathways, suggesting cargo-specific regulation. Here, we report that TTP (SH3BP4), a SH3-containing protein, specifically regulates the internalization of the transferrin receptor (TfR). TTP interacts with endocytic proteins, including clathrin, dynamin, and the TfR, and localizes selectively to TfR-containing coated-pits (CCP) and -vesicles (CCV). Overexpression of TTP specifically inhibits TfR internalization, and causes the formation of morphologically aberrant CCP, which are probably fission impaired. This effect is mediated by the SH3 of TTP, which can bind to dynamin, and it is rescued by overexpression of dynamin. Functional ablation of TTP causes a reduction in TfR internalization, and reduced cargo loading and size of TfR-CCV. Tyrosine phosphorylation of either TTP or dynamin prevents their interaction, pointing to a possible mechanism of exclusion of TTP from some CCP. Thus, TTP might represent one of the long sought for molecules that allow cargo-specific control of clathrin endocytosis.

    Funded by: NCI NIH HHS: CA46128

    Cell 2005;123;5;875-88

  • Mutations in dynamin 2 cause dominant centronuclear myopathy.

    Bitoun M, Maugenre S, Jeannet PY, Lacène E, Ferrer X, Laforêt P, Martin JJ, Laporte J, Lochmüller H, Beggs AH, Fardeau M, Eymard B, Romero NB and Guicheney P

    INSERM U582, Institute of Myology, IFR14, Groupe Hospitalier Pitié-Salpêtrière, UPMC, 47 Boulevard de l'Hôpital, 75651 Paris Cedex 13, France.

    Autosomal dominant centronuclear myopathy is a rare congenital myopathy characterized by delayed motor milestones and muscular weakness. In 11 families affected by centronuclear myopathy, we identified recurrent and de novo missense mutations in the gene dynamin 2 (DNM2, 19p13.2), which encodes a protein involved in endocytosis and membrane trafficking, actin assembly and centrosome cohesion. The transfected mutants showed reduced labeling in the centrosome, suggesting that DNM2 mutations might cause centronuclear myopathy by interfering with centrosome function.

    Nature genetics 2005;37;11;1207-9

  • Regulatory role of dynamin-2 in VEGFR-2/KDR-mediated endothelial signaling.

    Bhattacharya R, Kang-Decker N, Hughes DA, Mukherjee P, Shah V, McNiven MA and Mukhopadhyay D

    Department of Biochemistry and Molecular Biology, Mayo Clinic Cancer Center, Rochester, Minnesota 55905, USA.

    Vascular endothelial growth factor receptor-2 (VEGFR-2, also known as KDR) is a receptor tyrosine kinase (RTK) regulating mitogenic, chemotactic, permeability, and survival signals in vascular endothelial cells (EC) in response to its ligand, vascular permeability factor/VEGF (VPF/VEGF), arguably the most important angiogenic cytokine. However, the compartmentalization of KDR in EC and the mechanisms regulating this process have not been well defined. Here, we demonstrate that KDR is present on the plasma membrane, on endosomes, and in the perinuclear region of EC and colocalizes with early endosomal antigen (EEA1), caveolin-1, and dynamin-2, a signal transducing GTPase involved in receptor endocytosis. Furthermore, we also observed that dynamin-2 coimmunoprecipitates with KDR and is required for EC signaling/survival. Interestingly, EC overexpressing a mutant form of dynamin deficient in GTP binding (K44A) caused a selective inhibition in KDR protein level and endosomal vesicle formation and induced cell cycle arrest by inducing p21. Taken together, our findings suggest that dynamin-2 regulates KDR expression and function and hence plays an important role in VPF/VEGF mediated angiogenesis.

    Funded by: NCI NIH HHS: CA-78383; NHLBI NIH HHS: HL-072178, HL-70567

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2005;19;12;1692-4

  • 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

  • Kinetics of Src homology 3 domain association with the proline-rich domain of dynamins: specificity, occlusion, and the effects of phosphorylation.

    Solomaha E, Szeto FL, Yousef MA and Palfrey HC

    Department of Neurobiology, Pharamacology, and Physiology, University of Chicago, IL 60637, USA.

    Dynamin function is mediated in part through association of its proline-rich domain (PRD) with the Src homology 3 (SH3) domains of several putative binding proteins. To assess the specificity and kinetics of this process, we undertook surface plasmon resonance studies of the interaction between isolated PRDs of dynamin-1 and -2 and several purified SH3 domains. Glutathione S-transferase-linked SH3 domains bound with high affinity (K(D) approximately 10 nm to 1 microm) to both dynamin-1 and -2. The simplest interaction appeared to take place with the amphiphysin-SH3 domain; this bound to a single high affinity site (K(D) approximately 10 nm) in the C terminus of dynamin-1 PRD, as predicted by previous studies. Binding to the dynamin-2 PRD was also monophasic but with a slightly lower affinity (K(D) approximately 25 nm). Endophilin-SH3 binding to both dynamin-1 and -2 PRDs was biphasic, with one high affinity site (K(D) approximately 14 nm) in the N terminus of the PRD and another lower affinity site (K(D) approximately 60 nm) in the C terminus of dynamin-1. The N-terminal site in dynamin-2 PRD had a 10-fold lower affinity for endophilin-SH3. Preloading of dynamin-1 PRD with the amphiphysin-SH3 domain partially occluded binding of the endophilin-SH3 domain, indicating overlap between the binding sites in the C terminus, but endophilin was still able to interact with the high affinity N-terminal site. This shows that more than one SH3 domain can simultaneously bind to the PRD and suggests that competition probably occurs in vivo between different SH3-containing proteins for the limited number of PXXP motifs. Endophilin-SH3 binding to the high affinity site was disrupted when dynamin-1 PRD was phosphorylated with Cdk5, indicating that this site overlaps the phosphorylation sites, but amphiphysin-SH3 binding was unaffected. Other SH3 domains showed similarly complex binding characteristics, and substantial differences were noted between the PRDs from dynamin-1 and -2. For example, SH3 domains from c-Src, Grb2, and intersectin bound only to the C-terminal half of dynamin-2 PRD but to both the N- and C-terminal portions of dynamin-1 PRD. Thus, differential binding of SH3 domain-containing proteins to dynamin-1 and -2 may contribute to the distinct functions performed by these isoforms.

    The Journal of biological chemistry 2005;280;24;23147-56

  • Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase.

    Ezratty EJ, Partridge MA and Gundersen GG

    Department of Anatomy and Cell Biology, Columbia University, 630 West 168th Street, BB 1217, New York, NY 10032, USA.

    Imaging studies implicate microtubule targeting of focal adhesions in focal adhesion disassembly, although the molecular mechanism is unknown. Here, we develop a model system of focal adhesion disassembly based on the finding that microtubule regrowth after nocodazole washout induces disassembly of focal adhesions, and that this disassembly occurs independently of Rho and Rac, but depends on focal adhesion kinase (FAK) and dynamin. During disassembly, dynamin interacts with FAK and colocalizes with focal adhesions. Inhibition of dynamin prevents migration of cells with a focal adhesion phenotype. Our results show that focal adhesion disassembly involves microtubules, dynamin and FAK, and is not simply the reversal of focal adhesion formation.

    Funded by: NIGMS NIH HHS: GM68595

    Nature cell biology 2005;7;6;581-90

  • SNX9 regulates dynamin assembly and is required for efficient clathrin-mediated endocytosis.

    Soulet F, Yarar D, Leonard M and Schmid SL

    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

    Dynamin, a central player in clathrin-mediated endocytosis, interacts with several functionally diverse SH3 domain-containing proteins. However, the role of these interactions with regard to dynamin function is poorly defined. We have investigated a recently identified protein partner of dynamin, SNX9, sorting nexin 9. SNX9 binds directly to both dynamin-1 and dynamin-2. Moreover by stimulating dynamin assembly, SNX9 stimulates dynamin's basal GTPase activity and potentiates assembly-stimulated GTPase activity on liposomes. In fixed cells, we observe that SNX9 partially localizes to clathrin-coated pits. Using total internal reflection fluorescence microscopy in living cells, we detect a transient burst of EGFP-SNX9 recruitment to clathrin-coated pits that occurs during the late stages of vesicle formation and coincides spatially and temporally with a burst of dynamin-mRFP fluorescence. Transferrin internalization is inhibited in HeLa cells after siRNA-mediated knockdown of SNX9. Thus, our results establish that SNX9 is required for efficient clathrin-mediated endocytosis and suggest that it functions to regulate dynamin activity.

    Funded by: NIGMS NIH HHS: GM42455, R01 GM042455; NIMH NIH HHS: MH61345, R01 MH061345, R37 MH061345

    Molecular biology of the cell 2005;16;4;2058-67

  • Dynamin 2 regulates T cell activation by controlling actin polymerization at the immunological synapse.

    Gomez TS, Hamann MJ, McCarney S, Savoy DN, Lubking CM, Heldebrant MP, Labno CM, McKean DJ, McNiven MA, Burkhardt JK and Billadeau DD

    Department of Immunology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.

    Actin reorganization at the immunological synapse is required for the amplification and generation of a functional immune response. Using small interfering RNA, we show here that dynamin 2 (Dyn2), a large GTPase involved in receptor-mediated internalization, did not alter antibody-mediated T cell receptor internalization but considerably affected T cell receptor-stimulated T cell activation by regulating multiple biochemical signaling pathways and the accumulation of F-actin at the immunological synapse. Moreover, Dyn2 interacted directly with the Rho family guanine nucleotide exchange factor Vav1, and this interaction was required for T cell activation. These data identify a functionally important interaction between Dyn2 and Vav1 that regulates actin reorganization and multiple signaling pathways in T lymphocytes.

    Funded by: NCI NIH HHS: CA47752; NIAID NIH HHS: AI44959

    Nature immunology 2005;6;3;261-70

  • Mutations in the pleckstrin homology domain of dynamin 2 cause dominant intermediate Charcot-Marie-Tooth disease.

    Züchner S, Noureddine M, Kennerson M, Verhoeven K, Claeys K, De Jonghe P, Merory J, Oliveira SA, Speer MC, Stenger JE, Walizada G, Zhu D, Pericak-Vance MA, Nicholson G, Timmerman V and Vance JM

    Center for Human Genetics, Duke University Medical Center, Durham, North Carolina, USA.

    Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous group of peripheral neuropathies. Different chromosomal loci have been linked with three autosomal dominant, 'intermediate' types of CMT: DI-CMTA, DI-CMTB and DI-CMTC. We refined the locus associated with DI-CMTB on chromosome 19p12-13.2 to 4.2 Mb in three unrelated families with CMT originating from Australia, Belgium and North America. After screening candidate genes, we identified unique mutations in dynamin 2 (DNM2) in all families. DNM2 belongs to the family of large GTPases and is part of the cellular fusion-fission apparatus. In transiently transfected cell lines, mutations of DNM2 substantially diminish binding of DNM2 to membranes by altering the conformation of the beta3/beta4 loop of the pleckstrin homology domain. Additionally, in the Australian and Belgian pedigrees, which carry two different mutations affecting the same amino acid, Lys558, CMT cosegregated with neutropenia, which has not previously been associated with CMT neuropathies.

    Nature genetics 2005;37;3;289-94

  • Dynamin-2 regulates oxidized low-density lipoprotein-induced apoptosis of vascular smooth muscle cell.

    Kashiwakura Y, Watanabe M, Kusumi N, Sumiyoshi K, Nasu Y, Yamada H, Sawamura T, Kumon H, Takei K and Daida H

    Department of Cardiology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. yu-kashi@med.juntendo.ac.jp

    Background: On exposure to oxidized low-density lipoprotein (oxLDL), vascular cells generally undergo apoptosis, which is one of the major pathogenic factors of atherosclerosis. In this study, we examined the role of dynamin (a crucial GTPase protein in endocytosis) in oxLDL-induced apoptosis of vascular smooth muscle cells (VSMC).

    After oxLDL stimulation, dynamin-2 colocalized with LOX-1 around the cell surface, as well as oxLDL in the cytoplasm, suggesting that dynamin-2 was involved in scavenger receptor-mediated oxLDL endocytosis. Downregulation of dynamin-2 induced by dynamin-2 dominant negative plasmid (K44A) resulted in a decrease of oxLDL uptake and thereby in a reduction of apoptosis. These data demonstrated that dynamin-2 was involved in oxLDL-induced apoptosis via the oxLDL endocytotic pathway. On the other hand, dynamin-2 wild-type plasmid transfection promoted oxLDL-induced apoptosis without increasing oxLDL uptake. Interestingly, the p53 inhibitor pifithrin-alpha (PFT) significantly reduced apoptosis promoted by wild-type dynamin-2 (78% reduction compared with the PFT[-] condition). These results indicated that dynamin-2 enhanced oxLDL-induced apoptosis of VSMC by participating in the p53 pathway, probably as a signal transducer. Moreover, we demonstrated that, in advanced plaques of apolipoprotein E-/- mice, dynamin-2 expression was often enhanced in apoptotic VSMC, suggesting that dynamin-2 might participate in apoptosis of VSMC even in vivo.

    Conclusions: Our data demonstrated that dynamin-2 at least partially regulated oxLDL-induced apoptosis of VSMC by participating in 2 independent pathways: the oxLDL endocytotic pathway and the p53 pathway. These findings suggest that dynamin-2 may serve as a new research or therapeutic target in vascular disease.

    Circulation 2004;110;21;3329-34

  • 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

  • Regulated membrane recruitment of dynamin-2 mediated by sorting nexin 9.

    Lundmark R and Carlsson SR

    Department of Medical Biochemistry and Biophysics, Umeå University, S-901 87 Umeå, Sweden.

    The endocytic proteins sorting nexin 9 (SNX9) and dynamin-2 (Dyn2) assemble in the cytosol as a resting complex, together with a 41-kDa protein. We show here that the complex can be activated for membrane binding of SNX9 and Dyn2 by incubation of cytosol in the presence of ATP. SNX9 was essential for Dyn2 recruitment, whereas the reverse was not the case. RNA interference experiments confirmed that SNX9 functions as a mediator of Dyn2 recruitment to membranes in cells. The 41-kDa component was identified as the glycolytic enzyme aldolase. Aldolase bound with high affinity to a tryptophan-containing acidic sequence in SNX9 located close to its Phox homology domain, thereby blocking the membrane binding activity of SNX9. Phosphorylation of SNX9 released aldolase from the native cytosolic complex and rendered SNX9 competent for membrane binding. The results suggest that SNX9-dependent recruitment of Dyn2 to the membrane is regulated by an interaction between SNX9 and aldolase.

    The Journal of biological chemistry 2004;279;41;42694-702

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • A novel dynamin-associating molecule, formin-binding protein 17, induces tubular membrane invaginations and participates in endocytosis.

    Kamioka Y, Fukuhara S, Sawa H, Nagashima K, Masuda M, Matsuda M and Mochizuki N

    Department of Structural Analysis, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan.

    Dynamin associates with a variety of SH3 domain-containing molecules via a C-terminal proline-rich motif and takes part, with them, in endocytic processes. Here, we have investigated a new dynamin-associating molecule, formin-binding protein 17 (FBP17), involved in deforming the plasma membrane and in endocytosis. FBP17 formed tubular invaginations originating from the plasma membrane. Its N-terminal Fer/CIP4 homology domain, a coiled-coil domain, and a proline-rich motif were required for tubular invagination and self-assembly, by which tubular invagination might be induced. Using anti-FBP17 antibody, we detected positive immunoreactions in the testis that were restricted to the germ cells. We also detected FBP17 in the brain by immunoblotting and in situ hybridization. When COS cells expressing enhanced green fluorescent protein-tagged FBP17 were incubated with fluorescently labeled transferrin, epidermal growth factor, and cholera toxin, these molecules co-localized with FBP17-induced tubular invaginations, suggesting that FBP17 is involved in dynamin-mediated endocytosis in both a clathrin-dependent and -independent manner. These observations therefore indicate that FBP17 interacts with dynamin and regulates endocytosis by forming vesicotubular structures.

    The Journal of biological chemistry 2004;279;38;40091-9

  • 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

  • A protein interaction framework for human mRNA degradation.

    Lehner B and Sanderson CM

    MRC Rosalind Franklin Centre for Genomics Research, Hinxton, Cambridge CB10 1SB, United Kingdom.

    The degradation of mRNA is an important regulatory step in the control of gene expression. However, mammalian RNA decay pathways remain poorly characterized. To provide a framework for studying mammalian RNA decay, a two-hybrid protein interaction map was generated using 54 constructs from 38 human proteins predicted to function in mRNA decay. The results provide evidence for interactions between many different proteins required for mRNA decay. Of particular interest are interactions between the poly(A) ribonuclease and the exosome and between the Lsm complex, decapping factors, and 5'-->3' exonucleases. Moreover, multiple interactions connect 5'-->3' and 3'-->5' decay proteins to each other and to nonsense-mediated decay factors, providing the opportunity for coordination between decay pathways. The interaction network also predicts the internal organization of the exosome and Lsm complexes. Additional interactions connect mRNA decay factors to many novel proteins and to proteins required for other steps in gene expression. These results provide an experimental insight into the organization of proteins required for mRNA decay and their coupling to other cellular processes, and the physiological relevance of many of these interactions are supported by their evolutionary conservation. The interactions also provide a wealth of hypotheses to guide future research on mRNA degradation and demonstrate the power of exhaustive protein interaction mapping in aiding understanding of uncharacterized protein complexes and pathways.

    Genome research 2004;14;7;1315-23

  • Functional proteomics mapping of a human signaling pathway.

    Colland F, Jacq X, Trouplin V, Mougin C, Groizeleau C, Hamburger A, Meil A, Wojcik J, Legrain P and Gauthier JM

    Hybrigenics SA, 75014 Paris, France. fcolland@hybrigenics.fr

    Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.

    Genome research 2004;14;7;1324-32

  • Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference.

    Huang F, Khvorova A, Marshall W and Sorkin A

    Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80111, USA.

    To identify proteins that participate in clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR), 13 endocytic proteins were depleted in HeLa cells using highly efficient small interfering RNAs that were designed using a novel selection algorithm. The effects of small interfering RNAs on the ligand-induced endocytosis of EGFR were compared with those effects on the constitutive internalization of the transferrin receptor. The knock-downs of clathrin heavy chain and dynamin produced maximal inhibitory effects on the internalization of both receptors. Depletion of alpha, beta2, or micro2 subunits of AP-2 reduced EGF and transferrin internalization rates by 40-60%. Down-regulation of several accessory proteins individually had no effect on endocytosis but caused significant inhibition of EGF and transferrin endocytosis when the homologous proteins were depleted simultaneously. Surprisingly, knockdown of clathrin-assembly lymphoid myeloid leukemia protein, CALM, did not influence transferrin endocytosis but considerably affected EGFR internalization. Thus, CALM is the second protein besides Grb2 that appears to play a specific role in EGFR endocytosis. This study demonstrates that the efficient gene silencing by rationally designed small interfering RNA can be used as an approach to functionally analyze the entire cellular machineries, such as the clathrin-coated pits and vesicles.

    The Journal of biological chemistry 2004;279;16;16657-61

  • The DNA sequence and biology of human chromosome 19.

    Grimwood J, Gordon LA, Olsen A, Terry A, Schmutz J, Lamerdin J, Hellsten U, Goodstein D, Couronne O, Tran-Gyamfi M, Aerts A, Altherr M, Ashworth L, Bajorek E, Black S, Branscomb E, Caenepeel S, Carrano A, Caoile C, Chan YM, Christensen M, Cleland CA, Copeland A, Dalin E, Dehal P, Denys M, Detter JC, Escobar J, Flowers D, Fotopulos D, Garcia C, Georgescu AM, Glavina T, Gomez M, Gonzales E, Groza M, Hammon N, Hawkins T, Haydu L, Ho I, Huang W, Israni S, Jett J, Kadner K, Kimball H, Kobayashi A, Larionov V, Leem SH, Lopez F, Lou Y, Lowry S, Malfatti S, Martinez D, McCready P, Medina C, Morgan J, Nelson K, Nolan M, Ovcharenko I, Pitluck S, Pollard M, Popkie AP, Predki P, Quan G, Ramirez L, Rash S, Retterer J, Rodriguez A, Rogers S, Salamov A, Salazar A, She X, Smith D, Slezak T, Solovyev V, Thayer N, Tice H, Tsai M, Ustaszewska A, Vo N, Wagner M, Wheeler J, Wu K, Xie G, Yang J, Dubchak I, Furey TS, DeJong P, Dickson M, Gordon D, Eichler EE, Pennacchio LA, Richardson P, Stubbs L, Rokhsar DS, Myers RM, Rubin EM and Lucas SM

    Stanford Human Genome Center, Department of Genetics, Stanford University School of Medicine, 975 California Avenue, Palo Alto, California 94304, USA. jane@shgc.stanford.edu

    Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G + C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

    Nature 2004;428;6982;529-35

  • Proteomic identification of brain proteins that interact with dynein light chain LC8.

    Navarro-Lérida I, Martínez Moreno M, Roncal F, Gavilanes F, Albar JP and Rodríguez-Crespo I

    Departamento de Bioquímicay Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid, Spain. nacho@bbml.ucm.es

    Cytoplasmic dynein is a large minus end-directed microtubule motor that translocates cargos towards the minus end of microtubules. Light chain 8 of the dynein machinery (LC8) has been reported to interact with a large variety of proteins that possess K/RSTQT or GIQVD motifs in their sequence, hence permitting their transport in a retrograde manner. Yeast two-hybrid analysis has revealed that in brain, LC8 associates directly with several proteins such as neuronal nitric oxide synthase, guanylate kinase domain-associated protein and gephyrin. In this work, we report the identification of over 40 polypeptides, by means of a proteomic approach, that interact with LC8 either directly or indirectly. Many of the neuronal proteins that we identified cluster at the post-synaptic terminal, and some of them such as phosphofructokinase, lactate dehydrogenase or aldolase are directly involved in glutamate metabolism. Other pool of proteins identified displayed the LC8 consensus binding motif. Finally, recombinant LC8 was produced and a library of overlapping dodecapeptides (pepscan) was employed to map the LC8 binding site of some of the proteins that were previously identified using the proteomic approach, hence confirming binding to the consensus binding sites.

    Proteomics 2004;4;2;339-46

  • 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

  • Sorting nexin 9 participates in clathrin-mediated endocytosis through interactions with the core components.

    Lundmark R and Carlsson SR

    Department of Medical Biochemistry and Biophysics, Umeå University, S-901 87 Umeå, Sweden.

    Sorting nexin 9 (SNX9) belongs to a family of proteins, the sorting nexins, that are characterized by the presence of a subclass of the phosphoinositide-binding phox domain. SNX9 has in its amino terminus a Src homology 3 domain and a region with predicted low complexity followed by a carboxyl-terminal part containing the phox domain. We previously found that SNX9 is one of the major proteins in hematopoietic cells that binds to the alpha and beta2-appendages of adaptor protein complex 2 (AP-2), a protein with a critical role in the formation of clathrin-coated vesicles at the plasma membrane. In the present study we show that clathrin and dynamin-2, two other essential molecules in the endocytic process, also interact with SNX9. We found that both AP-2 and clathrin bind to the low complexity region in SNX9 in a cooperative manner, whereas dynamin-2 binds to the Src homology 3 domain. In the cytosol, SNX9 is present in a 14.5 S complex containing dynamin-2 and an unidentified 41-kDa protein. In HeLa cells, SNX9 co-localized with both AP-2 and dynamin-2 at the plasma membrane or on vesicular structures derived from it but not with the early endosomal marker EEA1 or with AP-1. The results suggest that SNX9 may be recruited together with dynamin-2 and become co-assembled with AP-2 and clathrin at the plasma membrane. Overexpression in both K562 and HeLa cells of truncated forms of SNX9 interfered with the uptake of transferrin, consistent with a role of SNX9 in endocytosis.

    The Journal of biological chemistry 2003;278;47;46772-81

  • The inhibitory gamma subunit of the type 6 retinal cGMP phosphodiesterase functions to link c-Src and G-protein-coupled receptor kinase 2 in a signaling unit that regulates p42/p44 mitogen-activated protein kinase by epidermal growth factor.

    Wan KF, Sambi BS, Tate R, Waters C and Pyne NJ

    Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 ONR, Scotland, United Kingdom.

    The inhibitory gamma subunit of the retinal photoreceptor type 6 cGMP phosphodiesterase (PDEgamma) is phosphorylated by G-protein-coupled receptor kinase 2 on threonine 62 and regulates the epidermal growth factor- dependent stimulation of p42/p44 mitogen-activated protein kinase in human embryonic kidney 293 cells. We report here that PDEgamma is in a pre-formed complex with c-Src and that stimulation of cells with epidermal growth factor promotes the association of GRK2 with this complex. c-Src has a critical role in the stimulation of the p42/p44 mitogen-activated protein kinase cascade by epidermal growth factor, because c-Src inhibitors block the activation of this kinase by the growth factor. Mutation of Thr-62 (to Ala) in PDEgamma produced a GRK2 phosphorylation-resistant mutant that was less effective in associating with GRK2 in response to epidermal growth factor and did not potentiate the stimulation of p42/p44 mitogen-activated protein kinase by this growth factor. The transcript for a short splice variant version of PDEgamma lacking the Thr-62 phosphorylation site is also expressed in certain mammalian cells and, in common with the Thr-62 mutant, failed to potentiate the stimulatory effect of epidermal growth factor on p42/p44 mitogen-activated protein kinase. The mutation of Thr-22 (to Ala) in PDEgamma, which is a site for phosphorylation by p42/p44 mitogen-activated protein kinase, resulted in a prolonged activation of p42/p44 mitogen-activated protein kinase by epidermal growth factor, suggesting a role for this phosphorylation event in the negative feedback control of PDEgamma.

    The Journal of biological chemistry 2003;278;20;18658-63

  • Agonist-induced endocytosis of lysophosphatidic acid-coupled LPA1/EDG-2 receptors via a dynamin2- and Rab5-dependent pathway.

    Murph MM, Scaccia LA, Volpicelli LA and Radhakrishna H

    School of Biology and Petit Institute for Biosciences and Bioengineering, Georgia Institute of Technology, Atlanta, GA 30332-0363, USA.

    Lysophosphatidic acid (LPA) is a serum-borne phospholipid that exerts a pleiotropic range of effects on cells through activation of three closely related G-protein-coupled receptors termed LPA1/EDG-2, LPA2/EDG-4 and LPA3/EDG-7. Of these receptors, the LPA1 receptor is the most widely expressed. In this study, we investigated the agonist-induced endocytosis of the human LPA1 receptor, bearing an N-terminal FLAG epitope tag, in stably transfected HeLa cells. Treatment with LPA induced the rapid endocytosis of approximately 40% of surface LPA1 within 15 minutes. Internalization was both dose dependent and LPA specific since neither lysophophatidylcholine nor sphingosine-1-phosphate induced LPA1 endocytosis. Removal of agonist following 30 minutes incubation resulted in recycling of LPA1 back to the cell surface. LPA1 internalization was strongly inhibited by dominant-inhibitory mutants of both dynamin2 (K44A) and Rab5a (S34N). In addition, both dynamin2 K44A and Rab5 S34N mildly inhibited LPA1-dependent activation of serum response factor. Finally, our results also indicate that LPA1 exhibits basal, LPA-dependent internalization in the presence of serum-containing medium.

    Funded by: NHLBI NIH HHS: HL 67134

    Journal of cell science 2003;116;Pt 10;1969-80

  • 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

  • Dynamin at the actin-membrane interface.

    Orth JD and McNiven MA

    Department of Biochemistry and Molecular Biology and the Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, MN 55905, USA.

    Many important cellular processes such as phagocytosis, cell motility and endocytosis require the participation of a dynamic and interactive actin cytoskeleton that acts to deform cellular membranes. The extensive family of non-traditional myosins has been implicated in linking the cortical actin gel with the plasma membrane. Recently, however, the dynamins have also been included in these cell processes as a second family of mechanochemical enzymes that self-associate and hydrolyze nucleotides to perform 'work' while linking cellular membranes to the actin cytoskeleton. The dynamins are believed to form large helical polymers from which extend many interactive proline-rich tail domains, and these domains bind to a variety of SH3-domain-containing proteins, many of which appear to be actin-binding proteins. Recent data support the concept that the dynamin family might act as a 'polymeric contractile scaffold' at the interface between biological membranes and filamentous actin.

    Current opinion in cell biology 2003;15;1;31-9

  • Dynamin2 and cortactin regulate actin assembly and filament organization.

    Schafer DA, Weed SA, Binns D, Karginov AV, Parsons JT and Cooper JA

    Department of Biology, University of Virginia, Charlottesville, VA 22904, USA. das9w@virginia.edu

    The GTPase dynamin is required for endocytic vesicle formation. Dynamin has also been implicated in regulating the actin cytoskeleton, but the mechanism by which it does so is unclear. Through interactions via its proline-rich domain (PRD), dynamin binds several proteins, including cortactin, profilin, syndapin, and murine Abp1, that regulate the actin cytoskeleton. We investigated the interaction of dynamin2 and cortactin in regulating actin assembly in vivo and in vitro. When expressed in cultured cells, a dynamin2 mutant with decreased affinity for GTP decreased actin dynamics within the cortical actin network. Expressed mutants of cortactin that have decreased binding of Arp2/3 complex or dynamin2 also decreased actin dynamics. Dynamin2 influenced actin nucleation by purified Arp2/3 complex and cortactin in vitro in a biphasic manner. Low concentrations of dynamin2 enhanced actin nucleation by Arp2/3 complex and cortactin, and high concentrations were inhibitory. Dynamin2 promoted the association of actin filaments nucleated by Arp2/3 complex and cortactin with phosphatidylinositol 4,5-bisphosphate (PIP2)-containing lipid vesicles. GTP hydrolysis altered the organization of the filaments and the lipid vesicles. We conclude that dynamin2, through an interaction with cortactin, regulates actin assembly and actin filament organization at membranes.

    Funded by: NCI NIH HHS: CA29243; NIGMS NIH HHS: GM 38542, GM55562, R01 GM038542, R01 GM038542-14

    Current biology : CB 2002;12;21;1852-7

  • Reduction in the minimum candidate interval in the dominant-intermediate form of Charcot-Marie-Tooth neuropathy to D19S586 to D19S432.

    Speer MC, Graham FL, Bonner E, Collier K, Stajich JM, Gaskell PC, Pericak-Vance MA and Vance JM

    Center for Human Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA. marcy@chg.mc.duke.edu

    As part of an on-going genomic screen of unlinked Charcot-Marie-Tooth disease type 2 (CMT2) families, we identified 11 regions in the genome with lod scores > or = 1.0. One of these regions was near the recently identified CMTDI1 locus on 19q. We show evidence of linkage of DUK 1118 to this region and our data reduce the minimum candidate interval for CMTDI1 to the 9-cM interval spanned by D19S586 and D19S432. We also demonstrate that five additional CMT2 families are unlinked to 19q markers, providing further evidence of CMT2 heterogeneity.

    Funded by: NINDS NIH HHS: NS26630

    Neurogenetics 2002;4;2;83-5

  • Identification of novel SH3 domain ligands for the Src family kinase Hck. Wiskott-Aldrich syndrome protein (WASP), WASP-interacting protein (WIP), and ELMO1.

    Scott MP, Zappacosta F, Kim EY, Annan RS and Miller WT

    Department of Physiology and Biophysics, School of Medicine, State University of New York, Stony Brook, New York 11794-8661, USA.

    The importance of the SH3 domain of Hck in kinase regulation, substrate phosphorylation, and ligand binding has been established. However, few in vivo ligands are known for the SH3 domain of Hck. In this study, we used mass spectrometry to identify approximately 25 potential binding partners for the SH3 domain of Hck from the monocyte cell line U937. Two major interacting proteins were the actin binding proteins Wiskott-Aldrich syndrome protein (WASP) and WASP-interacting protein (WIP). We also focused on a novel interaction between Hck and ELMO1, an 84-kDa protein that was recently identified as the mammalian ortholog of the Caenorhabditis elegans gene, ced-12. In mammalian cells, ELMO1 interacts with Dock180 as a component of the CrkII/Dock180/Rac pathway responsible for phagocytosis and cell migration. Using purified proteins, we confirmed that WASP-interacting protein and ELMO1 interact directly with the SH3 domain of Hck. We also show that Hck and ELMO1 interact in intact cells and that ELMO1 is heavily tyrosine-phosphorylated in cells that co-express Hck, suggesting that it is a substrate of Hck. The binding of ELMO1 to Hck is specifically dependent on the interaction of a polyproline motif with the SH3 domain of Hck. Our results suggest that these proteins may be novel activators/effectors of Hck.

    Funded by: NCI NIH HHS: CA58530

    The Journal of biological chemistry 2002;277;31;28238-46

  • Dynamin and endocytosis.

    Sever S

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Dana-Farber Cancer Institute, 1 Jimmy Fund Way, Boston, MA 02115, USA. sanja_sever@dfci.harvard.edu

    The GTPase dynamin is essential for endocytosis, but its mechanism of action remains uncertain. Structures of its GTPase domain, as well as that of assembled dynamin, have led to major advances in understanding the structural basis of its mode of action. Novel data point more clearly than ever towards a role for this protein in the actin cytoskeleton, mitogen-activated protein kinase signaling and apoptosis, suggesting that dynamin might be a signaling GTPase.

    Current opinion in cell biology 2002;14;4;463-7

  • Dynamin at actin tails.

    Lee E and De Camilli P

    Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, P.O. Box 9812, New Haven, CT 06536-0812, USA.

    Dynamin, the product of the shibire gene of Drosophila, is a GTPase critically required for endocytosis. Some studies have suggested a functional link between dynamin and the actin cytoskeleton. This link is of special interest, because there is evidence implicating actin dynamics in endocytosis. Here we show that endogenous dynamin 2, as well as green fluorescence protein fusion proteins of both dynamin 1 and 2, is present in actin comets generated by Listeria or by type I PIP kinase (PIPK) overexpression. In PIPK-induced tails, dynamin is further enriched at the interface between the tails and the moving organelles. Dynamin mutants harboring mutations in the GTPase domain inhibited nucleation of actin tails induced by PIPK and moderately reduced their speed. Although dynamin localization to the tails required its proline-rich domain, expression of a dynamin mutant lacking this domain also diminished tail formation. In addition, this mutant disrupted a membrane-associated actin scaffold (podosome rosette) previously shown to include dynamin. These findings suggest that dynamin is part of a protein network that controls nucleation of actin from membranes. At endocytic sites, dynamin may couple the fission reaction to the polymerization of an actin pool that functions in the separation of the endocytic vesicles from the plasma membrane.

    Funded by: NCI NIH HHS: CA46128, P01 CA046128; NINDS NIH HHS: NS36251, R01 NS036251, R37 NS036251

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;1;161-6

  • Dynamin: characteristics, mechanism of action and function.

    Wiejak J and Wyroba E

    Nencki Institute of Experimental Biology, Pasteura 3, 02-093 Warszawa, Poland.

    Dynamin - a member of the GTP-ase protein family - is essential for many intracellular membrane trafficking events in multiple endocytic processes. The unique biochemical features of dynamin - especially its propensity to assemble - enable severing the nascent vesicles from the membrane. The mechanism of dynamin's action is still a subject of debate - whether it functions as a mechanochemical enzyme or a regulatory GTPase. The GTPase domain of dynamin contains three GTP-binding motifs. This domain is very conservative across the species, including that recently cloned by us in the unicellular eukaryote Paramecium. Dynamin interacts with a number of partners such as endophilin and proteins involved in coordination of endocytosis with motor molecules. A growing body of evidence indicates that dynamin and dynamin-related proteins are involved both in pathology and protection against human diseases. The most interesting are dynamin-like Mx proteins exhibiting antiviral activity.

    Cellular & molecular biology letters 2002;7;4;1073-80

  • Dynamin isoform-specific interaction with the shank/ProSAP scaffolding proteins of the postsynaptic density and actin cytoskeleton.

    Okamoto PM, Gamby C, Wells D, Fallon J and Vallee RB

    Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

    Dynamin is a GTPase involved in endocytosis and other aspects of membrane trafficking. A critical function in the presynaptic compartment attributed to the brain-specific dynamin isoform, dynamin-1, is in synaptic vesicle recycling. We report that dynamin-2 specifically interacts with members of the Shank/ProSAP family of postsynaptic density scaffolding proteins and present evidence that dynamin-2 is specifically associated with the postsynaptic density. These data are consistent with a role for this otherwise broadly distributed form of dynamin in glutamate receptor down-regulation and other aspects of postsynaptic membrane turnover.

    Funded by: NCRR NIH HHS: P20 RR015578, P20 RR015578-020002; NIGMS NIH HHS: GM26701; NINDS NIH HHS: P01 NS039321, P01 NS039321-020002

    The Journal of biological chemistry 2001;276;51;48458-65

  • The inhibitory gamma subunit of the type 6 retinal cyclic guanosine monophosphate phosphodiesterase is a novel intermediate regulating p42/p44 mitogen-activated protein kinase signaling in human embryonic kidney 293 cells.

    Wan KF, Sambi BS, Frame M, Tate R and Pyne NJ

    Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 ONR, United Kingdom.

    The inhibitory gamma subunits of the retinal rod and cone photoreceptor type 6 retinal cyclic guanosine monophosphate phosphodiesterase (PDEgamma) are expressed in non-retinal tissues. Here, we show that PDEgamma interacts with the G-protein-coupled receptor kinase 2 signaling system to regulate the epidermal growth factor- and thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase in human embryonic kidney 293 cells. This is based upon several lines of evidence. First, the transfection of cells with an antisense rod PDEgamma plasmid construct, which reduced endogenous rod PDEgamma expression, ablated the epidermal growth factor- and thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase. Second, the transfection of cells with recombinant rod or cone PDEgamma and/or G-protein-coupled receptor kinase 2 increased the stimulation of p42/p44 mitogen-activated protein kinase by epidermal growth factor or thrombin. In contrast, a G-protein-coupled receptor kinase 2 phosphorylation-resistant rod PDEgamma mutant failed to increase the epidermal growth factor- or thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase and, in fact, functioned as a dominant negative. Thrombin also stimulated the association of endogenous rod PDEgamma with dynamin II, which was increased in cells transfected with rod PDEgamma or G-protein-coupled receptor kinase 2. Dynamin II plays a critical role in regulating endocytosis of receptor signal complexes required for activation of p42/p44 mitogen-activated protein kinase. Therefore, PDEgamma may have an important role in promoting endocytosis of receptor signal complexes leading to the activation of p42/p44 mitogen-activated protein kinase. We conclude that PDEgamma is an entirely novel intermediate regulating mitogenic signaling from both receptor tyrosine kinase and G-protein-coupled receptors in human embryonic kidney 293 cells.

    The Journal of biological chemistry 2001;276;41;37802-8

  • Dominant intermediate Charcot-Marie-Tooth neuropathy maps to chromosome 19p12-p13.2.

    Kennerson ML, Zhu D, Gardner RJ, Storey E, Merory J, Robertson SP and Nicholson GA

    Neurobiology Laboratory, ANZAC Research Institute, University of Sydney, New South Wales 2139, Australia. marinak@med.usyd.edu.au

    The hereditary disorders of peripheral nerve form one of the most common groups of human genetic diseases, collectively called Charcot-Marie-Tooth (CMT) neuropathy. Using linkage analysis we have identified a new locus for a form of CMT that we have called "dominant intermediate CMT" (DI-CMT). A genomewide screen using 383 microsatellite markers showed strong linkage to the short arm of chromosome 19 (maximum LOD score 4.3, with a recombination fraction (straight theta) of 0, at D19S221 and maximum LOD score 5.28, straight theta=0, at D19S226). Haplotype analysis performed with 14 additional markers placed the DI-CMT locus within a 16.8-cM region flanked by the markers D19S586 and D19S546. Multipoint linkage analysis suggested the most likely location at D19S226 (maximum multipoint LOD score 6.77), within a 10-cM confidence interval. This study establishes the presence of a locus for DI-CMT on chromosome 19p12-p13.2.

    American journal of human genetics 2001;69;4;883-8

  • The Eps15 C. elegans homologue EHS-1 is implicated in synaptic vesicle recycling.

    Salcini AE, Hilliard MA, Croce A, Arbucci S, Luzzi P, Tacchetti C, Daniell L, De Camilli P, Pelicci PG, Di Fiore PP and Bazzicalupo P

    Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy.

    Eps15 represents the prototype of a family of evolutionarily conserved proteins that are characterized by the presence of the EH domain, a protein-protein interaction module, and that are involved in many aspects of intracellular vesicular sorting. Although biochemical and functional studies have implicated Eps15 in endocytosis, its function in the endocytic machinery remains unclear. Here we show that the Caenorhabditis elegans gene, zk1248.3 (ehs-1), is the orthologue of Eps15 in nematodes, and that its product, EHS-1, localizes to synaptic-rich regions. ehs-1-impaired worms showed temperature-dependent depletion of synaptic vesicles and uncoordinated movement. These phenotypes could be correlated with a presynaptic defect in neurotransmission. Impairment of EHS-1 function in dyn-1(ky51) worms, which express a mutant form of dynamin and display a temperature-sensitive locomotion defect, resulted in a worsening of the dyn-1 phenotype and uncoordination at the permissive temperature. Thus, ehs-1 and dyn-1 interact genetically. Moreover, mammalian Eps15 and dynamin protein were shown to interact in vivo. Taken together, our results indicate that EHS-1 acts in synaptic vesicle recycling and that its function might be linked to that of dynamin.

    Funded by: Telethon: D.068, D.090, E.0942, GTF01018

    Nature cell biology 2001;3;8;755-60

  • Three ways to make a vesicle.

    Kirchhausen T

    Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA. kirchhausen@crystal.harvard.edu

    Cargo molecules have to be included in carrier vesicles of different forms and sizes to be transported between organelles. During this process, a limited set of proteins, including the coat proteins COPI, COPII and clathrin, carries out a programmed set of sequential interactions that lead to the budding of vesicles. A general model to explain the formation of coated vesicles is starting to emerge but the picture is more complex than we had imagined.

    Nature reviews. Molecular cell biology 2000;1;3;187-98

  • Evidence that dynamin-2 functions as a signal-transducing GTPase.

    Fish KN, Schmid SL and Damke H

    Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

    The role of dynamin GTPases in the regulation of receptor-mediated endocytosis is well established. Here, we present new evidence that the ubiquitously expressed isoform dynamin-2 (dyn2) can also function in a signal transduction pathway(s). A </=5-fold increase of dyn2 relative to endogenous levels activates the transcription factor p53 and induces apoptosis, as demonstrated by reduced cell proliferation, DNA fragmentation, and caspase-3 activation. Dyn2-triggered apoptosis occurs only in dividing cells and is p53 dependent. A mutant defective in GTP binding does not trigger apoptosis, indicating that increased levels of dyn2.GTP, rather than protein levels per se, are required to transduce signals that activate p53. A truncated dyn2 lacking the COOH-terminal proline/arginine-rich domain (PRD), which interacts with many SH3 domain-containing partners implicated in both endocytosis and signal transduction, triggers apoptosis even more potently than the wild-type. This observation provides additional support for the importance of the NH(2)-terminal GTPase domain for the apoptotic phenotype. All described effects are dyn2-specific because >200-fold overexpression of dyn1, the 70% identical neuronal isoform, has no effect. Our data suggest that dyn2 can act as a signal transducing GTPase affecting transcriptional regulation.

    Funded by: NCI NIH HHS: CA58689; NIGMS NIH HHS: F32 GM019689, F32 GM019689-01A1, F32 GM019689-02, F32 GM019689-03, GM19689, GM42455, R01 GM042455

    The Journal of cell biology 2000;150;1;145-54

  • Secretory protein trafficking and organelle dynamics in living cells.

    Lippincott-Schwartz J, Roberts TH and Hirschberg K

    Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA. jlippin@helix.nih.gov

    Green fluorescent protein chimerae acting as reporters for protein localization and trafficking within the secretory membrane system of living cells have been used in a wide variety of applications, including time-lapse imaging, double-labeling, energy transfer, quantitation, and photobleaching experiments. Results from this work are clarifying the steps involved in the formation, translocation, and fusion of transport intermediates; the organization and biogenesis of organelles; and the mechanisms of protein retention, sorting, and recycling in the secretory pathway. In so doing, they are broadening our thinking about the temporal and spatial relationships among secretory organelles and the membrane trafficking pathways that operate between them.

    Annual review of cell and developmental biology 2000;16;557-89

  • Characterization of RANTES- and aminooxypentane-RANTES-triggered desensitization signals reveals differences in recruitment of the G protein-coupled receptor complex.

    Vila-Coro AJ, Mellado M, Martín de Ana A, Martínez-A C and Rodríguez-Frade JM

    Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas/Universidad Autonoma de Madrid, Spain.

    The trafficking of lymphocyte populations is a complex process controlled by a vast array of molecules. In this process, cells must be able to sense small changes in chemoattractant gradients. Migration through a chemotactic gradient probably employs an on-off mechanism in which chemokine receptor desensitization, internalization, and recycling may be important steps. This multistep process requires the coordinated action of many factors, including G protein-coupled receptor kinases, arrestins, clathrin, and GTP-hydrolyzing proteins such as dynamin. In this report, we show that RANTES and its derivative, aminooxypentane (AOP)-RANTES, a potent RANTES antagonist as well as an inhibitor of HIV-1 infection, both promote CCR5 desensitization involving G protein-coupled receptor kinases-2 and beta-arrestin equally well. An important difference between the two molecules is that (AOP)-RANTES is more efficient than RANTES in promoting Ser/Thr phosphorylation of the receptor and association of G protein-coupled receptor kinases-2, beta-arrestin, and clathrin to the CCR5. After stimulation with either ligand, we observe rapid, transient association of dynamin to CCR5, implicating this protein in receptor sensitization, but this association is faster and longer-lasting following (AOP)-RANTES stimulation. In summary, we show that chemokine receptor internalization takes place through the formation of clathrin vesicles and involves dynamin activity. We provide compelling evidence that the differences between RANTES and (AOP)-RANTES in G alpha i activation condition subsequent signaling events, including internalization and receptor recycling.

    Journal of immunology (Baltimore, Md. : 1950) 1999;163;6;3037-44

  • Dynamin II is involved in endocytosis but not in the formation of transport vesicles from the trans-Golgi network.

    Kasai K, Shin HW, Shinotsuka C, Murakami K and Nakayama K

    Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City, Ibaraki, 305-8572, Japan.

    Dynamins are a family of approximately 100-kDa GTPases that are thought to play a pivotal role in the formation of endocytic coated vesicles. There are three dynamin genes in mammals: dynamin I is neuron-specific, dynamin II shows ubiquitous expression, and dynamin III is expressed in testis, brain, and lung. However, most studies on the functions of dynamins to date have been restricted to dynamin I. In the present study, we show that, like dynamin I, dynamin II is involved in receptor-mediated endocytosis. While this study was in progress, Jones et al. [Jones, S.M., Howell, K.E., Henley, J.R., Cao, H., and McNiven, M.A. (1998) Science 279, 573-577] reported that dynamin II is localized in the trans-Golgi network (TGN) and involved in the formation of constitutive transport vesicles and clathrin-coated vesicles from this compartment. However, immunofluorescence analyses and experiments using cells transfected with dominant-negative dynamin II failed to show any evidence for localization of dynamin II in the TGN or for its involvement in vesicle formation from this compartment. Our data thus indicate that dynamin II is involved in endocytosis but not in the formation of transport vesicles from the TGN.

    Journal of biochemistry 1999;125;4;780-9

  • Growth factor-dependent phosphorylation of the actin-binding protein cortactin is mediated by the cytoplasmic tyrosine kinase FER.

    Kim L and Wong TW

    Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.

    Previous characterization of the nonreceptor tyrosine kinase FER identified a tight physical association with the catenin pp120 and led to the suggestion that FER may be involved in cell-cell signaling. To further understand the function of FER, we have continued our analyses of the interaction of FER with pp120 and other proteins. The majority of FER is localized to the cytoplasmic fraction where it forms a complex with the actin-binding protein cortactin. The Src homology 2 sequence of FER is required for directly binding cortactin, and phosphorylation of the FER-cortactin complex is up-regulated in cells treated with peptide growth factors. Using a dominant-negative mutant of FER, we provided evidence that FER kinase activity is required for the growth factor-dependent phosphorylation of cortactin. These data suggest that cortactin is likely to be a direct substrate of FER. Our observations provide additional support for a role of FER in mediating signaling from the cell surface, via growth factor receptors, to the cytoskeleton. The nature of the FER-cortactin interaction, and their putative enzyme-substrate relationship, support the previous proposal that one of the functions of the Src homology 2 sequences of nonreceptor tyrosine kinases is to provide a binding site for their preferred substrates.

    The Journal of biological chemistry 1998;273;36;23542-8

  • Dynamin genes Dnm1 and Dnm2 are located on proximal mouse chromosomes 2 and 9, respectively.

    Klocke R, Augustin A, Ronsiek M, Stief A, van der Putten H and Jockusch H

    Developmental Biology Unit W7, University of Bielefeld, Germany.

    Dynamins, microtubule-binding GTPases, are encoded by at least three genes in mammals. Two distinct gene-specific cDNAs were used to analyze the segregation of dynamin genes Dnm1 and Dnm2 in a mouse interspecies backcross. The nervous system-expressed gene Dnm1 was localized to Chr 2 between the genes for vimentin and nebulin, within a chromosomal region of conserved synteny to human chromosome 9q, consistent with the localization of the human dynamin-1 gene by FISH (see accompanying paper by Newman-Smith et al., 1997, Genomics 41:286-289). The ubiquitously expressed Dnm2 gene was found to be closely linked to the intercellular adhesion molecule-1 gene, Icam1, in a region with homologies to human chromosomes 19p, 8q, and 11q. Potential relations of both loci to disease genes are discussed.

    Genomics 1997;41;2;290-2

  • Isolation of an ubiquitously expressed cDNA encoding human dynamin II, a member of the large GTP-binding protein family.

    Diatloff-Zito C, Gordon AJ, Duchaud E and Merlin G

    Institut Curie-Biologie, CNRS URA 1292, Paris, France.

    Dynamin (Dyn) is a member of a novel group of GTPases which was initially identified as a microtubule-binding protein with a role in vectorial movement. Three distinct Dyn-encoding genes (DYN I, II and III), with a neuronal-, ubiquitous or testis-specific expression, respectively, have been identified in rat. In man, only DYN I has so far been characterized. We have previously isolated a genomic DNA fragment implicated in the correction of mitomycin C hypersensitivity of cells from a Fanconi anemia patient belonging to genetic complementation group D (FA(D)). Using this probe, we have cloned a human complementary DNA designated hDYN II encoding a ubiquitous Dyn isoform. The predicted protein consists of 866 amino acids (97.5 kDa). Dyn proteins exhibit a high degree of evolutionary conservation: hDyn II is 98% identical to rat Dyn II and 73% identical to hDyn I. A unique 3.6-kb transcript is found in all human tissues examined and it is more abundant in skeletal muscle and heart. This transcript is also expressed in tissue-culture cells. The hDYN II message is present and not mutated in the FA(D) patient studied. In addition to the GTP-binding domain and motifs associated with regulatory function, the hDyn II protein contains a noticeable number of concensus motifs for p34Cdc2 kinase phosphorylation which may indicate a potential role at the G2/mitosis transition. The sequence reported here should allow a more complete analysis of Dyn function(s) in man.

    Gene 1995;163;2;301-6

Gene lists (9)

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
L00000013 G2C Homo sapiens Human mGluR5 Human orthologues of mouse mGluR5 complex adapted from Collins et al (2006) 52
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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