G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
ankyrin 3, node of Ranvier (ankyrin G)
G00000577 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000018288 (Vega human gene)
ENSG00000151150 (Ensembl human gene)
288 (Entrez Gene)
993 (G2Cdb plasticity & disease)
ANK3 (GeneCards)
600465 (OMIM)
Marker Symbol
Protein Sequence
Q12955 (UniProt)

Literature (33)

Pubmed - other

  • Two variants in Ankyrin 3 (ANK3) are independent genetic risk factors for bipolar disorder.

    Schulze TG, Detera-Wadleigh SD, Akula N, Gupta A, Kassem L, Steele J, Pearl J, Strohmaier J, Breuer R, Schwarz M, Propping P, Nöthen MM, Cichon S, Schumacher J, NIMH Genetics Initiative Bipolar Disorder Consortium, Rietschel M and McMahon FJ

    Unit on the Genetic Basis of Mood and Anxiety Disorders, National Institute of Mental Health, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD 20892-3719, USA. schulzet@mail.nih.gov

    Two recent reports have highlighted ANK3 as a susceptibility gene for bipolar disorder (BD). We first reported association between BD and the ANK3 marker rs9804190 in a genome-wide association study (GWAS) of two independent samples (Baum et al., 2008). Subsequently, a meta-analysis of GWAS data based on samples from the US and the UK reported association with a different ANK3 marker, rs10994336 (Ferreira et al., 2008). The markers lie about 340 kb apart in the gene. Here, we test both markers in additional samples and characterize the contribution of each marker to BD risk. Our previously reported findings at rs9804190, which had been based on DNA pooling, were confirmed by individual genotyping in the National Institute of Mental Health (NIMH) waves 1-4 (P=0.05; odds ratio (OR)=1.24) and German (P=0.0006; OR=1.34) samples. This association was replicated in an independent US sample known as NIMH wave 5 (466 cases, 212 controls; P=0.017; OR=1.38). A random-effects meta-analysis of all three samples was significant (P=3 x 10(-6); OR=1.32), with no heterogeneity. Individual genotyping of rs10994336 revealed a significant association in the German sample (P=0.0001; OR=1.70), and similar ORs in the NIMH 1-4 and NIMH 5 samples that were not significant at the P<0.05 level. Meta-analysis of all three samples supported an association with rs10994336 (P=1.7 x 10(-5); OR=1.54), again with no heterogeneity. There was little linkage disequilibrium between the two markers. Further analysis suggested that each marker contributed independently to BD, with no significant marker x marker interaction. Our findings strongly support ANK3 as a BD susceptibility gene and suggest true allelic heterogeneity.

    Funded by: Intramural NIH HHS: Z01 MH002810-05, Z99 MH999999; NIMH NIH HHS: R01 MH059533, R01 MH059534, R01 MH059535, R01 MH059545, R01 MH059548, R01 MH059553, R01 MH059556, R01 MH059565, R01 MH059566, R01 MH059567, R01 MH059571, R01 MH059586, R01 MH059587, R01 MH059588, R01 MH060068, R01 MH060870, R01 MH060879, R01 MH061675, R01 MH067257, Z01 MH002810

    Molecular psychiatry 2009;14;5;487-91

  • Ankyrin facilitates intracellular trafficking of alpha1-Na+-K+-ATPase in polarized cells.

    Stabach PR, Devarajan P, Stankewich MC, Bannykh S and Morrow JS

    Dept. of Pathology, Yale Univ., 310 Cedar St., New Haven, CT 06520, USA.

    Defects in ankyrin underlie many hereditary disorders involving the mislocalization of membrane proteins. Such phenotypes are usually attributed to ankyrin's role in stabilizing a plasma membrane scaffold, but this assumption may not be accurate. We found in Madin-Darby canine kidney cells and in other cultured cells that the 25-residue ankyrin-binding sequence of alpha(1)-Na(+)-K(+)-ATPase facilitates the entry of alpha(1),beta(1)-Na(+)-K(+)-ATPase into the secretory pathway and that replacement of the cytoplasmic domain of vesicular stomatitis virus G protein (VSV-G) with this ankyrin-binding sequence bestows ankyrin dependency on the endoplasmic reticulum (ER) to Golgi trafficking of VSV-G. Expression of the ankyrin-binding sequence of alpha(1)-Na(+)-K(+)-ATPase alone as a soluble cytosolic peptide acts in trans to selectively block ER to Golgi transport of both wild-type alpha(1)-Na(+)-K(+)-ATPase and a VSV-G fusion protein that includes the ankyrin-binding sequence, whereas the trafficking of other proteins remains unaffected. Similar phenotypes are also generated by small hairpin RNA-mediated knockdown of ankyrin R or the depletion of ankyrin in semipermeabilized cells. These data indicate that the adapter protein ankyrin acts not only at the plasma membrane but also early in the secretory pathway to facilitate the intracellular trafficking of alpha(1)-Na(+)-K(+)-ATPase and presumably other selected proteins. This novel ankyrin-dependent assembly pathway suggests a mechanism whereby hereditary disorders of ankyrin may be manifested as diseases of membrane protein ER retention or mislocalization.

    Funded by: NHLBI NIH HHS: R01-HL-28560; NIDDK NIH HHS: P01-DK-55389, R01-DK-43812

    American journal of physiology. Cell physiology 2008;295;5;C1202-14

  • Phosphorylation and ankyrin-G binding of the C-terminal domain regulate targeting and function of the ammonium transporter RhBG.

    Sohet F, Colin Y, Genetet S, Ripoche P, Métral S, Le Van Kim C and Lopez C

    INSERM, U665, Paris F-75015, the Institut National de la Transfusion Sanguine, 6 Rue Alexandre Cabanel, Paris F-75015, France.

    RhBG, a human member of the Amt/Mep/Rh/superfamily of ammonium transporters, has been shown to facilitate NH(3) transport and to be anchored to the basolateral plasma membrane of kidney epithelial cells, via ankyrin-G. We showed here that triple alanine substitution of the (419)FLD(421) sequence, which links the cytoplasmic C-terminal domain of RhBG to ankyrin-G, not only disrupted the interaction of RhBG with the spectrin-based skeleton but also delayed its cell surface expression, decreased its plasma membrane stability, and abolished its NH(3) transport function in epithelial cell lines. Similarly, we demonstrated that both anchoring to the membrane skeleton and ammonium transport activity are regulated by the phosphorylation status of the C-terminal tail of RhBG. Tyrosine 429, which belongs to the previously reported YED basolateral targeting signal of RhBG, was demonstrated to be phosphorylated in vitro using purified Src and Syk kinases and ex vivo by analyzing the effect of pervanadate treatment on wild-type RhBG or Y429A mutants. Then, we showed that Y429D and Y429E mutations, mimicking constitutive phosphorylation, abolished NH(3) transport and enhanced Triton X-100 solubilization of RhBG from the cell membrane. In contrast, the nonphosphorylated/nonphosphorylatable Y429A and Y429F mutants behaved the same as wild-type RhBG. Conversely, Y/A or Y/F but not Y/E or Y/D mutations of residue 429 abolished the exclusive basolateral localization of RhBG in polarized epithelial cells. All these results led to a model in which targeting and ammonium transport function of RhBG are regulated by both phosphorylation and membrane skeleton binding of the C-terminal cytoplasmic domain.

    The Journal of biological chemistry 2008;283;39;26557-67

  • Association analysis of 528 intra-genic SNPs in a region of chromosome 10 linked to late onset Alzheimer's disease.

    Morgan AR, Hamilton G, Turic D, Jehu L, Harold D, Abraham R, Hollingworth P, Moskvina V, Brayne C, Rubinsztein DC, Lynch A, Lawlor B, Gill M, O'Donovan M, Powell J, Lovestone S, Williams J and Owen MJ

    Department of Psychological Medicine, School of Medicine, Cardiff University, Cardiff, UK. morganar1@cardiff.ac.uk

    Late-onset Alzheimer's disease (LOAD) is a genetically complex neurodegenerative disorder. Currently, only the epsilon4 allele of the Apolipoprotein E gene has been identified unequivocally as a genetic susceptibility factor for LOAD. Others remain to be found. In 2002 we observed genome-wide significant evidence of linkage to a region on chromosome 10q11.23-q21.3 [Myers et al. (2002) Am J Med Genet 114:235-244]. Our objective in this study was to test every gene within the maximum LOD-1 linkage region, for association with LOAD. We obtained results for 528 SNPs from 67 genes, with an average density of 1 SNP every 10 kb within the genes. We demonstrated nominally significant association with LOAD for 4 SNPs: rs1881747 near DKK1 (P = 0.011, OR = 1.24), rs2279420 in ANK3 (P = 0.022, OR = 0.79), rs2306402 in CTNNA3 (P = 0.024, OR = 1.18), and rs5030882 in CXXC6 (P = 0.046, OR = 1.29) in 1,160 cases and 1,389 controls. These results would not survive correction for multiple testing but warrant attempts at confirmation in independent samples.

    Funded by: Medical Research Council: G0300429, G9810900; Wellcome Trust: 064354

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2008;147B;6;727-31

  • Collaborative genome-wide association analysis supports a role for ANK3 and CACNA1C in bipolar disorder.

    Ferreira MA, O'Donovan MC, Meng YA, Jones IR, Ruderfer DM, Jones L, Fan J, Kirov G, Perlis RH, Green EK, Smoller JW, Grozeva D, Stone J, Nikolov I, Chambert K, Hamshere ML, Nimgaonkar VL, Moskvina V, Thase ME, Caesar S, Sachs GS, Franklin J, Gordon-Smith K, Ardlie KG, Gabriel SB, Fraser C, Blumenstiel B, Defelice M, Breen G, Gill M, Morris DW, Elkin A, Muir WJ, McGhee KA, Williamson R, MacIntyre DJ, MacLean AW, St CD, Robinson M, Van Beck M, Pereira AC, Kandaswamy R, McQuillin A, Collier DA, Bass NJ, Young AH, Lawrence J, Ferrier IN, Anjorin A, Farmer A, Curtis D, Scolnick EM, McGuffin P, Daly MJ, Corvin AP, Holmans PA, Blackwood DH, Gurling HM, Owen MJ, Purcell SM, Sklar P, Craddock N and Wellcome Trust Case Control Consortium

    Department of Psychiatry, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.

    To identify susceptibility loci for bipolar disorder, we tested 1.8 million variants in 4,387 cases and 6,209 controls and identified a region of strong association (rs10994336, P = 9.1 x 10(-9)) in ANK3 (ankyrin G). We also found further support for the previously reported CACNA1C (alpha 1C subunit of the L-type voltage-gated calcium channel; combined P = 7.0 x 10(-8), rs1006737). Our results suggest that ion channelopathies may be involved in the pathogenesis of bipolar disorder.

    Funded by: Chief Scientist Office; Medical Research Council: G0500791, G0701003, G9309834, G9623693N; NCRR NIH HHS: U54 RR020278; NIMH NIH HHS: MH062137, MH063445, MH067288, MH63420, N01MH80001, R01 MH062137, R01 MH063420, R01 MH063445, R01 MH067288; Wellcome Trust: 076113, 077011, 082371

    Nature genetics 2008;40;9;1056-8

  • Ankyrin-G is a molecular partner of E-cadherin in epithelial cells and early embryos.

    Kizhatil K, Davis JQ, Davis L, Hoffman J, Hogan BL and Bennett V

    Howard Hughes Medical Institute, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    E-cadherin is a ubiquitous component of lateral membranes in epithelial tissues and is required to form the first lateral membrane domains in development. Here, we identify ankyrin-G as a molecular partner of E-cadherin and demonstrate that ankyrin-G and beta-2-spectrin are required for accumulation of E-cadherin at the lateral membrane in both epithelial cells and early embryos. Ankyrin-G binds to the cytoplasmic domain of E-cadherin at a conserved site distinct from that of beta-catenin. Ankyrin-G also recruits beta-2-spectrin to E-cadherin-beta-catenin complexes, thus providing a direct connection between E-cadherin and the spectrin/actin skeleton. In addition to restricting the membrane mobility of E-cadherin, ankyrin-G and beta-2-spectrin also are required for exit of E-cadherin from the trans-Golgi network in a microtubule-dependent pathway. Ankyrin-G and beta-2-spectrin co-localize with E-cadherin in preimplantation mouse embryos. Moreover, knockdown of either ankyrin-G or beta-2-spectrin in one cell of a two-cell embryo blocks accumulation of E-cadherin at sites of cell-cell contact. E-cadherin thus requires both ankyrin-G and beta-2-spectrin for its cellular localization in early embryos as well as cultured epithelial cells. We have recently reported that ankyrin-G and beta-2-spectrin collaborate in biogenesis of the lateral membrane ( Kizhatil, K., Yoon, W., Mohler, P. J., Davis, L. H., Hoffman, J. A., and Bennett, V. (2007) J. Biol. Chem. 282, 2029-2037 ). Together with the current findings, these data suggest a ankyrin/spectrin-based mechanism for coordinating membrane assembly with extracellular interactions of E-cadherin at sites of cell-cell contact.

    The Journal of biological chemistry 2007;282;36;26552-61

  • Association studies of 23 positional/functional candidate genes on chromosome 10 in late-onset Alzheimer's disease.

    Morgan AR, Turic D, Jehu L, Hamilton G, Hollingworth P, Moskvina V, Jones L, Lovestone S, Brayne C, Rubinsztein DC, Lawlor B, Gill M, O'Donovan MC, Owen MJ and Williams J

    Department of Psychological Medicine, School of Medicine, Cardiff University, Cardiff, UK.

    Late-onset Alzheimer's disease (LOAD) is a common neurodegenerative disorder, with a complex etiology. APOE is the only confirmed susceptibility gene for LOAD. Others remain yet to be found. Evidence from linkage studies suggests that a gene (or genes) conferring susceptibility for LOAD resides on chromosome 10. We studied 23 positional/functional candidate genes from our linkage region on chromosome 10 (APBB1IP, ALOX5, AD037, SLC18A3, DKK1, ZWINT, ANK3, UBE2D1, CDC2, SIRT1, JDP1, NET7, SUPV3L1, NEN3, SAR1, SGPL1, SEC24C, CAMK2G, PP3CB, SNCG, CH25H, PLCE1, ANXV111) in the MRC genetic resource for LOAD. These candidates were screened for sequence polymorphisms in a sample of 14 LOAD subjects and detected polymorphisms tested for association with LOAD in a three-stage design involving two stages of genotyping pooled DNA samples followed by a third stage in which markers showing evidence for association in the first stages were subjected to individual genotyping. One hundred and twenty polymorphisms were identified and tested in stage 1 (4 case + 4 control pools totaling 366 case and 366 control individuals). Single nucleotide polymorphisms (SNPs) showing evidence of association with LOAD were then studied in stage 2 (8 case + 4 control pools totaling 1,001 case and 1,001 control individuals). Five SNPs, in four genes, showed evidence for association (P < 0.1) at stage 2 and were individually genotyped in the complete dataset, comprising 1,160 LOAD cases and 1,389 normal controls. Two SNPs in SGPL1 demonstrated marginal evidence of association, with uncorrected P values of 0.042 and 0.056, suggesting that variation in SGPL1 may confer susceptibility to LOAD.

    Funded by: Medical Research Council: G0300429, G9810900; Wellcome Trust: 064354

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2007;144B;6;762-70

  • Ankyrin-G and beta2-spectrin collaborate in biogenesis of lateral membrane of human bronchial epithelial cells.

    Kizhatil K, Yoon W, Mohler PJ, Davis LH, Hoffman JA and Bennett V

    Howard Hughes Medical Institute and Departments of Cell Biology, Biochemistry, and Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Ankyrins are a family of adapter proteins required for localization of membrane proteins to diverse specialized membrane domains including axon initial segments, specialized sites at the transverse tubule/sarcoplasmic reticulum in cardiomyocytes, and lateral membrane domains of epithelial cells. Little is currently known regarding the molecular basis for specific roles of different ankyrin isoforms. In this study, we systematically generated alanine mutants of clusters of charged residues in the spectrin-binding domains of both ankyrin-B and -G. The corresponding mutants were evaluated for activity in either restoration of abnormal localization of the inositol trisphosphate receptor in the sarcoplasmic reticulum in mutant mouse cardiomyocytes deficient in ankyrin-B or in prevention of loss of lateral membrane in human bronchial epithelial cells depleted of ankyrin-G by small interfering RNA. Interestingly, ankyrin-B and -G share two homologous sites that result in loss of function in both systems, suggesting that common molecular interactions underlie diverse roles of these isoforms. Ankyrins G and B also exhibit differences; mutations affecting spectrin binding had no effect on ankyrin-B function but did abolish activity of ankyrin-G in restoring lateral membrane biogenesis. Depletion of beta(2)-spectrin by small interfering RNA phenocopied depletion of ankyrin-G and resulted in a failure to form new lateral membrane in interphase and mitotic cells. These results demonstrate that ankyrin-G and beta(2)-spectrin are functional partners in biogenesis of the lateral membrane of epithelial cells.

    The Journal of biological chemistry 2007;282;3;2029-37

  • Ankyrin-G regulates inactivation gating of the neuronal sodium channel, Nav1.6.

    Shirahata E, Iwasaki H, Takagi M, Lin C, Bennett V, Okamura Y and Hayasaka K

    Department of Pediatrics, Yamagata University School of Medicine, Yamagata, Japan.

    Ankyrin-G, a modular protein, plays a critical role in clustering voltage-gated sodium channels (Nav channels) in nodes of Ranvier and initial segments of mammalian neurons. However, direct effects of ankyrin-G on electrophysiological properties of Nav channels remain elusive. In this study, we explored whether ankyrin-G has a role in modifying gating properties of the neuronal Nav1.6 channel that is predominantly localized at nodes of Ranvier and initial segments. TsA201 cells transfected with the human Nav1.6 cDNA alone exhibited significant persistent sodium current (Ina-p). On the other hand, Ina-p was barely detected on co-expression with ankyrin-G. Ankyrin-B, another ankyrin, did not show such an effect. Expression of chimeras between the two isoforms of ankyrin suggests that the membrane-binding domain of ankyrin-G is critical for reducing the Ina-p of Nav1.6. These results suggest that ankyrin-G regulates neuronal excitability not only through clustering Nav channels but also by directly modifying their channel gating.

    Journal of neurophysiology 2006;96;3;1347-57

  • The smaller isoforms of ankyrin 3 bind to the p85 subunit of phosphatidylinositol 3'-kinase and enhance platelet-derived growth factor receptor down-regulation.

    Ignatiuk A, Quickfall JP, Hawrysh AD, Chamberlain MD and Anderson DH

    Cancer Research Unit, Health Research Division, Saskatchewan Cancer Agency, 20 Campus Drive, Saskatoon, Saskatchewan S7N 4H4, Canada.

    The Src homology 2 (SH2) domains of the p85 subunit of phosphatidylinositol 3'-kinase have been shown to bind to the tyrosine-phosphorylated platelet-derived growth factor receptor (PDGFR). Previously, we have demonstrated that p85 SH2 domains can also bind to the serine/threonine kinase A-Raf via a unique phosphorylation-independent interaction. In this report, we describe a new phosphotyrosine-independent p85 SH2-binding protein, ankyrin 3 (Ank3). In general, ankyrins serve a structural role by binding to both integral membrane proteins at the plasma membrane and spectrin/fodrin proteins of the cytoskeleton. However, smaller isoforms of Ank3 lack the membrane domain and are localized to late endosomes and lysosomes. We found that p85 binds directly to these smaller 120- and 105-kDa Ank3 isoforms. Both the spectrin domain and the regulatory domain of Ank3 are involved in binding to p85. At least two domains of p85 can bind to Ank3, and the interaction involving the p85 C-SH2 domain was found to be phosphotyrosine-independent. Overexpression of the 120- or 105-kDa Ank3 proteins resulted in significantly enhanced PDGFR degradation and a reduced ability to proliferate in response to PDGF. Ank3 overexpression also differentially regulated signaling pathways downstream from the PDGFR. Chloroquine, an inhibitor of lysosomal-mediated degradation pathways, blocked the ability of Ank3 to enhance PDGFR degradation. Immunofluorescence experiments demonstrated that both small Ank3 isoforms colocalized with the lysosomal-associated membrane protein and with p85 and the PDGFR. These results suggest that Ank3 plays an important role in lysosomal-mediated receptor down-regulation, likely through a p85-Ank3 interaction.

    The Journal of biological chemistry 2006;281;9;5956-64

  • A scan of chromosome 10 identifies a novel locus showing strong association with late-onset Alzheimer disease.

    Grupe A, Li Y, Rowland C, Nowotny P, Hinrichs AL, Smemo S, Kauwe JS, Maxwell TJ, Cherny S, Doil L, Tacey K, van Luchene R, Myers A, Wavrant-De Vrièze F, Kaleem M, Hollingworth P, Jehu L, Foy C, Archer N, Hamilton G, Holmans P, Morris CM, Catanese J, Sninsky J, White TJ, Powell J, Hardy J, O'Donovan M, Lovestone S, Jones L, Morris JC, Thal L, Owen M, Williams J and Goate A

    Celera Diagnostics, Alameda, CA, USA.

    Strong evidence of linkage to late-onset Alzheimer disease (LOAD) has been observed on chromosome 10, which implicates a wide region and at least one disease-susceptibility locus. Although significant associations with several biological candidate genes on chromosome 10 have been reported, these findings have not been consistently replicated, and they remain controversial. We performed a chromosome 10-specific association study with 1,412 gene-based single-nucleotide polymorphisms (SNPs), to identify susceptibility genes for developing LOAD. The scan included SNPs in 677 of 1,270 known or predicted genes; each gene contained one or more markers, about half (48%) of which represented putative functional mutations. In general, the initial testing was performed in a white case-control sample from the St. Louis area, with 419 LOAD cases and 377 age-matched controls. Markers that showed significant association in the exploratory analysis were followed up in several other white case-control sample sets to confirm the initial association. Of the 1,397 markers tested in the exploratory sample, 69 reached significance (P < .05). Five of these markers replicated at P < .05 in the validation sample sets. One marker, rs498055, located in a gene homologous to RPS3A (LOC439999), was significantly associated with Alzheimer disease in four of six case-control series, with an allelic P value of .0001 for a meta-analysis of all six samples. One of the case-control samples with significant association to rs498055 was derived from the linkage sample (P = .0165). These results indicate that variants in the RPS3A homologue are associated with LOAD and implicate this gene, adjacent genes, or other functional variants (e.g., noncoding RNAs) in the pathogenesis of this disorder.

    Funded by: Intramural NIH HHS; Medical Research Council: G0300429, G0701075, G9810900; NHGRI NIH HHS: T32 HG000045; NIA NIH HHS: AG 05146, AG05128, P01 AG003991, P01 AG03991, P50 AG005128, P50 AG005131, P50 AG005146, P50 AG005681, P50 AG008671, P50 AG016570, P50 AG05131, P50 AG05681, P50 AG16570, P50-AG08671, R01 AG016208, R01 AG16208, U24 AG021886; NIGMS NIH HHS: GM065509, P50 GM065509; NIMH NIH HHS: MH60451, P50 MH060451, U01 MH046281, U01 MH046290, U01 MH046373; NINDS NIH HHS: NS39764, P50 NS039764

    American journal of human genetics 2006;78;1;78-88

  • Ankyrin G overexpression in Hutchinson-Gilford progeria syndrome fibroblasts identified through biological filtering of expression profiles.

    Wang J, Robinson JF, O'Neil CH, Edwards JY, Williams CM, Huff MW, Pickering JG and Hegele RA

    Vascular Biology Research Group, Robarts Research Institute, 100 Perth Drive, London, ON, Canada, N6A 5K8.

    Hutchinson-Gilford progeria syndrome (HGPS; MIM 176670) is a rare disease characterized by accelerated aging. In this study, light and immunofluorescence microscopy were used to assess morphological changes, measures of cell growth kinetics and gene expression profiles in HGPS cells and normal fibroblasts in culture. A filtering strategy was developed based on differentially expressed transcripts seen consistently across three culture stages based on cell passage number. This filtering strategy produced a list of 66 unique differentially expressed genes, of which approximately 40% were upregulated in HGPS cells compared to normal fibroblasts. The increased mRNA expression in HGPS cells that was seen for one gene defined using this strategy--namely ANK3--was validated using quantitative reverse-transcriptase amplification, Western analysis and immunofluorescence microscopy, all of which showed significantly increased ankyrin G expression. These findings demonstrate differences in morphology, growth kinetics and mRNA expression profiles in HGPS cells compared to normal fibroblasts in culture, including increased expression of ANK3/ankyrin G. Furthermore, other genes that co-clustered with ANK3 might provide mechanistic clues regarding senescence in cultured HGPS cells.

    Journal of human genetics 2006;51;11;934-42

  • Microarray analysis identifies a death-from-cancer signature predicting therapy failure in patients with multiple types of cancer.

    Glinsky GV, Berezovska O and Glinskii AB

    Sidney Kimmel Cancer Center, San Diego, California 92121, USA. gglinsky@skcc.org

    Activation in transformed cells of normal stem cells' self-renewal pathways might contribute to the survival life cycle of cancer stem cells and promote tumor progression. The BMI-1 oncogene-driven gene expression pathway is essential for the self-renewal of hematopoietic and neural stem cells. We applied a mouse/human comparative translational genomics approach to identify an 11-gene signature that consistently displays a stem cell-resembling expression profile in distant metastatic lesions as revealed by the analysis of metastases and primary tumors from a transgenic mouse model of prostate cancer and cancer patients. To further validate these results, we examined the prognostic power of the 11-gene signature in several independent therapy-outcome sets of clinical samples obtained from 1,153 cancer patients diagnosed with 11 different types of cancer, including 5 epithelial malignancies (prostate, breast, lung, ovarian, and bladder cancers) and 5 nonepithelial malignancies (lymphoma, mesothelioma, medulloblastoma, glioma, and acute myeloid leukemia). Kaplan-Meier analysis demonstrated that a stem cell-like expression profile of the 11-gene signature in primary tumors is a consistent powerful predictor of a short interval to disease recurrence, distant metastasis, and death after therapy in cancer patients diagnosed with 11 distinct types of cancer. These data suggest the presence of a conserved BMI-1-driven pathway, which is similarly engaged in both normal stem cells and a highly malignant subset of human cancers diagnosed in a wide range of organs and uniformly exhibiting a marked propensity toward metastatic dissemination as well as a high probability of unfavorable therapy outcome.

    Funded by: NCI NIH HHS: 5R01 CA89827, R01 CA089827

    The Journal of clinical investigation 2005;115;6;1503-21

  • Elevation of Hook1 in a disease model of Batten disease does not affect a novel interaction between Ankyrin G and Hook1.

    Weimer JM, Chattopadhyay S, Custer AW and Pearce DA

    Center for Aging and Developmental Biology, Aab Institute of Biomedical Sciences, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.

    Hook1 is a member of a family of microtubule-binding proteins. Studies on the Drosophila homolog of Hook1 have suggested a role in the maturation and trafficking of internalized proteins to the late endosome. A weak interaction between Hook1 and the lysosomal/late endosomal protein, CLN3, was recently reported. Mutations in CLN3 result in the neurological disorder Batten disease. Here we show a novel interaction between Hook1 and Ankyrin G, an adaptor protein that binds the spectrin-actin cytoskeleton and targets proteins to the peripheral membrane. Although we demonstrate co-localization of Hook1 and Ankyrin G, Hook1 also localizes to additional regions of the cell devoid of Ankyrin G where it likely interacts with other proteins. There is no disruption of the Hook1-Ankyrin G interaction or localization in tissue derived from a Cln3-knockout mouse despite a nearly threefold increase in the expression of Hook1. However, mutation of CLN3 could lead to alterations in the functioning and positioning of organelles and membrane proteins through this Hook1-Ankyrin G interaction.

    Funded by: NIMH NIH HHS: T32 MH065181; NINDS NIH HHS: NS36610

    Biochemical and biophysical research communications 2005;330;4;1176-81

  • The ammonium transporter RhBG: requirement of a tyrosine-based signal and ankyrin-G for basolateral targeting and membrane anchorage in polarized kidney epithelial cells.

    Lopez C, Métral S, Eladari D, Drevensek S, Gane P, Chambrey R, Bennett V, Cartron JP, Le Van Kim C and Colin Y

    INSERM, U665, Institut National de la Transfusion Sanguine, Paris F-75015, France.

    RhBG is a nonerythroid member of the Rhesus (Rh) protein family, mainly expressed in the kidney and belonging to the Amt/Mep/Rh superfamily of ammonium transporters. The epithelial expression of renal RhBG is restricted to the basolateral membrane of the connecting tubule and collecting duct cells. We report here that sorting and anchoring of RhBG to the basolateral plasma membrane require a cis-tyrosine-based signal and an association with ankyrin-G, respectively. First, we show by using a model of polarized epithelial Madin-Darby canine kidney cells that the targeting of transfected RhBG depends on a YED motif localized in the cytoplasmic C terminus of the protein. Second, we reveal by yeast two-hybrid analysis a direct interaction between an FLD determinant in the cytoplasmic C-terminal tail of RhBG and the third and fourth repeat domains of ankyrin-G. The biological relevance of this interaction is supported by two observations. (i) RhBG and ankyrin-G were colocalized in vivo in the basolateral domain of epithelial cells from the distal nephron by immunohistochemistry on kidney sections. (ii) The disruption of the FLD-binding motif impaired the membrane expression of RhBG leading to retention on cytoplasmic structures in transfected Madin-Darby canine kidney cells. Mutation of both targeting signal and ankyrin-G-binding site resulted in the same cell surface but nonpolarized expression pattern as observed for the protein mutated on the targeting signal alone, suggesting the existence of a close relationship between sorting and anchoring of RhBG to the basolateral domain of epithelial cells.

    The Journal of biological chemistry 2005;280;9;8221-8

  • 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

  • Lateral membrane biogenesis in human bronchial epithelial cells requires 190-kDa ankyrin-G.

    Kizhatil K and Bennett V

    Howard Hughes Medical Institute and Departments of Cell Biology, Biochemistry, and Neuroscience, Duke University Medical Center, Durham, North Carolina 27710, USA. k.kizhatil@cellbio.duke.edu

    Ankyrin-G polypeptides are required for restriction of voltage-gated sodium channels, L1 cell adhesion molecules, and beta IV spectrin to axon initial segments and are believed to couple the Na/K-ATPase to the spectrin-actin network at the lateral membrane in epithelial cells. We report here that depletion of 190-kDa ankyrin-G in human bronchial epithelial cells by small interfering RNA results in nearly complete loss of lateral plasma membrane in interphase cells, and also blocks de novo lateral membrane biogenesis following mitosis. Loss of the lateral membrane domain is accompanied by an expansion of apical and basal plasma membranes and preservation of apical-basal polarity. Expression of rat 190-kDa ankyrin-G, which is resistant to human small interfering RNA, prevents loss of the lateral membrane following depletion of human 190-kDa ankyrin-G. Human 220-kDa ankyrin-B, a closely related ankyrin isoform, is incapable of preserving the lateral membrane following 190-kDa ankyrin-G depletion. Moreover, analysis of rat 190-kDa ankyrin G/ankyrin B chimeras shows that all three domains of 190-kDa ankyrin-G are required for preservation of the lateral membrane. These results demonstrate that 190-kDa ankyrin-G plays a pleiotropic role in assembly of lateral membranes of bronchial epithelial cells.

    The Journal of biological chemistry 2004;279;16;16706-14

  • Sodium channel beta1 subunit-mediated modulation of Nav1.2 currents and cell surface density is dependent on interactions with contactin and ankyrin.

    McEwen DP, Meadows LS, Chen C, Thyagarajan V and Isom LL

    Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109-0632, USA.

    Voltage-gated sodium channels are composed of a pore-forming alpha subunit and at least one auxiliary beta subunit. Both beta1 and beta2 are cell adhesion molecules that interact homophilically, resulting in ankyrin recruitment. In contrast, beta1, but not beta2, interacts heterophilically with contactin, resulting in increased levels of cell surface sodium channels. We took advantage of these results to investigate the molecular basis of beta1-mediated enhancement of sodium channel cell surface density, including elucidating structure-function relationships for beta1 association with contactin, ankyrin, and Nav1.2. beta1/beta2 subunit chimeras were used to assign putative sites of contactin interaction to two regions of the beta1 Ig loop. Recent studies have shown that glutathione S-transferase fusion proteins containing portions of Nav1.2 intracellular domains interact directly with ankyrinG. We show that native Nav1.2 associates with ankyrinG in cells in the absence of beta subunits and that this interaction is enhanced in the presence of beta1 but not beta1Y181E, a mutant that does not interact with ankyrinG. beta1Y181E does not modulate Nav1.2 channel function despite efficient association with Nav1.2 and contactin. beta1Y181E increases Nav1.2 cell surface expression, but not as efficiently as wild type beta1. beta1/beta2 chimeras exchanging various regions of the beta1 Ig loop were all ineffective in increasing Nav1.2 cell surface density. Our results demonstrate that full-length beta1 is required for channel modulation and enhancement of sodium channel cell surface expression.

    Funded by: NIGMS NIH HHS: GM07767; NIMH NIH HHS: R01MH59980; NINDS NIH HHS: NS43067, R01NS17965-17

    The Journal of biological chemistry 2004;279;16;16044-9

  • The death domain of kidney ankyrin interacts with Fas and promotes Fas-mediated cell death in renal epithelia.

    Del Rio M, Imam A, DeLeon M, Gomez G, Mishra J, Ma Q, Parikh S and Devarajan P

    Department of Nephrology, Children's Hospital at Montefiore, Albert Einstein College of Medicine, Bronx, New York, USA.

    Ankyrins are a ubiquitously expressed family of conserved proteins that mediate the linkage of integral membrane proteins such as transporters and channels with the underlying cytoskeleton. Ankyrins possess a conserved death domain, the functional significance of which has remained puzzling. In this study, the death domain of AnkG190, the isoform of ankyrin expressed in kidney tubules, was used as bait in a yeast two-hybrid screen to identify interacting partners. One of these interactions was with the proapoptotic molecule Fas. This was confirmed by coimmunoprecipitation, colocalization, and glutathione S-transferase pull-down assays in cultured renal epithelial (MDCK) cells. Site-directed mutagenesis of a conserved arginine (R1496 in AnkG190), previously shown to be critical for the binding of Fas (R234 in Fas) to FADD, abolished the interaction of ankyrin's death domain with Fas. Overexpression of constructs containing ankyrin's death domain promoted Fas-mediated apoptosis in MDCK cells. The linkage between ankyrin and Fas was confirmed in vivo in mouse kidney tubule cells by coimmunoprecipitation and colocalization. In an established mouse model of renal ischemia-reperfusion injury characterized by apoptotic tubule cell death, the expression of both ankyrin and Fas was markedly induced, and the interaction between these molecules remained intact. The results identify a novel tethering interaction between ankyrin and Fas in kidney epithelia and suggest that AnkG190 may play a role as an adapter molecule in renal tubule cell death.

    Funded by: NIDDK NIH HHS: DK52612, DK53289

    Journal of the American Society of Nephrology : JASN 2004;15;1;41-51

  • 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

  • Kv3.1b is a novel component of CNS nodes.

    Devaux J, Alcaraz G, Grinspan J, Bennett V, Joho R, Crest M and Scherer SS

    Department of Neurology, The University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6077, USA. jdevaux@mail.med.upenn.edu

    We herein demonstrate that Kv3.1b subunits are present at nodes of Ranvier in the CNS of both rats and mice. Kv3.1b colocalizes with voltage-gated Na+ channels in a subset of nodes in the spinal cord, particularly those of large myelinated axons. Kv3.1b is abundantly expressed in the gray matter of the spinal cord, but does not colocalize with Na+ channels in initial segments. In the PNS, few nodes are Kv3.1b-positive. During the development of the CNS, Kv3.1b clustering at nodes occurs later than that of Na+ channels, but precedes the juxtaparanodal clustering of Kv1.2. Moreover, in myelin-deficient rats, which have severe CNS dysmyelination, node-like clusters of Kv3.1b and Na+ channels are observed even in regions devoid of oligodendrocytes. Ankyrin G coimmunoprecipitates Kv3.1b in vivo, indicating that these two proteins may interact in the CNS at nodes. 4-Aminopyridine, a K+ channel blocker, broadened the compound action potential recorded from adult rat optic nerve and spinal cord, but not from the sciatic nerve. These effects were also observed in Kv3.1-deficient mice. In conclusion, Kv3.1b is the first K+ channel subunit to be identified in CNS nodes; but Kv3.1b does not account for the effects of 4-aminopyridine on central myelinated tracts.

    Funded by: NINDS NIH HHS: R01 NS43174

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;11;4509-18

  • Painful neuromas: a potential role for a structural transmembrane protein, ankyrin G.

    Kretschmer T, Nguyen DH, Beuerman RW, Happel LT, England JD, Tiel RL and Kline DG

    Center for Neuroscience, Louisiana State University Health Science Center, New Orleans, Louisiana, USA. kretschmerthomas@hotmail.com

    Object: Severe nerve injury induces the formation of a neuroma. Some neuromas cause excruciating pain. Overexpression of Na+ channels leads to hyperexcitability and painful phenomena. Ankyrin G, a multifunctional transmembrane protein of the axolemma, might be a key protein in neuroma formation because it binds Na+ channels in the initial segments of a regenerating axon and links with neuronal cell adhesion molecules. The authors wanted to determine if ankyrin G could be detected in neuroma, and if present, whether there would be differences in distribution between nonpainful neuromas, painful neuromas, and normal nerve.

    Methods: First, frozen sections of nine nerve specimens obtained from six patients (six nonpainful neuromas, one painful neuroma, and two normal nerves) were immunocytochemically screened for ankyrin G by using confocal laser scanning microscopy. Second, specimens from 29 patients (seven painful neuromas, 15 nonpainful neuromas, and seven normal nerves) were examined using immunoblot analysis for their ankyrin G content. Western blot analysis detected ankyrin G, which was visualized by applying the enhanced chemiluminescence technique. Computerized densitometry was used to quantitate ankyrin G expression by comparing band intensities. Normal nerve served as control. Neurofilament was used as a marker for nerve tissue content. Ankyrin G could be detected and was found to be increased in neuromas. The mean band intensity values were 1838 for painful neuromas, 1166 for nonpainful neuromas, and 411 for normal nerves. In two cases the authors were able to compare specimens of painful neuroma and normal nerve from the same patient. The painful neuromas exhibited considerably higher levels of ankyrin G. Painful neuroma and normal nerve densitometry values were 499 and 165, respectively, for one patient, and 4254 and 821, respectively, for the other patient. Painful neuromas were also found to have higher neurofilament values than nonpainful neuromas.

    Conclusions: Altered regulation of ankyrin G after nerve injury may lead to hyperexcitability and painful phenomena via clustering of Na+ channels. A propensity to overexpress ankyrin G after peripheral nerve trauma may turn out to be a factor in the development of painful neuromas and neuropathic pain. The relevant literature regarding the importance of ankyrin G for nerve regeneration and nerve membrane remodeling is reviewed.

    Journal of neurosurgery 2002;97;6;1424-31

  • Interaction of the Nav1.2a subunit of the voltage-dependent sodium channel with nodal ankyrinG. In vitro mapping of the interacting domains and association in synaptosomes.

    Bouzidi M, Tricaud N, Giraud P, Kordeli E, Caillol G, Deleuze C, Couraud F and Alcaraz G

    INSERM U 464 Neurobiologie des Canaux Ioniques, Institut Jean Roche, Université de la Méditerranée, 13916 Marseille Cedex 20, France.

    Voltage-dependant sodium channels at the axon initial segment and nodes of Ranvier colocalize with the nodal isoforms of ankyrin(G) (Ank(G) node). Using fusion proteins derived from the intracellular regions of the Nav1.2a subunit and the Ank repeat domain of Ank(G) node, we mapped a major interaction site in the intracellular loop separating alpha subunit domains I-II. This 57-amino acid region binds the Ank repeat region with a K(D) value of 69 nm. We identified another site in intracellular loop III-IV, and we mapped both Nav1.2a binding sites on the ankyrin repeat domain to the region encompassing repeats 12-22. The ankyrin repeat domain did not bind the beta(1) and beta(2) subunit cytoplasmic regions. We showed that in cultured embryonic motoneurons, expression of the beta(2) subunit is not necessary for the colocalization of Ank(G) node with functional sodium channels at the axon initial segment. Antibodies directed against the beta(1) subunit intracellular region, alpha subunit loop III-IV, and Ank(G) node could not co-immunoprecipitate Ank(G) node and sodium channels from Triton X-100 solubilisates of rat brain synaptosomes. Co-immunoprecipitation of sodium channel alpha subunit and of the 270- and 480-kDa AnkG node isoforms was obtained when solubilization conditions that maximize membrane protein extraction were used. However, we could not find conditions that allowed for co-immunoprecipitation of ankyrin with the sodium channel beta(1) subunit.

    The Journal of biological chemistry 2002;277;32;28996-9004

  • Structural requirements for interaction of sodium channel beta 1 subunits with ankyrin.

    Malhotra JD, Koopmann MC, Kazen-Gillespie KA, Fettman N, Hortsch M and Isom LL

    Department of Pharmacology, the University of Michigan, Ann Arbor, Michigan 48109, USA.

    Sodium channel beta subunits modulate channel kinetic properties and cell surface expression levels and function as cell adhesion molecules. beta 1 and beta 2 participate in homophilic cell adhesion resulting in ankyrin recruitment to cell contact sites. We hypothesized that a tyrosine residue in the cytoplasmic domain of beta 1 may be important for ankyrin recruitment and tested our hypothesis using beta 1 mutants replacing Tyr(181) with alanine (beta 1Y181A), phenylalanine (beta 1Y181F), or glutamate (beta 1Y181E), or a truncated construct deleting all residues beyond Tyr(181) (beta 1L182(STOP)). Ankyrin recruitment was observed in beta 1L182(STOP), showing that residues Ile(166)-Tyr(181) contain the major ankyrin recruiting activity of beta 1. Ankyrin recruitment was abolished in beta 1Y181E, suggesting that tyrosine phosphorylation of beta 1 may inhibit beta 1-ankyrin interactions. Ankyrin(G) and beta 1 associate in rat brain membranes and in transfected cells expressing beta 1 and ankyrin(G) in the absence of sodium channel alpha subunits. beta 1 subunits are recognized by anti-phosphotyrosine antibodies following treatment of these cell lines with fibroblast growth factor. beta 1 and ankryin(G) association is not detectable in cells following treatment with fibroblast growth factor. Ankyrin(G) and beta 1Y181E do not associate even in the absence of fibroblast growth factor treatment. beta 1 subunit-mediated cell adhesion and ankyrin recruitment may contribute to sodium channel placement at nodes of Ranvier. The phosphorylation state of beta 1Y181 may be a critical regulatory step in these developmental processes.

    Funded by: NICHD NIH HHS: R01HD29388; NIMH NIH HHS: R01MH59980

    The Journal of biological chemistry 2002;277;29;26681-8

  • Ankyrin G and voltage gated sodium channels colocalize in human neuroma--key proteins of membrane remodeling after axonal injury.

    Kretschmer T, England JD, Happel LT, Liu ZP, Thouron CL, Nguyen DH, Beuerman RW and Kline DG

    Department of Neurosurgery, University of Ulm, BKH Neurochirurgie, Ludwig-Heilmeyer-Strasse 2, D-89312 Günzburg, Germany. kretschmerthomas@hotmail.com

    We tested if ankyrin G could be detected in human neuroma, if it colocalized with site-specific peripheral nerve sodium channels that accumulate at axon tips of injured nerve, and if there are differences in the distribution of these proteins in non-painful neuroma and painful neuroma tissue vs. normal nerve. Frozen sections from one painful, six non-painful, and three normal nerves were immunocytochemically examined. A double labeling technique with highly specific antibodies against peripheral nerve type 1 (Na(v)1.7), and peripheral nerve type 3 (Na(v)1.8) sodium channels and anti-ankyrin G antibodies detected sodium channels and ankyrin G on the same section, using confocal laser scanning microscopy. Ankyrin G colocalized with both types of sodium channels. Neuroma specimens exhibited considerably larger immunofluorescence for both sodium channels and ankyrin G compared with normal nerve. The painful neuroma presented an even more pronounced immunolabeling in clusters. Findings support results from animal models that link ankyrin G with clustering of sodium channels at axon tips of unmyelinated, sprouting fibers. A common (repair-) mechanism that exists throughout the human nervous system for clustering sodium channels at a high density is assumed. A dysregulation in this membrane remodeling mechanism might be an initial step in a cascade that leads to a painful rather than a non-painful neuroma.

    Neuroscience letters 2002;323;2;151-5

  • The ankyrin-B C-terminal domain determines activity of ankyrin-B/G chimeras in rescue of abnormal inositol 1,4,5-trisphosphate and ryanodine receptor distribution in ankyrin-B (-/-) neonatal cardiomyocytes.

    Mohler PJ, Gramolini AO and Bennett V

    Howard Hughes Medical Institute and Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA. p.mohler@cellbio.duke.edu

    Ankyrins are a closely related family of membrane adaptor proteins that are believed to participate in targeting diverse membrane proteins to specialized domains in the plasma membrane and endoplasmic reticulum. This study addresses the question of how individual ankyrin isoforms achieve functional specificity when co-expressed in the same cell. Cardiomyocytes from ankyrin-B (-/-) mice display mis-localization of inositol 1,4,5-trisphosphate receptors and ryanodine receptors along with reduced contraction rates that can be rescued by expression of green fluorescent protein (GFP)-ankyrin-B but not GFP-ankyrin-G. We developed chimeric GFP expression constructs containing all combinations of the three major domains of ankyrin-B and ankyrin-G to determine which domain(s) of ankyrin-B are required for ankyrin-B-specific functions. The death/C-terminal domain of ankyrin-B determined activity of ankyrin-B/G chimeras in localization in a striated pattern in cardiomyocytes and in restoration of a normal striated distribution of both ryanodine and inositol 1,4,5-trisphosphate receptors as well as normal beat frequency of contracting cardiomyocytes. Further deletions within the death/C-terminal domain demonstrated that the C-terminal domain determines ankyrin-B activity, whereas deletion of the death domain had no effect. C-terminal domains are the most divergent between ankyrin isoforms and are candidates to encode the signal(s) that enable ankyrins to selectively target proteins to diverse cellular sites.

    The Journal of biological chemistry 2002;277;12;10599-607

  • [Beta]IV-spectrin regulates sodium channel clustering through ankyrin-G at axon initial segments and nodes of Ranvier.

    Komada M and Soriano P

    Program in Developmental Biology and Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. makomada@bio.titech.ac.jp

    beta-Spectrin and ankyrin are major components of the membrane cytoskeleton. We have generated mice carrying a null mutation in the betaIV-spectrin gene using gene trapping in embryonic stem cells. Mice homozygous for the mutation exhibit tremors and contraction of hindlimbs. betaIV-spectrin expression is mostly restricted to neurons, where it colocalizes with and binds to ankyrin-G at axon initial segments (AISs) and nodes of Ranvier (NR). In betaIV-spectrin-null neurons, neither ankyrin-G nor voltage-gated sodium channels (VGSC) are correctly clustered at these sites, suggesting that impaired action potential caused by mislocalization of VGSC leads to the phenotype. Conversely, in ankyrin-G-null neurons, betaIV-spectrin is not localized to these sites. These results indicate that betaIV-spectrin and ankyrin-G mutually stabilize the membrane protein cluster and the linked membrane cytoskeleton at AIS and NR.

    Funded by: NICHD NIH HHS: HD24875, R01 HD024875

    The Journal of cell biology 2002;156;2;337-48

  • Ankyrin-Tiam1 interaction promotes Rac1 signaling and metastatic breast tumor cell invasion and migration.

    Bourguignon LY, Zhu H, Shao L and Chen YW

    Department of Cell Biology and Anatomy, School of Medicine, University of Miami, Miami, Florida 33136, USA. Lbourgui@med.miami.edu

    Tiam1 (T-lymphoma invasion and metastasis 1) is one of the known guanine nucleotide (GDP/GTP) exchange factors (GEFs) for Rho GTPases (e.g., Rac1) and is expressed in breast tumor cells (e.g., SP-1 cell line). Immunoprecipitation and immunoblot analyses indicate that Tiam1 and the cytoskeletal protein, ankyrin, are physically associated as a complex in vivo. In particular, the ankyrin repeat domain (ARD) of ankyrin is responsible for Tiam1 binding. Biochemical studies and deletion mutation analyses indicate that the 11-amino acid sequence between amino acids 717 and 727 of Tiam1 ((717)GEGTDAVKRS(727)L) is the ankyrin-binding domain. Most importantly, ankyrin binding to Tiam1 activates GDP/GTP exchange on Rho GTPases (e.g., Rac1). Using an Escherichia coli-derived calmodulin-binding peptide (CBP)-tagged recombinant Tiam1 (amino acids 393-728) fragment that contains the ankyrin-binding domain, we have detected a specific binding interaction between the Tiam1 (amino acids 393-738) fragment and ankyrin in vitro. This Tiam1 fragment also acts as a potent competitive inhibitor for Tiam1 binding to ankyrin. Transfection of SP-1 cell with Tiam1 cDNAs stimulates all of the following: (1) Tiam1-ankyrin association in the membrane projection; (2) Rac1 activation; and (3) breast tumor cell invasion and migration. Cotransfection of SP1 cells with green fluorescent protein (GFP)-tagged Tiam1 fragment cDNA and Tiam1 cDNA effectively blocks Tiam1-ankyrin colocalization in the cell membrane, and inhibits GDP/GTP exchange on Rac1 by ankyrin-associated Tiam1 and tumor-specific phenotypes. These findings suggest that ankyrin-Tiam1 interaction plays a pivotal role in regulating Rac1 signaling and cytoskeleton function required for oncogenic signaling and metastatic breast tumor cell progression.

    Funded by: NCI NIH HHS: CA 78633, CA66163, R01 CA066163, R01 CA078633

    The Journal of cell biology 2000;150;1;177-91

  • All-trans-retinoic acid induces tyrosine phosphorylation of the CrkL adapter in acute promyelocytic leukemia cells.

    Alsayed Y, Modi S, Uddin S, Mahmud N, Druker BJ, Fish EN, Hoffman R and Platanias LC

    Section of Hematology-Oncology, University of Illinois at Chicago and West Side Veterans Administration Medical Center, USA.

    Objective: All-trans-retinoic acid (RA) is a potent inducer of differentiation of acute promyelocytic leukemia (APL) cells in vitro and in vivo. It also exhibits synergistic effects with interferons on the induction of differentiation and growth inhibition in vitro. Recent studies showed that interferons engage a signaling pathway involving the CBL proto-oncogene and the CrkL adapter, which mediates interferon-induced growth inhibitory signals. The objective of this study was to determine whether the CBL-CrkL pathway is activated by treatment of the NB-4 and HL-60 acute leukemia cell lines with RA.

    The effects of RA treatment on CBL and CrkL phosphorylation, as well as on protein-protein interactions, were determined in studies involving immunoprecipitations of cell extracts with specific antibodies and Western blots. In addition, glutathione-S-transferase fusion proteins were used in binding studies to determine whether the SH2 domain of CrkL interacts with CBL in a RA-dependent manner and whether Rapl is activated by RA.

    Results: Treatment of NB-4 or HL-60 cells with RA resulted in strong tyrosine phosphorylation of CBL, which was time and dose dependent. Similarly, RA induced tyrosine phosphorylation of the CrkL adapter and the association of CrkL with CBL. The RA-dependent interaction of CrkL with CBL was mediated by binding of the SH2 domain of CrkL to tyrosine phosphorylated CBL, suggesting that CBL provides a docking site for engagement of CrkL in a RA-activated cellular pathway. The guanine exchange factor C3G was found to be associated with CrkL at similar levels before and after RA treatment, but Rapl activation downstream of C3G was not inducible by RA.

    Conclusions: These findings demonstrate that the CBL-CrkL pathway is one of the mediators of the effects of RA on APL cells and suggest that one of the mechanisms of synergy between RA and interferons may involve regulation of components of this signaling cascade.

    Funded by: NCI NIH HHS: CA73381, CA77816

    Experimental hematology 2000;28;7;826-32

  • Identification of a small cytoplasmic ankyrin (AnkG119) in the kidney and muscle that binds beta I sigma spectrin and associates with the Golgi apparatus.

    Devarajan P, Stabach PR, Mann AS, Ardito T, Kashgarian M and Morrow JS

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

    Ankyrins are a family of large, membrane-associated proteins that mediate the linkage of the cytoskeleton to a variety of membrane transport and receptor proteins. A repetitive 33-residue motif characteristic of domain I of ankyrin has also been identified in proteins involved with cell cycle control and development. We have cloned and characterized a novel ankyrin isoform, AnkG119 (GenBank accession No. U43965), from the human kidney which lacks part of this repetitive domain and associates in MDCK cells with beta I sigma spectrin and the Golgi apparatus, but not the plasma membrane. Sequence comparison reveals this ankyrin to be an alternative transcript of AnkG, a much larger ankyrin recently cloned from brain. AnkG119 has a predicted size of 119,201 D, and contains a 47-kD domain I consisting of 13 ankyrin repeat units, a 67-kD domain II with a highly conserved spectrin-binding motif, and a truncated 5-kD putative regulatory domain. An AnkG119 cDNA probe hybridized to a 6.0-kb message in human and rat kidney, placenta, and skeletal muscle. An antibody raised to AnkG119 recognized an apparent 116-kD peptide in rat kidney cortical tissue and MDCK cell lysates, and did not react with larger isoforms of ankyrin at 190 and 210 kD in these tissues, nor in bovine brain, nor with ankyrin from human erythrocytes. AnkG119 remains extractable in 0.5% Triton X-100, and assumes a punctuate cytoplasmic distribution in mature MDCK cells, in contrast to the Triton-stable plasma membrane localization of all previously described renal ankyrins. AnkG119 immunocreativity in subconfluent MDCK cells distributes with the Golgi complex in a pattern coincident with beta -COP and beta I sigma spectrin immunoreactivity. A fusion peptide containing residues 669-860 of AnkG119 interacts with beta I sigma 1 spectrin in vitro with a Kd = 4.2 +/- 4.0 ( +/- 2 SD) nM, and avidly binds the beta spectrin in MDCK cell lysates. Collectively, these data identify AnkG119 as a novel small ankyrin that binds and colocalizes with beta I sigma spectrin in the ER and Golgi apparatus, and possible on a subset of endosomes during the early stages of polarity development. We hypothesize that AnkG119 and beta I spectrin form a vesicular Golgi-associated membrane skeleton, promote the organization of protein microdomains within the Golgi and trans-Golgi networks, and contribute to polarized vesicle transport.

    The Journal of cell biology 1996;133;4;819-30

  • Chromosomal localization of the ankyrinG gene (ANK3/Ank3) to human 10q21 and mouse 10.

    Kapfhamer D, Miller DE, Lambert S, Bennett V, Glover TW and Burmeister M

    Department of Human Genetics, University of Michigan, Ann Arbor, USA.

    The ankyrin3 gene encodes a novel form of ankyrin, AnkyrinG, expressed in multiple tissues but characteristically present at the axonal initial segment and nodes of Ranvier of neurons in the central and peripheral nervous systems. We have localized ANK3 to human Chromosome 10q21 by fluorescence in situ hybridization. The position of the murine homologue was determined by intersubspecific backcross analysis, mapping Ank3 to mouse Chromosome 10, between microsatellite marker D10Mit31 and the Bcr gene. This interval is known to comprise a region syntenic to human 10q. The localization of ANK3 is a preliminary step in identifying neurological disorders potentially associated with the gene.

    Genomics 1995;27;1;189-91

  • AnkyrinG. A new ankyrin gene with neural-specific isoforms localized at the axonal initial segment and node of Ranvier.

    Kordeli E, Lambert S and Bennett V

    Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.

    We have characterized a new ankyrin gene, expressed in brain and other tissues, that is subject to extensive tissue-specific alternative mRNA processing. The full-length polypeptide has a molecular mass of 480 kDa and includes a predicted globular head domain, with membrane- and spectrin-binding activities, as well as an extended "tail" domain. We term this gene ankyrinG based on its giant size and general expression. Two brain-specific isoforms of 480 kDa and 270 kDa were identified that contain a unique stretch of sequence highly enriched in serine and threonine residues immediately following the globular head domain. Antibodies against the serine-rich domain and spectrin-binding domain revealed labeling of nodes of Ranvier and axonal initial segments. Ankyrin-binding proteins also known to be localized in these specialized membrane domains include the voltage-dependent sodium channel, the sodium/potassium ATPase, sodium/calcium exchanger, and members of the neurofascin/L1 family of cell adhesion molecules. The neural-specific ankyrinG polypeptides are candidates to participate in maintenance/targeting of ion channels and cell adhesion molecules to nodes of Ranvier and axonal initial segments.

    Funded by: NIDDK NIH HHS: 537DK29808

    The Journal of biological chemistry 1995;270;5;2352-9

Gene lists (6)

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
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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