G2Cdb::Gene report

Gene id
G00001838
Gene symbol
ANK1 (HGNC)
Species
Homo sapiens
Description
ankyrin 1, erythrocytic
Orthologue
G00000589 (Mus musculus)

Databases (7)

Gene
ENSG00000029534 (Ensembl human gene)
286 (Entrez Gene)
991 (G2Cdb plasticity & disease)
ANK1 (GeneCards)
Literature
182900 (OMIM)
Marker Symbol
HGNC:492 (HGNC)
Protein Sequence
P16157 (UniProt)

Synonyms (1)

  • SPH1

Literature (52)

Pubmed - other

  • Screening for replication of genome-wide SNP associations in sporadic ALS.

    Cronin S, Tomik B, Bradley DG, Slowik A and Hardiman O

    Department of Clinical Neurological Sciences, Royal College of Surgeons in Ireland, Dublin 2, Ireland. scronin@rcsi.ie

    We recently reported a joint analysis of genome-wide association (GWA) data on 958 sporadic amyotrophic lateral sclerosis (ALS) cases and 932 controls from Ireland and the publicly available data sets from the United States and the Netherlands. The strongest pooled association was rs10260404 in the dipeptidyl-peptidase 6 (DPP6) gene. Here, we sought confirmation of joint analysis signals in both an expanded Irish and a Polish ALS cohort. Among 287 522 autosomal single-nucleotide polymorphisms (SNPs), 27 were commonly associated on joint analysis of the Irish, US and Dutch GWAs. These 27 SNPs were genotyped in an expanded Irish cohort (312 patients with SALS; 259 controls) and an additional Polish cohort (218 patients; 356 controls). Eleven SNPs, including rs10260404, reached a final P-value below 0.05 in the Irish cohort. In the Polish cohort, only one SNP, rs6299711, showed nominal association with ALS. Pooling of data for 1267 patients with ALS and 1336 control subjects did not identify any association reaching Bonferroni significance (P<1.74 x 10(-7)). The present strategy did not reveal any consistently associated SNP across four populations. The result for DPP6 is surprising, as it has been replicated elsewhere. We discuss the possible interpretations and implications of these findings for future ALS GWA studies both within and between populations.

    European journal of human genetics : EJHG 2009;17;2;213-8

  • 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

  • Lipid-binding role of betaII-spectrin ankyrin-binding domain.

    Bok E, Plazuk E, Hryniewicz-Jankowska A, Chorzalska A, Szmaj A, Dubielecka PM, Stebelska K, Diakowski W, Lisowski M, Langner M and Sikorski AF

    Institute of Biochemistry and Molecular Biology, University of Wrocław, ul. Przybyszewskiego 63/77, 51-149 Wrocław, Poland.

    It is known that erythroid and non-erythroid spectrins binding of vesicles and monolayers containing PE proved sensitive to inhibition by red blood cell ankyrin. We now show that the bacterially-expressed recombinant peptides representing betaII(brain)-spectrin's ankyrin-binding domain and its truncated mutants showed lipid-binding activity, although only those containing a full-length amino terminal fragment showed high to moderate affinity towards phospholipid mono- and bilayers and a substantial sensitivity of this binding to inhibition by ankyrin. These results are in accordance with our published data on betaI-spectrin's ankyrin-binding domain [Hryniewicz-Jankowska A, et al. Mapping of ankyrin-sensitive, PE/PC mono- and bilayer binding site in erythroid beta-spectrin. Biochem J 2004;382:677-85]. Moreover, we tested also the effect of transient transfection of living cells of several cell-lines with vectors coding for GFP-conjugates including betaII and also betaI full-length ankyrin-binding domain and their truncated fragments on the membrane skeleton organization. The transfection with constructs encoding full-length ankyrin-binding domain of betaII and betaI spectrin resulted in increased aggregation of membrane skeleton and its punctate appearance in contrast to near normal appearance of membrane skeleton of cells transiently transfected with GFP control or construct encoding ankyrin-binding domain truncated at their N-terminal region. Our results therefore indicate the importance of N-terminal region for lipid-binding activity of the beta-spectrin ankyrin-binding domain and its substantial role in maintaining the spectrin-based skeleton distribution.

    Cell biology international 2007;31;12;1482-94

  • Mapping the binding site on small ankyrin 1 for obscurin.

    Borzok MA, Catino DH, Nicholson JD, Kontrogianni-Konstantopoulos A and Bloch RJ

    Department of Biochemistry and Molecular Biology, University of Maryland, School of Medicine, Baltimore 21201, USA.

    Small ankyrin 1 (sAnk1), an integral protein of the sarcoplasmic reticulum encoded by the ANK1 gene, binds with nanomolar affinity to the C terminus of obscurin, a giant protein surrounding the contractile apparatus in striated muscle. We used site-directed mutagenesis to characterize the binding site on sAnk1, specifically addressing the role of two putative amphipathic, positively charged helices. We measured binding qualitatively by blot overlay assays and quantitatively by surface plasmon resonance and showed that both positively charged sequences are required for activity. We showed further that substitution of a lysine or arginine with an alanine or glutamate located at the same position along either of the two putative helices has similar inhibitory or stimulatory effects on binding and that the effects of a particular mutation depended on the position of the mutated amino acid in each helix. We modeled the structure of the binding region of sAnk1 by homology with ankyrin repeats of human Notch1, which have a similar pattern of charged and hydrophobic residues. Our modeling suggested that each of the two positively charged sequences forms pairs of amphipathic, anti-parallel alpha-helices flanked by beta-hairpin-like turns. Most of the residues in homologous positions along each helical unit have similar, though not identical, orientations. CD spectroscopy confirmed the alpha-helical content of sAnk1, approximately 33%, predicted by the model. Thus, structural and mutational studies of the binding region on sAnk1 for obscurin suggest that it consists of two ankyrin repeats with very similar structures.

    Funded by: NHLBI NIH HHS: R01 HL64304; NIAMS NIH HHS: R01 AR52768

    The Journal of biological chemistry 2007;282;44;32384-96

  • Structural insight into an ankyrin-sensitive lipid-binding site of erythroid beta-spectrin.

    Czogalla A, Jaszewski AR, Diakowski W, Bok E, Jezierski A and Sikorski AF

    Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland.

    It was recently shown that the region within beta-spectrin responsible for interactions with ankyrin includes a lipid-binding site which displayed sensitivity to inhibition by ankyrin. We studied its structure by constructing a series of single and double spin-labeled beta-spectrin-derived peptides and analyzing their spin-spin distances via electron paramagnetic resonance spectroscopy and the Fourier deconvolution method. The results indicate that the whole ankyrin-sensitive lipid-binding site of beta-spectrin exhibits a helical conformation revealing a distinct 3(10)-helix contribution at its N-terminus. The start of the helix was located five residues upstream along the sequence compared to the theoretical predictions. A model based on the obtained data provides direct evidence that the examined lipid-binding site is a highly amphip f7e athic helix, which is correlated with the specific conformation of its N-terminal fragment.

    Molecular membrane biology 2007;24;3;215-24

  • Molecular interactions with obscurin are involved in the localization of muscle-specific small ankyrin1 isoforms to subcompartments of the sarcoplasmic reticulum.

    Armani A, Galli S, Giacomello E, Bagnato P, Barone V, Rossi D and Sorrentino V

    Molecular Medicine Section, Department of Neuroscience, University of Siena, 53100 Siena, Italy.

    We report here on experiments aimed to characterise the molecular basis of the interactions between muscle-specific ankyrin1 isoforms localized on the sarcoplasmic reticulum and obscurin a protein associated with the contractile apparatus. A novel small muscle-specific ankyrin isoform, ank1.9 was identified that, similarly to the known ank1.5 isoform, was able to bind to obscurin in yeast two-hybrid assay and in pull-down experiments. Two distinct binding sites in the C-terminus of obscurin were found to mediate binding with ank1.5 and ank1.9. Interactions between ank1.5 and ank1.9 with recombinant proteins containing one or two of the binding sites of obscurin were confirmed by expressing recombinant proteins in NIH3T3 cells. In cultured myotubes, ank1.5 and ank1.9 colocalized with endogenous obscurin at the M-band region. In contrast with evidence of efficient binding between small ank1 isoforms and obscurin, in vitro interaction studies and transfection experiments in myotubes indicated that small ank1 isoforms do not efficiently interact with titin. Altogether, these results support a role of obscurin in mediating the subcellular localization of small ank1 isoforms in striated muscle cells. Given that the localization of small muscle-specific ank1 isoforms mirrors that of obscurin, we propose that obscurin and small ank1 isoforms may form stable interactions that may be relevant to connect the sarcoplasmic reticulum and the contractile apparatus in skeletal muscle cells.

    Funded by: Telethon: GGP02168

    Experimental cell research 2006;312;18;3546-58

  • Associations of protein 4.2 with band 3 and ankyrin.

    Su Y, Ding Y, Jiang M, Jiang W, Hu X and Zhang Z

    Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China.

    Protein-protein and protein-lipid interactions are thought to play the vital role in maintenance and deformation of red blood cell (RBC) membrane. Protein 4.2, a 76-KDa peripheral protein, binds to the cytoplasmic domain of band 3 (CDB3) and also interacts with ankyrin in RBCs. In order to explore the characteristics of protein 4.2-CDB3-ankyrin interactions, three protein 4.2-derived recombinant proteins encompassing amino acid residues 31-200, 1-300, and 187-260 respectively were expressed in Escherichia coli. Their interactions with CDB3 and ankyrin were investigated by using Far-Western blot and pull-down assay. The results showed that the CDB3-binding site of protein 4.2 is located in the region of residues 200-211 and the ankyrin-binding site is located in the region of residues 187-200 of protein 4.2. Our findings also suggested that the ankyrin D34 domain can interact directly with protein 4.2. The proper tertiary structures of these protein 4.2 fragments are essential for protein 4.2-ankyrin interaction. Meanwhile, ankyrin can enhance the interaction between protein 4.2 and CDB3.

    Molecular and cellular biochemistry 2006;289;1-2;159-66

  • [Polymorphism analysis of G199A, Ncol in ANK1 and Memphis I in SLC4A1 genes in Mexican healthy individuals and subjects affected with hereditary spherocytosis].

    Camacho-Torres AL, Sánchez-López JY, Mesa-Cornejo VM, Ibarra B and Perea-Díaz FJ

    División de Genética Humana, Centro de Investigación Biomédica de Occidente, Centro Médico Nacional de Occidente, Instituto Mexicano del Seguro Social, Guadalajara, Jal., México.

    Background: In Mexico, Hereditary Spherocytosis (HS) is the main cause of hereditary hemolytic anemia, due to mutations of one or more genes involved in the erythrocyte membrane, making it difficult to identify the primary gene.

    Objective: With the purpose of estimating the use of the polymorphisms G199A and NcoI of ANK1 gene, and Memphis I of SLC4A1 gene, as genetic markers to screen this disease, we searched the allelic and genotypic frequencies in 45 DNA samples of HS patients and 28 from healthy individuals.

    Results: Allelic and genotypic frequencies were similar in both studied groups for the G199A and Memphis I polymorphisms, with low frequency of heterozygosis showing its limited use as a genetic marker. The allelic and genotypic frequencies of the NcoI polymorphism were also similar in both groups, however a higher heterozygote frequency was observed (0.49 and 0.43 in patients and healthy individuals), a feature that may turn it into a useful genetic marker.

    Conclusions: Since there are other genes implicated in the molecular pathology of the HS, we consider it necessary to continue analyzing other polymorphisms of the genes involved in Hereditary Spherocytosis among the Mexican population.

    Gaceta medica de Mexico 2006;142;5;435-7

  • Rate of rupture and reattachment of the band 3-ankyrin bridge on the human erythrocyte membrane.

    Anong WA, Weis TL and Low PS

    Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.

    The principal bridge connecting the erythrocyte membrane to the spectrin-based skeleton is established by band 3 and ankyrin; mutations leading to reduced bridge formation or increased bridge rupture result in morphological and mechanical abnormalities. Because membrane mechanical properties are determined in part by the protein interactions that stabilize the membrane, we have evaluated the rates of rupture and reattachment of band 3-ankyrin bridges under both resting and mechanically stressed conditions. To accomplish this, we have examined the rate of ankyrin displacement from inside-out vesicles by the hexahistidine-tagged cytoplasmic domain of band 3, cdb3-(His)6 and the rate of substitution of cdb3-(His)6 into endogenous band 3-ankyrin bridges in resealed erythrocytes in the presence and absence of shear stress. We demonstrate that 1) exogenous cdb3-(His)6 displaces endogenous ankyrin from IOVs with a half-time and first order rate constant of 42 +/- 14 min and 0.017 +/- 0.0058 min(-1), respectively; 2) exogenous cdb3-(His)6 substitutes endogenous band 3 in its linkage to ankyrin in resealed cells with a half-time and first order rate constant of 12 +/- 3.6 min and 0.060 +/- 0.019 min(-1), respectively; 3) cdb3-(His)6-mediated rupture of the band 3-ankyrin bridge in resealed cells results in decreased membrane mechanical stability, decreased deformability, abnormal morphology, and spontaneous vesiculation of the cells; and 4) the above on/off rates are not significantly accelerated by mechanical shear stress. We conclude that the off rates of the band 3-ankyrin interaction are sufficiently slow to allow sustained erythrocyte deformation without loss of elasticity.

    Funded by: NIGMS NIH HHS: GM24417-26, T32-GM008296-17

    The Journal of biological chemistry 2006;281;31;22360-6

  • Functional interaction between Rh proteins and the spectrin-based skeleton in erythroid and epithelial cells.

    Nicolas V, Mouro-Chanteloup I, Lopez C, Gane P, Gimm A, Mohandas N, Cartron JP, Le Van Kim C and Colin Y

    Inserm, U665, F-75015 Paris, France.

    We summarize the different experimental approaches which provide evidence that direct interaction of Rh and RhAG to ankyrin-R constitutes, together with the AE-1 (Band 3)-ankyrin-protein 4.2 and GPC-protein 4.1-p55 complexes, another major anchoring site between the red cell membrane bilayer and the underlying spectrin-based skeleton. The observations that some residues of the ankyrin binding site are mutated in Rh and RhAG proteins from some weak D and Rh(null) variants, respectively, suggest that the Rh-RhAG/ankyrin-R interaction plays a crucial role in the biosynthesis and/or the stability of the Rh complex in the red cell membrane. Similarly, binding to ankyrin G is required for cell surface expression of the non-erythroid member of the Rh protein family, RhBG, at the basolateral membrane domain of polarized epithelial cells. The next challenge will be to determine whether binding to the membrane skeleton may be critical for the emerging ammonium transport function of Rh proteins in erythroid and non-erythroid cells.

    Transfusion clinique et biologique : journal de la Societe francaise de transfusion sanguine 2006;13;1-2;23-8

  • 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

  • CD44 interaction with ankyrin and IP3 receptor in lipid rafts promotes hyaluronan-mediated Ca2+ signaling leading to nitric oxide production and endothelial cell adhesion and proliferation.

    Singleton PA and Bourguignon LY

    Endocrine Unit, Department of Medicine, University of California-San Francisco and VA Medical Center, San Francisco, CA 94121, USA.

    In this study, we have showed that aortic endothelial cells (GM7372A cell line) express CD44v10 [a hyaluronan (HA) receptor], which is significantly enriched in cholesterol-containing lipid rafts (characterized as caveolin-rich plasma membrane microdomains). HA binding to CD44v10 promotes recruitment of the cytoskeletal protein, ankyrin and inositol 1,4,5-triphosphate (IP3) receptor into cholesterol-containing lipid rafts. The ankyrin repeat domain (ARD) of ankyrin is responsible for binding IP3 receptor to CD44v10 at lipid rafts and subsequently triggering HA/CD44v10-mediated intracellular calcium (Ca2+) mobilization leading to a variety of endothelial cell functions such as nitric oxide (NO) production, cell adhesion and proliferation. Further analyses indicate (i) disruption of lipid rafts by depleting cholesterol from the membranes of GM7372A cells (using methyl-beta-cyclodextrin treatment) or (ii) interference of endogenous ankyrin binding to CD44 and IP3 receptor using overexpression of ARD fragments (by transfecting cells with ARDcDNA) not only abolishes ankyrin/IP3 receptor accumulation into CD44v10/cholesterol-containing lipid rafts, but also blocks HA-mediated Ca2+ signaling and endothelial cell functions. Taken together, our findings suggest that CD44v10 interaction with ankyrin and IP3 receptor in cholesterol-containing lipid rafts plays an important role in regulating HA-mediated Ca2+ signaling and endothelial cell functions such as NO production, cell adhesion and proliferation.

    Funded by: NCI NIH HHS: R01CA 78633, R01CA66163; NIAMS NIH HHS: P01 AR039448

    Experimental cell research 2004;295;1;102-18

  • Ankyrin is a target of spectrin's E2/E3 ubiquitin-conjugating/ligating activity.

    Chang TL, Cubillos FF, Kakhniashvili DG and Goodman SR

    Department of Molecular and Cell Biology, UTD Sickle Cell Disease Research Center, Institute of Biomedical Sciences and Technology, The University of Texas at Dallas, Richardson, TX 75083, USA.

    Ubiquitin is a small protein of 8.6 kDa molecular weight. When polyubiquitin is attached to target proteins, they are tagged for destruction by cytoplasmic organelles called proteasomes. We now know that ubiquitination of target proteins also regulates functions as diverse as the sorting of proteins to different intracellular destinations, cell signaling, cell division, gene transcription, and protein-protein interactions. The ubiquitination of target proteins requires a cascade of enzymes: E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme and E3 ubiquitin ligating enzyme. Recently we have demonstrated that the red blood cell (RBC) membrane skeletal protein, spectrin, has E2/E3 enzymatic activities in its alpha-subunit, that can transfer ubiquitin to itself. We have now created a cell free assay using biotinylated ubiquitin that allows detection of target proteins by streptavidin peroxidase. This approach coupled with immunoprecipitation, purification and micro liquid chromatography coupled to tandem mass spectrometry has identified ankyrin as a target of spectrin's E2/E3 activity. Western blotting, with ubiquitin antibody, of purified ankyrin and its well characterized functional domains, has demonstrated that both the spectrin and band 3 binding domains are ubiquitinated in vivo.

    Funded by: NHLBI NIH HHS: HL 070588

    Cellular and molecular biology (Noisy-le-Grand, France) 2004;50;1;59-66

  • Rh-RhAG/ankyrin-R, a new interaction site between the membrane bilayer and the red cell skeleton, is impaired by Rh(null)-associated mutation.

    Nicolas V, Le Van Kim C, Gane P, Birkenmeier C, Cartron JP, Colin Y and Mouro-Chanteloup I

    INSERM U76, Institut National de la Transfusion Sanguine, 6 Rue Alexandre Cabanel, 75015 Paris, France.

    Several studies suggest that the Rh complex represents a major interaction site between the membrane lipid bilayer and the red cell skeleton, but little is known about the molecular basis of this interaction. We report here that ankyrin-R is capable of interacting directly with the C-terminal cytoplasmic domain of Rh and RhAG polypeptides. We first show that the primary defect of ankyrin-R in normoblastosis (nb/nb) spherocytosis mutant mice is associated with a sharp reduction of RhAG and Rh polypeptides. Secondly, our flow cytometric analysis of the Triton X-100 extractability of recombinant fusion proteins expressed in erythroleukemic cell lines suggests that the C-terminal cytoplasmic domains of Rh and RhAG are sufficient to mediate interaction with the erythroid membrane skeleton. Using the yeast two-hybrid system, we demonstrate a direct interaction between the cytoplasmic tails of Rh and RhAG and the second repeat domain (D2) of ankyrin-R. This finding is supported by the demonstration that the substitution of Asp-399 in the cytoplasmic tail of RhAG, a mutation associated with the deficiency of the Rh complex in one Rhnull patient, totally impaired interaction with domain D2 of ankyrin-R. These results identify the Rh/RhAG-ankyrin complex as a new interaction site between the red cell membrane and the spectrin-based skeleton, the disruption of which might result in the stomato-spherocytosis typical of Rhnull red cells.

    The Journal of biological chemistry 2003;278;28;25526-33

  • Obscurin is a ligand for small ankyrin 1 in skeletal muscle.

    Kontrogianni-Konstantopoulos A, Jones EM, Van Rossum DB and Bloch RJ

    Department of Physiology, University of Maryland, School of Medicine, Baltimore 21201, USA. akons001@umaryland.edu

    The factors that organize the internal membranes of cells are still poorly understood. We have been addressing this question using striated muscle cells, which have regular arrays of membranes that associate with the contractile apparatus in stereotypic patterns. Here we examine links between contractile structures and the sarcoplasmic reticulum (SR) established by small ankyrin 1 (sAnk1), a approximately 17.5-kDa integral protein of network SR. We used yeast two-hybrid to identify obscurin, a giant Rho-GEF protein, as the major cytoplasmic ligand for sAnk1. The binding of obscurin to the cytoplasmic sequence of sAnk1 is mediated by a sequence of obscurin that is C-terminal to its last Ig-like domain. Binding was confirmed in two in vitro assays. In one, GST-obscurin, bound to glutathione-matrix, specifically adsorbed native sAnk1 from muscle homogenates. In the second, MBP-obscurin bound recombinant GST-sAnk1 in nitrocellulose blots. Kinetic studies using surface plasmon resonance yielded a K(D) = 130 nM. On subcellular fractionation, obscurin was concentrated in the myofibrillar fraction, consistent with its identification as sarcomeric protein. Nevertheless, obscurin, like sAnk1, concentrated around Z-disks and M-lines of striated muscle. Our findings suggest that obscurin binds sAnk1, and are the first to document a specific and direct interaction between proteins of the sarcomere and the SR.

    Funded by: NHLBI NIH HHS: R01 HL 64304, R01 HL064304; NIAMS NIH HHS: T32 AR 07293

    Molecular biology of the cell 2003;14;3;1138-48

  • The hydrophilic domain of small ankyrin-1 interacts with the two N-terminal immunoglobulin domains of titin.

    Kontrogianni-Konstantopoulos A and Bloch RJ

    Department of Physiology, University of Maryland School of Medicine, Baltimore 21201, USA. akons001@umaryland.edu

    Little is known about the mechanisms that organize the internal membrane systems in eukaryotic cells. We are addressing this question in striated muscle, which contains two novel systems of internal membranes, the transverse tubules and the sarcoplasmic reticulum (SR). Small ankyrin-1 (sAnk1) is an approximately 17-kDa transmembrane protein of the SR that concentrates around the Z-disks and M-lines of each sarcomere. We used the yeast two-hybrid assay to determine whether sAnk1 interacts with titin, a giant myofibrillar protein that organizes the sarcomere. We found that the hydrophilic cytoplasmic domain of sAnk1 interacted with the two most N-terminal Ig domains of titin, ZIg1 and ZIg2, which are present at the Z-line in situ. Both ZIg1 and ZIg2 were required for binding activity. sAnk1 did not interact with other sequences of titin that span the Z-disk or with Ig domains of titin near the M-line. Titin ZIg1/2 also bound T-cap/telethonin, a 19-kDa protein of the Z-line. We show that titin ZIg1/2 could form a three-way complex with sAnk1 and T-cap. Our results indicate that titin ZIg1/2 can bind sAnk1 in muscle homogenates and suggest a role for these proteins in organizing the SR around the contractile apparatus at the Z-line.

    Funded by: NHLBI NIH HHS: R01 HL64304

    The Journal of biological chemistry 2003;278;6;3985-91

  • Dissociation of spectrin-ankyrin complex as a basis for loss of Na-K-ATPase polarity after ischemia.

    Woroniecki R, Ferdinand JR, Morrow JS and Devarajan P

    Division of Pediatric Nephrology, Albert Einstein College of Medicine, New York, New York 10467, USA.

    The polarized distribution of Na-K-ATPase at the basolateral membranes of renal tubule epithelial cells is maintained via a tethering interaction with the underlying spectrin-ankyrin cytoskeleton. In this study, we have explored the mechanism underlying the loss of Na-K-ATPase polarity after ischemic injury in Madin-Darby canine kidney (MDCK) cells, utilizing a novel antibody raised against a recently described kidney-specific isoform of ankyrin. In control MDCK cells, ankyrin was colocalized with Na-K-ATPase at the basolateral membrane. ATP depletion resulted in a duration-dependent mislocation of Na-K-ATPase and ankyrin throughout the cytoplasm. Colocalization studies showed a partial overlap between the distribution of ankyrin and Na-K-ATPase at all periods after ATP depletion. By immunoprecipitation with anti-ankyrin antibody, the mislocated Na-K-ATPase remained bound to ankyrin at all time points after ATP depletion. However, the interaction between ankyrin and spectrin was markedly diminished within 3 h of ATP depletion and was completely lost after 6 h. In solution binding assays using a fusion peptide of glutathione S-transferase with the ankyrin binding domain of Na-K-ATPase, a complex with ankyrin was detected at all time points after ATP depletion, but spectrin was lost from the complex in a duration-dependent manner. The loss of spectrin binding was not attributable to spectrin degradation but was associated with hyperphosphorylation of ankyrin. The results suggest that a dissociation of the membrane-cytoskeleton complex at the spectrin-ankyrin interface may contribute to the loss of Na-K-ATPase polarity after ischemic injury and reaffirm a critical adapter role for ankyrin in the normal maintenance of Na-K-ATPase polarity.

    Funded by: NIDDK NIH HHS: DK-07110, DK-43812, DK-53289

    American journal of physiology. Renal physiology 2003;284;2;F358-64

  • Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles.

    Bagnato P, Barone V, Giacomello E, Rossi D and Sorrentino V

    Molecular Medicine Section, Department of Neuroscience, University of Siena, 53100 Siena, Italy.

    Assembly of specialized membrane domains, both of the plasma membrane and of the ER, is necessary for the physiological activity of striated muscle cells. The mechanisms that mediate the structural organization of the sarcoplasmic reticulum with respect to the myofibrils are, however, not known. We report here that ank1.5, a small splice variant of the ank1 gene localized on the sarcoplasmic reticulum membrane, is capable of interacting with a sequence of 25 aa located at the COOH terminus of obscurin. Obscurin is a giant sarcomeric protein of approximately 800 kD that binds to titin and has been proposed to mediate interactions between myofibrils and other cellular structures. The binding sites and the critical aa required in the interaction between ank1.5 and obscurin were characterized using the yeast two-hybrid system, in in vitro pull-down assays and in experiments in heterologous cells. In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin. The M line localization of ank1.5 required a functional obscurin-binding site, because mutations of this domain resulted in a diffused distribution of the mutant ank1.5 protein in skeletal muscle cells. The interaction between ank1.5 and obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils. In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum. Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin. Based on reported evidence showing that the COOH terminus of ank2.2 is necessary for the localization of ryanodine receptors and InsP3 receptors in the sarcoplasmic reticulum, we propose that obscurin, through multiple interactions with ank1.5 and ank2.2 isoforms, may assemble a large protein complex that, in addition to a structural function, may play a role in the organization of specific subdomains in the sarcoplasmic reticulum.

    Funded by: Telethon: TI_GGP02168

    The Journal of cell biology 2003;160;2;245-53

  • Crystal structure of a 12 ANK repeat stack from human ankyrinR.

    Michaely P, Tomchick DR, Machius M and Anderson RG

    Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9039, USA. peter.michaely@utsouthwestern.edu

    Ankyrins are multifunctional adaptors that link specific proteins to the membrane-associated, spectrin- actin cytoskeleton. The N-terminal, 'membrane-binding' domain of ankyrins contains 24 ANK repeats and mediates most binding activities. Repeats 13-24 are especially active, with known sites of interaction for the Na/K ATPase, Cl/HCO(3) anion exchanger, voltage-gated sodium channel, clathrin heavy chain and L1 family cell adhesion molecules. Here we report the crystal structure of a human ankyrinR construct containing ANK repeats 13-24 and a portion of the spectrin-binding domain. The ANK repeats are observed to form a contiguous spiral stack with which the spectrin-binding domain fragment associates as an extended strand. The structural information has been used to construct models of all 24 repeats of the membrane-binding domain as well as the interactions of the repeats with the Cl/HCO(3) anion exchanger and clathrin. These models, together with available binding studies, suggest that ion transporters such as the anion exchanger associate in a large central cavity formed by the ANK repeat spiral, while clathrin and cell adhesion molecules associate with specific regions outside this cavity.

    Funded by: NHLBI NIH HHS: HL 20948, P01 HL020948; NIGMS NIH HHS: GM52016, R01 GM052016

    The EMBO journal 2002;21;23;6387-96

  • BLOC-1, a novel complex containing the pallidin and muted proteins involved in the biogenesis of melanosomes and platelet-dense granules.

    Falcón-Pérez JM, Starcevic M, Gautam R and Dell'Angelica EC

    Department of Human Genetics, UCLA School of Medicine, Los Angeles, California 90095, USA.

    Recent studies have led to the identification of a group of genes required for normal biogenesis of lysosome-related organelles such as melanosomes and platelet-dense granules. Two of these genes, which are defective in the pallid and muted mutant mouse strains, encode small, coiled-coil-forming proteins that display no homology to each other or to any known protein. We report that these two proteins, pallidin and muted, are components of a novel protein complex. We raised antibodies that allow for detection of pallidin from a wide variety of mammalian cells. Endogenous pallidin was distributed in both soluble and peripheral membrane protein fractions. Size-exclusion chromatography and sedimentation velocity analyses indicated that the bulk of cytosolic pallidin is a component of an asymmetric protein complex with a molecular mass of approximately 200 kDa. We named this complex BLOC-1 (for biogenesis of lysosome-related organelles complex 1). Steady-state pallidin protein levels were reduced in fibroblasts derived from muted and reduced pigmentation mice, suggesting that the genes defective in these two mutant strains could encode components of BLOC-1 that are required for pallidin stability. Co-immunoprecipitation and immunodepletion experiments using an antibody to muted confirmed that this protein is a subunit of BLOC-1. Yeast two-hybrid analyses revealed that pallidin is capable of self-association through a region that contains its two coiled-coil forming domains. Unlike AP-3-deficient pearl fibroblasts, which display defects in intracellular zinc storage, zinc distribution was not noticeably affected in pallid or muted fibroblasts. Interestingly, immunofluorescence and in vitro binding experiments demonstrated that pallidin/BLOC-1 is able to associate with actin filaments. We propose that BLOC-1 mediates the biogenesis of lysosome-related organelles by a mechanism that may involve self-assembly and interaction with the actin cytoskeleton.

    Funded by: NHLBI NIH HHS: HL68117; NIGMS NIH HHS: GM07104

    The Journal of biological chemistry 2002;277;31;28191-9

  • Plasmodium falciparum cysteine protease falcipain-2 cleaves erythrocyte membrane skeletal proteins at late stages of parasite development.

    Hanspal M, Dua M, Takakuwa Y, Chishti AH and Mizuno A

    Department of Biomedical Research, St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA 02135, USA. manjit_hanspal@cchcs.org

    Plasmodium falciparum-derived cysteine protease falcipain-2 cleaves host erythrocyte hemoglobin at acidic pH and specific components of the membrane skeleton at neutral pH. Analysis of stage-specific expression of these 2 proteolytic activities of falcipain-2 shows that hemoglobin-hydrolyzing activity is maximum in early trophozoites and declines rapidly at late stages, whereas the membrane skeletal protein hydrolyzing activity is markedly increased at the late trophozoite and schizont stages. Among the erythrocyte membrane skeletal proteins, ankyrin and protein 4.1 are cleaved by native and recombinant falcipain-2 near their C-termini. To identify the precise peptide sequence at the hydrolysis site of protein 4.1, we used a recombinant construct of protein 4.1 as substrate followed by MALDI-MS analysis of the cleaved product. We show that falcipain-2-mediated cleavage of protein 4.1 occurs immediately after lysine 437, which lies within a region of the spectrin-actin-binding domain critical for erythrocyte membrane stability. A 16-mer peptide containing the cleavage site completely inhibited the enzyme activity and blocked falcipain-2-induced fragmentation of erythrocyte ghosts. Based on these results, we propose that falcipain-2 cleaves hemoglobin in the acidic food vacuole at the early trophozoite stage, whereas it cleaves specific components of the red cell skeleton at the late trophozoite and schizont stages. It is the proteolysis of skeletal proteins that causes membrane instability, which, in turn, facilitates parasite release in vivo.

    Funded by: NHLBI NIH HHS: HL60152

    Blood 2002;100;3;1048-54

  • Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues.

    Bennett V and Baines AJ

    Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA. benne012@mc.duke.edu

    The spectrin-based membrane skeleton of the humble mammalian erythrocyte has provided biologists with a set of interacting proteins with diverse roles in organization and survival of cells in metazoan organisms. This review deals with the molecular physiology of spectrin, ankyrin, which links spectrin to the anion exchanger, and two spectrin-associated proteins that promote spectrin interactions with actin: adducin and protein 4.1. The lack of essential functions for these proteins in generic cells grown in culture and the absence of their genes in the yeast genome have, until recently, limited advances in understanding their roles outside of erythrocytes. However, completion of the genomes of simple metazoans and application of homologous recombination in mice now are providing the first glimpses of the full scope of physiological roles for spectrin, ankyrin, and their associated proteins. These functions now include targeting of ion channels and cell adhesion molecules to specialized compartments within the plasma membrane and endoplasmic reticulum of striated muscle and the nervous system, mechanical stabilization at the tissue level based on transcellular protein assemblies, participation in epithelial morphogenesis, and orientation of mitotic spindles in asymmetric cell divisions. These studies, in addition to stretching the erythrocyte paradigm beyond recognition, also are revealing novel cellular pathways essential for metazoan life. Examples are ankyrin-dependent targeting of proteins to excitable membrane domains in the plasma membrane and the Ca(2+) homeostasis compartment of the endoplasmic reticulum. Exciting questions for the future relate to the molecular basis for these pathways and their roles in a clinical context, either as the basis for disease or more positively as therapeutic targets.

    Physiological reviews 2001;81;3;1353-92

  • Cytoplasmic domain mutations of the L1 cell adhesion molecule reduce L1-ankyrin interactions.

    Needham LK, Thelen K and Maness PF

    Department of Biochemistry, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.

    The neural adhesion molecule L1 mediates the axon outgrowth, adhesion, and fasciculation that are necessary for proper development of synaptic connections. L1 gene mutations are present in humans with the X-linked mental retardation syndrome CRASH (corpus callosum hypoplasia, retardation, aphasia, spastic paraplegia, hydrocephalus). Three missense mutations associated with CRASH syndrome reside in the cytoplasmic domain of L1, which contains a highly conserved binding region for the cytoskeletal protein ankyrin. In a cellular ankyrin recruitment assay that uses transfected human embryonic kidney (HEK) 293 cells, two of the pathologic mutations located within the conserved SFIGQY sequence (S1224L and Y1229H) strikingly reduced the ability of L1 to recruit 270 kDa ankyrinG protein that was tagged with green fluorescent protein (ankyrin-GFP) to the plasma membrane. In contrast, the L1 missense mutation S1194L and an L1 isoform lacking the neuron-specific sequence RSLE in the cytoplasmic domain were as effective as RSLE-containing neuronal L1 in the recruitment of ankyrin-GFP. Ankyrin binding by L1 was independent of cell-cell interactions. Receptor-mediated endocytosis of L1 regulates intracellular signal transduction, which is necessary for neurite outgrowth. In rat B35 neuroblastoma cell lines stably expressing L1 missense mutants, antibody-induced endocytosis was unaffected by S1224L or S1194L mutations but appeared to be enhanced by the Y1229H mutation. These results suggested a critical role for tyrosine residue 1229 in the regulation of L1 endocytosis. In conclusion, specific mutations within key residues of the cytoplasmic domain of L1 (Ser(1224), Tyr(1229)) destabilize normal L1-ankyrin interactions and may influence L1 endocytosis to contribute to the mechanism of neuronal dysfunction in human X-linked mental retardation.

    Funded by: NIAAA NIH HHS: AA 11605; NICHD NIH HHS: HD 35170; NINDS NIH HHS: NS 26620

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;5;1490-500

  • Ankyrin gene mutations in japanese patients with hereditary spherocytosis.

    Nakanishi H, Kanzaki A, Yawata A, Yamada O and Yawata Y

    Department of Medicine, Kawasaki Medical School, Kurashiki City, Japan.

    We studied mutations of the ankyrin-1 (ANK-1) gene of genomic DNA from Japanese patients with hereditary spherocytosis (HS). Forty-nine patients from 46 unrelated families were included in this study. Of these patients, 19 cases from 16 unrelated families had HS of autosomal-dominant inheritance, and 30 patients had non-autosomal-dominant HS. Fifteen mutations of the ANK-1 gene pathognomonic for HS were identified: 4 nonsense mutations, 7 frameshift mutations, and 4 abnormal splicing mutations. These 15 mutations have not been previously reported. The frameshift mutations were found from exon 1 to exon 26, corresponding particularly to the band 3-binding domain of ankyrin. The nonsense mutations, on the contrary, were present mostly at the 3'-terminal side, especially in the spectrin-binding domain and the regulatory domain. The patients with ankyrin gene mutations tended to be more anemic with a higher level of reticulocytosis than those without these mutations. Fifteen silent mutations of the ANK-1 gene, most of which have previously been detected in HS patients in Western populations, were also found. The allele frequency of these silent mutations in the HS patients was nearly identical to that in normal subjects. There was no difference between the Japanese and Western populations in the allele frequency of these gene polymorphisms in healthy subjects or HS patients.

    International journal of hematology 2001;73;1;54-63

  • The human ankyrin-1 gene is selectively transcribed in erythroid cell lines despite the presence of a housekeeping-like promoter.

    Gallagher PG, Romana M, Tse WT, Lux SE and Forget BG

    Departments of Pediatrics, Internal Medicine and Genetics, Yale University School of Medicine, New Haven, CT 06520-8064, USA. patrick.gallagher@yale.edu

    To begin to study the sequence variations identified in the 5' flanking genomic DNA of the ankyrin gene in ankyrin-deficient hereditary spherocytosis patients and to provide additional insight into our understanding of the regulation of genes encoding erythrocyte membrane proteins, we have identified and characterized the erythroid promoter of the human ankyrin-1 gene. This compact promoter has characteristics of a housekeeping gene promoter, including very high G+C content and enzyme restriction sites characteristic of an HTF-island, no TATA, InR, or CCAAT consensus sequences, and multiple transcription initiation sites. In vitro DNAseI footprinting analyses revealed binding sites for GATA-1, CACCC-binding, and CGCCC-binding proteins. Transfection of ankyrin promoter/reporter plasmids into tissue culture cell lines yielded expression in erythroid, but not muscle, neural, or HeLa cells. Electrophoretic mobility shift assays, including competition and antibody supershift experiments, demonstrated binding of GATA-1, BKLF, and Sp1 to core ankyrin promoter sequences. In transfection assays, mutation of the Sp1 site had no effect on reporter gene expression, mutation of the CACCC site decreased expression by half, and mutation of the GATA-1 site completely abolished activity. The ankyrin gene erythroid promoter was transactivated in heterologous cells by forced expression of GATA-1 and to a lesser degree BKLF.

    Blood 2000;96;3;1136-43

  • 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

  • Structural requirements for association of neurofascin with ankyrin.

    Zhang X, Davis JQ, Carpenter S and Bennett V

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

    This paper presents the first structural analysis of the cytoplasmic domain of neurofascin, which is highly conserved among the L1CAM family of cell adhesion molecules, and describes sequence requirements for neurofascin-ankyrin interactions in living cells. The cytoplasmic domain of neurofascin dimerizes in solution, has an asymmetric shape, and exhibits a reversible temperature-dependent beta-structure. Residues Ser56-Tyr81 are necessary for ankyrin binding but do not contribute to either dimerization or formation of structure. Transfected neurofascin recruits GFP-tagged 270-kDa ankyrinG to the plasma membrane of human embryo kidney 293 cells. Deletion mutants demonstrate that the sequence Ser56-Tyr81 contains the major ankyrin-recruiting activity of neurofascin. Mutations of the FIGQY tyrosine (Y81H/A/E) greatly impair neurofascin-ankyrin interactions. Mutation of human L1 at the equivalent tyrosine (Y1229H) is responsible for certain cases of mental retardation (Van Camp, G., Fransen, E., Vits, L., Raes, G., and Willems, P. J. (1996) Hum. Mutat. 8, 391). Mutations F77A and E73Q greatly impair ankyrin binding activity, whereas mutation D74N and a triple mutation of D57N/D58N/D62N result in less loss of ankyrin binding activity. These results provide evidence for a highly specific interaction between ankyrin and neurofascin and suggest that ankyrin association with L1 is required for L1 function in humans.

    The Journal of biological chemistry 1998;273;46;30785-94

  • An alternative first exon in the distal end of the erythroid ankyrin gene leads to production of a small isoform containing an NH2-terminal membrane anchor.

    Birkenmeier CS, Sharp JJ, Gifford EJ, Deveau SA and Barker JE

    The Jackson Laboratory, Bar Harbor, Maine 04609, USA. cb@aretha.jax.org

    Mouse erythroid ankyrin is encoded by the Ank1 gene on Chromosome 8. The best studied isoform is 210 kDa and contains three large functional domains. We have recently reported a small Ank1 isoform (relative mobility 25 kDa) that localizes to the M and Z lines in skeletal muscle. Analyses of cDNA and genomic clones show that three transcripts of 3.5, 2.0, and 1.6 kb code for this protein. The different transcript sizes are due to their 3'-untranslated regions. They are encoded by a new first exon located in intron 39 of the Ank1 gene and three previously described Ank1 exons (40, 41, and 42). The 5'-flanking region contains a putative muscle-specific promoter. The sequence of the first 72 amino acids is novel and is predicted to form a transmembrane helix at the NH2-terminus. Functional testing of the putative transmembrane segment indicates that it acts as a membrane anchor, suggesting that the new Ank1 isoform may play an important role in organizing the contractile apparatus within the cell.

    Funded by: NCI NIH HHS: CA34196; NHLBI NIH HHS: HL29305; NIDDK NIH HHS: DK27726

    Genomics 1998;50;1;79-88

  • Anion exchanger 2 (AE2) binds to erythrocyte ankyrin and is colocalized with ankyrin along the basolateral plasma membrane of human gastric parietal cells.

    Jöns T and Drenckhahn D

    Institute of Anatomy of the Charité of the Humboldt-University of Berlin, Germany.

    The hydrochloric acid secreting parietal cells of the human stomach mucosa have been shown to express anion exchanger 2 (AE2). AE2 is restricted to the basolateral membrane domain and is responsible for the basolateral uptake of Cl- and release of HCO3-. It is unknown which mechanism is responsible for the basolateral positioning of AE2 in parietal cells. We raised the question whether AE2 might be immobilized at the cell surface by linkage via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we communicate two observations that support this hypothesis, namely that in parietal cells ankyrin is localized with AE2 along the basolateral cell surface and, secondly, that purified erythrocyte ankyrin binds to the in vitro-translated cytoplasmic domain of AE2. We conclude from these observations that AE2 in parietal cells might be linked via ankyrin to the basolateral membrane cytoskeleton and that this type of linkage might play a role in immobilizing AE2 in a non-random fashion along the basolateral membrane domain.

    European journal of cell biology 1998;75;3;232-6

  • An alternate promoter directs expression of a truncated, muscle-specific isoform of the human ankyrin 1 gene.

    Gallagher PG and Forget BG

    Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520-8021, USA. Patrick_Gallagher@QM.Yale.edu

    Ankyrin 1, an erythrocyte membrane protein that links the underlying cytoskeleton to the plasma membrane, is also expressed in brain and muscle. We cloned a truncated, muscle-specific ankyrin 1 cDNA composed of novel 5' sequences and 3' sequences previously identified in the last 3 exons of the human ankyrin 1 erythroid gene. Northern blot analysis revealed expression restricted to cardiac and skeletal muscle tissues. Deduced amino acid sequence of this muscle cDNA predicted a peptide of 155 amino acids in length with a hydrophobic NH2 terminus. Cloning of the corresponding chromosomal gene revealed that the ankyrin 1 muscle transcript is composed of four exons spread over approximately 10 kilobase pairs of DNA. Reverse transcriptase-polymerase chain reaction of skeletal muscle cDNA identified multiple cDNA isoforms created by alternative splicing. The ankyrin 1 muscle promoter was identified as a (G + C)-rich promoter located > 200 kilobase pairs from the ankyrin 1 erythroid promoter. An ankyrin 1 muscle promoter fragment directed high level expression of a reporter gene in cultured C2C12 muscle cells, but not in HeLa or K562 (erythroid) cells. DNA-protein interactions were identified in vitro at a single Sp1 and two E box consensus binding sites contained within the promoter. A MyoD cDNA expression plasmid transactivated an ankyrin 1 muscle promoter fragment/reporter gene plasmid in a dose-dependent fashion in both HeLa and K562 cells. A polyclonal antibody raised to human ankyrin 1 muscle-specific sequences reacted with peptides of 28 and 30 kDa on immunoblots of human skeletal muscle.

    The Journal of biological chemistry 1998;273;3;1339-48

  • Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library.

    Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A and Sugano S

    International and Interdisciplinary Studies, The University of Tokyo, Japan.

    Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.

    Gene 1997;200;1-2;149-56

  • Structure and organization of the human ankyrin-1 gene. Basis for complexity of pre-mRNA processing.

    Gallagher PG, Tse WT, Scarpa AL, Lux SE and Forget BG

    Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510, USA. Patrick_Gallagher@QM.Yale.edu

    Ankyrin-1 (ANK-1) is an erythrocyte membrane protein that is defective in many patients with hereditary spherocytosis, a common hemolytic anemia. In the red cell, ankyrin-1 provides the primary linkage between the membrane skeleton and the plasma membrane. To gain additional insight into the structure and function of this protein and to provide the necessary tools for further genetic studies of hereditary spherocytosis patients, we cloned the human ANK-1 chromosomal gene. Characterization of the ANK-1 gene genomic structure revealed that the erythroid transcript is composed of 42 exons distributed over approximately 160 kilobase pairs of DNA. Comparison of the genomic structure with the protein domains reveals a near-absolute correlation between the tandem repeats encoding the membrane-binding domain of ankyrin with the location of the intron/exon boundaries in the corresponding part of the gene. Erythroid stage-specific, complex patterns of alternative splicing were identified in the region encoding the regulatory domain of ankyrin-1. Novel brain-specific transcripts were also identified in this region, as well as in the "hinge" region between the membrane-binding and spectrin-binding domains. Utilization of alternative polyadenylation signals was found to be the basis for the previously described, stage-specific 9.0- and 7.2-kilobase pair transcripts of the ANK-1 gene.

    The Journal of biological chemistry 1997;272;31;19220-8

  • Small, membrane-bound, alternatively spliced forms of ankyrin 1 associated with the sarcoplasmic reticulum of mammalian skeletal muscle.

    Zhou D, Birkenmeier CS, Williams MW, Sharp JJ, Barker JE and Bloch RJ

    Department of Physiology, University of Maryland School of Medicine, Baltimore 21201, USA.

    We have recently found that the erythroid ankyrin gene, Ank1, expresses isoforms in mouse skeletal muscle, several of which share COOH-terminal sequence with previously known Ank1 isoforms but have a novel, highly hydrophobic 72-amino acid segment at their NH2 termini. Here, through the use of domain-specific peptide antibodies, we report the presence of the small ankyrins in rat and rabbit skeletal muscle and demonstrate their selective association with the sarcoplasmic reticulum. In frozen sections of rat skeletal muscle, antibodies to the spectrin-binding domain (anti-p65) react only with a 210-kD Ank1 and label the sarcolemma and nuclei, while antibodies to the COOH terminus of the small ankyrin (anti-p6) react with peptides of 20 to 26 kD on immunoblots and decorate the myoplasm in a reticular pattern. Mice homozygous for the normoblastosis mutation (gene symbol nb) are deficient in the 210-kD ankyrin but contain normal levels of the small ankyrins in the myoplasm. In nb/nb skeletal muscle, anti-p65 label is absent from the sarcolemma, whereas anti-p6 label shows the same distribution as in control skeletal muscle. In normal skeletal muscle of the rat, anti-p6 decorates Z lines, as defined by antidesmin distribution, and is also present at M lines where it surrounds the thick myosin filaments. Immunoblots of the proteins isolated with rabbit sarcoplasmic reticulum indicate that the small ankyrins are highly enriched in this fraction. When expressed in transfected HEK 293 cells, the small ankyrins are distributed in a reticular pattern resembling the ER if the NH2-terminal hydrophobic domain is present, but they are uniformly distributed in the cytosol if this domain is absent. These results suggest that the small ankyrins are integral membrane proteins of the sarcoplasmic reticulum. We propose that, unlike the 210-kD form of Ank1, previously localized to the sarcolemma and believed to be a part of the supporting cytoskeleton, the small Ank1 isoforms may stabilize the sarcoplasmic reticulum by linking it to the contractile apparatus.

    Funded by: NHLBI NIH HHS: HL29305, R01 HL029305; NICHD NIH HHS: HD16596, P01 HD016596; NINDS NIH HHS: NS17282, R01 NS017282

    The Journal of cell biology 1997;136;3;621-31

  • Ankyrin-1 mutations are a major cause of dominant and recessive hereditary spherocytosis.

    Eber SW, Gonzalez JM, Lux ML, Scarpa AL, Tse WT, Dornwell M, Herbers J, Kugler W, Ozcan R, Pekrun A, Gallagher PG, Schröter W, Forget BG and Lux SE

    Division of Hematology/Oncology, Children's Hospital, Boston, Massachusetts 02115, USA.

    Hereditary spherocytosis (HS) is the most common inherited haemolytic anaemia in Northern Europeans. The primary molecular defects reside in the red blood cell (RBC) membrane, particularly in proteins that link the membrane skeleton to the overlying lipid bilayer and its integral membrane constituents. Ankyrin-1 is the predominant linker molecule. It attaches spectrin, the major skeletal protein, to the cytoplasmic domain of band 3, the RBC anion exchanger. Two-thirds of patients with HS have combined spectrin and ankyrin-1 deficiency; deficiency of band 3 occurs in about 15 to 20% (ref.1). These data suggest that ankyrin-1 or band 3 defects may be common in HS. To test this we screened all 42 coding exons plus the 5' untranslated/promoter region of ankyrin-1 and the 19 coding exons of band 3 in 46 HS families. Twelve ankyrin-1 mutations and five band 3 mutations were identified. Missense mutations and a mutation in the putative ankyrin-1 promoter were common in recessive HS. In contrast, ankyrin-1 and band 3 frameshift and nonsense null mutations prevailed in dominant HS. Increased accumulation of the normal protein product partially compensated for the ankyrin-1 or band 3 defects in some of these null mutations. Our findings indicate that ankyrin-1 mutations are a major cause of dominant and recessive HS (approximately 35 to 65%), that band 3 mutations are less common (approximately 15 to 25%), and that the severity of HS is modified by factors other than the primary gene defect.

    Funded by: NHLBI NIH HHS: HL15157, HL32262; NIDDK NIH HHS: DK34083; ...

    Nature genetics 1996;13;2;214-8

  • Ankyrin Napoli: a de novo deletional frameshift mutation in exon 16 of ankyrin gene (ANK1) associated with spherocytosis.

    del Giudice EM, Hayette S, Bozon M, Perrotta S, Alloisio N, Vallier A, Iolascon A, Delaunay J and Morlé L

    Dipartimento di Pediatria, Seconda Università di Napoli, Italy.

    We report a case of apparently recessive hereditary spherocytosis in an Italian child. The proband exhibited a reduction of overall ankyrin in the red cell membrane. The parents were free of any haematological manifestations. The VNDR associated with the ankyrin gene (ANK1) were consistent with the following diplotypes: AC11/ AC14 (father), AC14/AC14 (mother) and AC11/AC14 (child). The cDNA of the patient disclosed the expression of the AC11 allele only. As a consequence, we put forward the hypothesis of a de novo inactivation affecting the ankyrin allele of maternal origin (AC14) and accounting for the disease. PCR amplification of exons, SSCP analysis and nucleotide sequencing disclosed a polymorphism: GAC --> AAC; Asp --> Asn in codon 328 of exon 10, and a one-nucleotide deletion : CTG --> CG in codon 573 of the exon 16. This frameshift mutation placed in phase the TGA triplet that normally overlaps codons 636 and 637. Termination of translation near the middle of ankyrin mRNA coding sequence resulted, presumably, in its premature degadation. The present allele has been designated allele Napoli.

    British journal of haematology 1996;93;4;828-34

  • Identification of 4370 expressed sequence tags from a 3'-end-specific cDNA library of human skeletal muscle by DNA sequencing and filter hybridization.

    Lanfranchi G, Muraro T, Caldara F, Pacchioni B, Pallavicini A, Pandolfo D, Toppo S, Trevisan S, Scarso S and Valle G

    A systematic study on the mRNA species expressed in the human skeletal muscle is presented in this paper. To carry on this study, a new method has been developed for the construction of unbiased cDNA libraries specially designed for the production of ESTs corresponding to the 3'-end portion of the mRNAs. The method has been applied to human skeletal muscle, where the analysis of the transcription profile is particularly difficult for the presence of several very abundant transcripts. To detect and quantify high-level mRNAs, the first 1054 ESTs were obtained from randomly selected clones. The 10 most abundant transcripts accounted for > 45% of the clones. Subsequently, these transcripts were identified by filter hybridization, thus making DNA sequencing more productive. Overall, 4370 clones were identified: 3372 by DNA sequencing and 998 by filter hybridization. The number of groups of sequences identifying individual transcripts was relatively low compared with other tissues, resulting in a total of 934 groups out of 4370 ESTs. Of these, 719 groups were represented by only one sequence.

    Genome research 1996;6;1;35-42

  • Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.

    Maruyama K and Sugano S

    Institute of Medical Science, University of Tokyo, Japan.

    We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

    Gene 1994;138;1-2;171-4

  • Hyaluronic acid-induced lymphocyte signal transduction and HA receptor (GP85/CD44)-cytoskeleton interaction.

    Bourguignon LY, Lokeshwar VB, Chen X and Kerrick WG

    Department of Cell Biology and Anatomy, University of Miami Medical School, FL 33101.

    The purposes of this study are to characterize the binding of hyaluronic acid (HA) to mouse T lymphoma cells, to measure changes in intracellular Ca2+ after HA binding, to elucidate the interaction between the HA receptor, GP85(CD44), and ankyrin in the membrane skeleton, and finally to correlate these events with HA receptor patching/capping and cell adhesion to HA. First, we established an in vivo assay using [3H]HA to measure the binding of HA to mouse T lymphoma cells, and found that the binding of [3H]HA to these cells is readily inhibited by the addition of anti-GP85(CD44) antibody suggesting that GP85(CD44) is the HA receptor. Next, we examined various signal transducing events that occur after HA binds to its receptor on mouse T lymphoma cells. The results of these studies indicate that the concentration of intracellular Ca2+ (as measured by Fura-2 fluorescence) begins to increase within seconds, and reaches a maximal level 5 min after the addition of HA to the cells. After this increase of intracellular Ca2+, HA induces both its receptors, GP85(CD44), to form patched/capped structures, and cell adhesion to HA-coated plates. Furthermore, we have determined that GP85(CD44) binds directly and specifically to ankyrin (Kd approximately 1.94 nM) in a saturable manner; and that ankyrin is preferentially accumulated underneath the HA-induced GP85(CD44) capped structures. The Ca2+ ionophore, ionomycin, was found to stimulate HA-induced receptor capping and adhesion while EGTA (a Ca2+ chelator), nefedipine/bepridil (Ca2+ channel blockers), W-7 (a calmodulin antagonist), and cytochalasin D (a microfilament inhibitor), but not colchicine (a microtubule disrupting agent), inhibit HA-induced receptor redistribution and adhesion to HA-coated plates. These findings strongly suggest that ankyrin plays an important role in linking the HA receptor, GP85(CD44), to the membrane-associated actomyosin contractile system during hyaluronic acid-mediated lymphocyte activation.

    Funded by: NCI NIH HHS: 1F32CA06057; NIGMS NIH HHS: GM 36353

    Journal of immunology (Baltimore, Md. : 1950) 1993;151;12;6634-44

  • Association of the brain anion exchanger, AE3, with the repeat domain of ankyrin.

    Morgans CW and Kopito RR

    Department of Biological Sciences, Stanford University, CA 94305-5020.

    The 89 kDa NH2-terminal domain of erythrocyte ankyrin is composed almost entirely of 22 tandem repeats of a 33 amino acid sequence and constitutes the binding site for the cytoplasmic NH2-terminal domain of the erythrocyte anion exchanger, AE1. We have developed an assay to evaluate the in vivo interaction between a fragment of ankyrin corresponding to this domain (ANK90) and two non-erythroid anion exchangers, AE2 and AE3, that share considerable structural homology with AE1. Association was assessed by co-immunoprecipitation of ANK90-anion exchanger complexes from detergent extracts of cells cotransfected with plasmids encoding the ankyrin fragment and the anion exchanger or mutants thereof. ANK90 was co-immunoprecipitated with AE1 but not with an AE1 deletion mutant lacking the cytoplasmic NH2-terminal domain. Using this assay, we show that the brain anion exchanger AE3, but not the closely related homologue, AE2, is capable of binding to ankyrin.

    Funded by: NIDDK NIH HHS: R01DK43994; NIGMS NIH HHS: R01GM38543

    Journal of cell science 1993;105 ( Pt 4);1137-42

  • The involvement of ankyrin in the regulation of inositol 1,4,5-trisphosphate receptor-mediated internal Ca2+ release from Ca2+ storage vesicles in mouse T-lymphoma cells.

    Bourguignon LY, Jin H, Iida N, Brandt NR and Zhang SH

    Department of Cell Biology and Anatomy, School of Medicine, University of Miami, Florida 33101.

    Mouse T-lymphoma cells contain a unique type of internal vesicle which bands at the relatively light density of 1.07 g/cc. These vesicles do not contain any detectable Golgi, endoplasmic reticulum, plasma membrane, or lysosomal marker protein activities. Binding of [3H]inositol 1,4,5-trisphosphate (IP3) to these internal vesicles reveals the presence of a single, high affinity class of IP3 receptor with a dissociation constant (Kd) of 1.6 +/- 0.3 nM. Using a panel of monoclonal and polyclonal antibodies against IP3 receptor, we have established that the IP3 receptor (approximately 260 kDa) displays immunological cross-reactivity with the rat brain IP3 receptor. Polymerase chain reaction analysis of first-strand cDNAs from both mouse T-lymphoma cells and rat brain tissues reveals that the IP3 receptor transcript in mouse T-lymphoma cells belongs to the short form (non-neuronal form) and not the long form (neuronal form) detected in rat brain tissue. Scatchard plot analysis shows that high affinity binding occurs between ankyrin and the IP3 receptor with a Kd of 0.2 nM. Most importantly, the binding of ankyrin to the light density vesicles significantly inhibits IP3 binding and IP3-induced internal Ca2+ release. These findings suggest that the cytoskeleton plays a pivotal role in the regulation of IP3 receptor-mediated internal Ca2+ release during lymphocyte activation.

    Funded by: NIGMS NIH HHS: GM 36353

    The Journal of biological chemistry 1993;268;10;7290-7

  • Immunocytochemical localization of fodrin and ankyrin in bovine chromaffin cells in vitro.

    Fujimoto T, Lee K, Miwa S and Ogawa K

    Department of Anatomy, Faculty of Medicine, Kyoto University, Japan.

    We raised antibodies to brain fodrin and erythrocyte ankyrin and examined the distribution of the antigens in cultured bovine chromaffin cells by immunocytochemical techniques. Immunofluorescence microscopy of whole cells showed intense labeling for both proteins, but fine localization could not be determined. In contrast, in cell specimens mechanically unroofed before fixation, the distribution of the two proteins revealed an apparent difference in the ventral plasma membrane: immunofluorescence for fodrin was dense and mostly even, whereas that for ankyrin appeared as scattered dots. Immunogold electron microscopy of the unroofed cells showed that labeling for fodrin was localized in a network of thin filaments, the diameter of which was 2-3 nm at the thinnest portion. Ankyrin labeling was mostly associated with filaments 5-10 nm in diameter. Notably, labeling for both fodrin and ankyrin was found over the coated membrane. The present results indicate that fodrin and ankyrin in the chromaffin cell do not constitute a submembranous network as spectrin and ankyrin do in the erythrocyte; whereas fodrin is closely associated with the plasma membrane, ankyrin is mostly linked to the cytoskeleton. The existence of both proteins in the coated region implies that they are functionally related to exocytosis and/or to ensuing membrane retrieval in the chromaffin cell.

    The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 1991;39;11;1485-93

  • Mapping the binding sites of human erythrocyte ankyrin for the anion exchanger and spectrin.

    Davis LH and Bennett V

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

    This report describes initial characterization of the binding sites of ankyrin for spectrin and the anion exchanger using defined subfragments isolated from purified ankyrin domains. The spectrin-binding domain of ankyrin is comprised of two subdomains: an acidic, proline-rich region (pI = 4) involving the amino-terminal 80 residues from 828 to 908 and a basic region (pI = 8.8) that extends from 898 to 1386. The amino-terminal 70 amino acids of the spectrin-binding domain are critical for association with spectrin, since a subfragment missing this region is only 5% as active as the intact domain in displacing binding of spectrin to inside-out membrane vesicles, while deletion of the 1d2f first 38 residues of the acidic domain results in a 10-fold reduction in activity. The anion exchanger-binding site is confined to an 89-kDa domain that was isolated and characterized as a globular molecule with approximately 30% alpha-helical configuration. A subfragment of the 89-kDa domain extending from residues 403 to 779 (or possibly 740) retains ability to associate with the anion exchanger. The 89-kDa domain is comprised of a series of tandem repeats of 33 amino acids that extend from residues 35 to 778 (Lux, S., John, K., and Bennett, V. (1990) Nature 344, 36-42). The activity of residues 403-779 demonstrates that the 33-amino acid repeats of the 89-kDa domain are responsible for association between ankyrin and the anion exchanger. The 33-amino acid repeating sequence of ankyrin represents an ancient motif also found in proteins of Drosophila, yeast, and Caenor habditis elegans. The finding that the 33-amino acid repeating sequence is involved in interaction with the anion exchanger implies that this motif may perform a role in molecular recognition in diverse proteins.

    The Journal of biological chemistry 1990;265;18;10589-96

  • Analysis of cDNA for human erythrocyte ankyrin indicates a repeated structure with homology to tissue-differentiation and cell-cycle control proteins.

    Lux SE, John KM and Bennett V

    Division of Hematology/Oncology, Children's Hospital, Boston, Massachusetts.

    Analysis of complementary DNA for human erythroid ankyrin indicates that the mature protein contains 1,880 amino acids comprising an N-terminal domain binding integral membrane proteins and tubulin, a central domain binding spectrin and vimentin, and an acidic C-terminal 'regulatory' domain containing an alternatively spliced sequence missing from ankyrin variant 2.2. The N-terminal domain is almost entirely composed of 22 tandem 33-amino-acid repeats. Similar repeats are found in yeast and invertebrate proteins involved in cell-cycle control and tissue differentiation.

    Nature 1990;344;6261;36-42

  • cDNA sequence for human erythrocyte ankyrin.

    Lambert S, Yu H, Prchal JT, Lawler J, Ruff P, Speicher D, Cheung MC, Kan YW and Palek J

    Department of Biomedical Research, Saint Elizabeth's Hospital of Boston, Tufts University Medical School, MA 02135.

    The cDNA for human erythrocyte ankyrin has been isolated from a series of overlapping clones obtained from a reticulocyte cDNA library. The composite cDNA sequence has a large open reading frame of 5636 base pairs (bp) with the complete coding sequence for a polypeptide of 1879 amino acids with a predicted molecular mass of 206 kDa. The derived amino acid sequence contained 194 residues that were identical to those obtained by direct amino acid sequencing of 11 ankyrin proteolytic peptides. The primary sequence contained 23 highly homologous repeat units of 33 amino acids within the 90-kDa band 3 binding domain. Two cDNA clones showed evidence of apparent mRNA processing, resulting in the deletions of 486 bp and 135 bp, respectively. The 486-bp deletion resulted in the removal of a 16-kDa highly acidic peptide, and the smaller deletion had the effect of altering the COOH terminus of the molecule. Radiolabeled ankyrin cDNAs recognized two erythroid message sizes by RNA blot analysis, one of which was predominantly associated with early erythroid cell types. An ankyrin message was also observed in RNA from the human cerebellum by the same method. The ankyrin gene is assigned to chromosome 8 using genomic DNA from a panel of sorted human chromosomes.

    Funded by: NHLBI NIH HHS: HL 27215, HL 37462-01

    Proceedings of the National Academy of Sciences of the United States of America 1990;87;5;1730-4

  • Ankyrin-independent membrane protein-binding sites for brain and erythrocyte spectrin.

    Steiner JP and Bennett V

    Howard Hughes Medical Institute, Durham, North Carolina.

    Brain spectrin reassociates in in vitro binding assays with protein(s) in highly extracted brain membranes quantitatively depleted of ankyrin and spectrin. These newly described membrane sites for spectrin are biologically significant and involve a protein since (a) binding occurs optimally at physiological pH (6.7-6.9) and salt concentrations (50 mM), (b) binding is abolished by digestion of membranes with alpha-chymotrypsin, (c) Scatchard analysis is consistent with a binding capacity of at least 50 pmol/mg total membrane protein, and highest affinity of 3 nM. The major ankyrin-independent binding activity of brain spectrin is localized to the beta subunit of spectrin. Brain membranes also contain high affinity binding sites for erythrocyte spectrin, but a 3-4 fold lower capacity than for brain spectrin. Some spectrin-binding sites associate preferentially with brain spectrin, some with erythrocyte spectrin, and some associate with both types of spectrin. Erythrocyte spectrin contains distinct binding domains for ankyrin and brain membrane protein sites, since the Mr = 72,000 spectrin-binding fragment of ankyrin does not compete for binding of spectrin to brain membranes. Spectrin binds to a small number of ankyrin-independent sites in erythrocyte membranes present in about 10,000-15,000 copies/cell or 10% of the number of sites for ankyrin. Brain spectrin binds to these sites better than erythrocyte spectrin suggesting that erythrocytes have residual binding sites for nonerythroid spectrin. Ankyrin-independent-binding proteins that selectively bind to certain isoforms of spectrin provide a potentially important flexibility in cellular localization and time of synthesis of proteins involved in spectrin-membrane interactions. This flexibility has implications for assembly of the membrane skeleton and targeting of spectrin isoforms to specialized regions of cells.

    Funded by: NIADDK NIH HHS: AM 19808; NIGMS NIH HHS: GM 33996

    The Journal of biological chemistry 1988;263;28;14417-25

  • Associations of human erythrocyte band 4.2. Binding to ankyrin and to the cytoplasmic domain of band 3.

    Korsgren C and Cohen CM

    Department of Biomedical Research, St. Elizabeth's Hospital, Boston, Massachusetts 02135.

    We have examined the associations of purified red cell band 4.2 with red cell membrane and membrane skeletal proteins using in vitro binding assays. Band 4.2 bound to the purified cytoplasmic domain of band 3 with a Kd between 2 and 8 X 10(-7) M. Binding was saturable and slow, requiring 2-4 h to reach equilibrium. This finding confirms previous work suggesting that the principal membrane-binding site for band 4.2 lies within the 43-kDa cytoplasmic domain of band 3 (Korsgren, C., and Cohen, C. M. (1986) J. Biol. Chem. 261, 5536-5543). Band 4.2 also bound to purified ankyrin in solution with a Kd between 1 and 3.5 X 10(-7) M. As with the cytoplasmic domain of band 3, binding was saturable and required 4-5 h to reach equilibrium. Reconstitution with ankyrin of inside-out vesicles stripped of all peripheral proteins had no effect upon band 4.2 binding to membranes; similarly, reconstitution with band 4.2 had no effect upon ankyrin binding. This shows that ankyrin and band 4.2 bind to distinct loci within the 43-kDa band 3 cytoplasmic domain. Coincubation of ankyrin and band 4.2 in solution partially blocked the binding of both proteins to the membrane. Similarly, coincubation of bands 4.1 and 4.2 in solution partially blocked binding of both to membranes. In all cases, the data suggest the possibility that domains on each of these proteins responsible for low affinity membrane binding are principally affected. The data also provide evidence for an association of band 4.2 with band 4.1. Our results show that band 4.2 can form multiple associations with red cell membrane proteins and may therefore play an as yet unrecognized structural role on the membrane.

    Funded by: NHLBI NIH HHS: HL 36090, HL 37462

    The Journal of biological chemistry 1988;263;21;10212-8

  • Phosphorylation of ankyrin down-regulates its cooperative interaction with spectrin and protein 3.

    Cianci CD, Giorgi M and Morrow JS

    Department of Pathology, Yale University School of Medicine, New Haven, CT 06510.

    Ankyrin mediates the primary attachment between beta spectrin and protein 3. Ankyrin and spectrin interact in a positively cooperative fashion such that ankyrin binding increases the extent of spectrin tetramer and oligomer formation (Giorgi and Morrow: submitted, 1988). This cooperative interaction is enhanced by the cytoplasmic domain of protein 3, which is prepared as a 45-41-kDa fragment generated by chymotryptic digestion of erythrocyte membranes. Using sensitive isotope-ratio methods and nondenaturing PAGE, we now demonstrate directly (1) the enhanced affinity of ankyrin for spectrin oligomers compared to spectrin dimers; (2) a selective stimulation of the affinity of ankyrin for spectrin oligomer by the 43-kDa cytoplasmic domain of protein 3; and (3) a selective reduction in the affinity of ankyrin for spectrin tetramer and oligomer after its phosphorylation by the erythrocyte cAMP-independent membrane kinase. The phosphorylation of ankyrin does not affect its binding to spectrin dimer. Ankyrin also enhances the rate of interconversion between dimer-tetramer-oligomer by 2-3-fold at 30 degrees C, and in the presence of the 43-kDa fragment, ankyrin stimulates the rate of oligomer interconversions by nearly 40-fold at this temperature. These results demonstrate a long-range cooperative interaction between an integral membrane protein and the peripheral cytoskeleton and indicate that this linkage may be regulated by covalent protein phosphorylation. Such interactions may be of general importance in nonerythroid cells.

    Funded by: NHLBI NIH HHS: HL28560

    Journal of cellular biochemistry 1988;37;3;301-15

  • Reassociation of ankyrin with band 3 in erythrocyte membranes and in lipid vesicles.

    Hargreaves WR, Giedd KN, Verkleij A and Branton D

    The binding of human erythrocyte ankyrin (band 2.1) to the erythrocyte membrane has been characterized by reassociating purified ankyrin with ankyrin-depleted inside-out vesicles. Ankyrin reassociates at high affinity with a limited number of protease-sensitive sites located only on the cytoplasmic side of the erythrocyte membrane. Depleting the vesicles of band 4.2 does not affect their binding capacity. A 45,000-dalton polypeptide derived from the cytoplasmic portion of band 3 competitively inhibits the binding of ankyrin to inside-out vesicles. Although the bulk of band 3 molecules appear to have the potential for binding ankyrin, nly a fraction of the band 3 molecules in native membranes or in reconstituted liposomes actually provides accessible high affinity ankyrin binding sites.

    Funded by: NHLBI NIH HHS: HL-17411

    The Journal of biological chemistry 1980;255;24;11965-72

  • Immunoreactive forms of human erythrocyte ankyrin are present in diverse cells and tissues.

    Bennett V

    Ankyrin is a polypeptide of molecular weight (MW) 200,000 which is tightly bound to the cytoplasmic surface of the human erythrocyte membrane and has been identified as the high-affinity membrane attachment protein for spectrin. This protein has also been shown to be associated with band 3 (ref. 4), the major transmembrane protein which links a cytoplasmic structural protein to an integral membrane protein. A water-soluble, 72,000-MW, proteolytic fragment of ankyrin has been purified which retains the ability to bind to spectrin, and competitively inhibits reassociation of spectrin with membranes. Monospecific antibodies directed against this fragment have been prepared and demonstrated to cross-react only with ankyrin among the erythrocyte membrane proteins. The present study reports the use of these antibodies to develop a radioimmunoassay capable of detecting femtomolar quantities of ankyrin, and demonstrates the presence of small but significant amounts of immunoreactivity in a variety of types of cells and tissues.

    Nature 1979;281;5732;597-9

  • The membrane attachment protein for spectrin is associated with band 3 in human erythrocyte membranes.

    Bennett V and Stenbuck PJ

    Ankyrin, the membrane attachment protein for human erythrocyte spectrin, is tightly linked in a 1:1 molar ratio with band 3 in detergent extracts of spectrin-depleted membranes. Ankyrin-linked band 3, which represents 10--15% of the total band 3, spans the membrane, and is nearly identical to the major band 3 by peptide analysis. Spectrin binds to solubilised ankyrin-linked band 3, but not to free band 3. A portion of band 3 remains firmly associated with detergent-extracted cytoskeletal proteins. It is concluded that a fraction of band 3 is attached to the erythrocyte cytoskeleton through association with ankyrin, which in turn is bound to spectrin.

    Nature 1979;280;5722;468-73

OMIM - other

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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