Cell Science at a Glance 1565 Ankyrins distribution, and the prototypic ankyrin Therefore, ankyrins appear to have first characterized in erythrocytes; also evolved early in metazoan evolution and Peter J. Mohler*,‡, Anthony O. expressed in a subset of neurons and might perform functions unique to Gramolini* and Vann Bennett striated muscle) encoded by Ank1 on multicellular animals. Howard Hughes Medical Institute, Departments of human chromosome 8p11; ankyrins-B Cell Biology and Biochemistry, Duke University (B for broadly expressed; first Canonical ankyrins are 190-220 kDa Medical Center, Durham, NC 27513, USA proteins expressed in most tissues and *These authors contributed equally to this work characterized in brain, but now ‡Author for correspondence recognized in most cell types) encoded cell types and comprise a membrane- (e-mail: [email protected]) by Ank2 on human chromosome 4q25- binding domain (MBD) of 24 ANK 27; and ankyrins-G (G for giant size and repeats, a spectrin-binding domain, a Journal of Cell Science 115, 1565-1566 (2002) © The Company of Biologists Ltd general expression, first characterized as death domain and a C-terminal domain. a 480 kDa polypeptide in the nervous Whereas death domains in other proteins Ankyrins are a ubiquitously expressed system; expressed in most cell types) may function in activation of NF-κB, family of intracellular adaptor proteins encoded by Ank3, on human caspase proteases and cell death, this involved in targeting diverse proteins to chromosome 10q21. Although ankyrin domain has no known role within specialized membrane domains in both genes are not present in the completed ankyrins. Ankyrin expression is the plasma membrane and the genomes of yeast or plants, simple regulated by both tissue- and endoplasmic reticulum (Bennett and metazoans including Drosophila developmental-stage-specific cues that Baines, 2001). Vertebrate ankyrin melanogaster (Dank1, Dank2) and also give rise to numerous ankyrin polypeptides fall into three classes, each Caenorhabditis elegans (unc44) express polypeptides due to alternative splicing. containing multiple alternatively spliced ankyrin(s) that exhibit a high level of 440 kDa ankyrin-B and 480 kDa variants: ankyrins-R (R for restricted similarity to their vertebrate orthologues. ankyrin-G polypeptides result from an Ankyrin complexes Domain organization Erythrocyte membrane Anion-exchanger dimers Canonical ankyrins 190-220 kDa ankyrins (most cells and tissues) Death domain β α-Spectrin -Spectrin Spectrin-binding domain C-terminal domain Small ankyrins 26-120 kDa ankyrins Unique N-terminus 26-120 kDa ankyrin-R Actin (sarcoplasmic reticulum) 210 kDa Ankyrin-R 100/120 kDa ankyrin-G (lysosomes) 119 kDa ankyrin-G (Golgi) Axon initial segment L1 CAMs Large ankyrins 270-480 kDa ankyrins Na+ channel 440 kDa ankyrin-B β α β β 1/ 2 (unmyelinated axons) Tail domain Y Y 480 kDa/270 ankyrin-G (axon initial segments, nodes of Ranvier) Ser/Thr domain Actin O-GlucNAc 480/270 kDa Ankyrin-G αIIβIV spectrin GFP-AnkB transfected Ankyrin-associated proteins Wild-type cardiomyocytes AnkB -/- cardiomyocytes AnkB -/- cardiomyocytes Cell adhesion molecules CD44, L1CAMs: L1 LAD-1, NrCAM, NgCAM, neuroglian, neurofascin Ion channels Anion exchanger (1-3) β1,2) Na+ K+-ATPase H+ +- Na+ 2+ exchanger Calcium-release channels Ryanodine receptor Ins(1,4,5)P3 receptor Cytoplasmic Tubulin Clathrin β spectrin I-IV Journal of Cell Science 2002 (115, pp. 1565-1566) (See poster insert) 1566 Journal of Cell Science 115 (8) insertion of a 220 kDa random coil exposed loops arranged perpendicular to degeneration of a set of Purkinje neurons between the spectrin-binding domain the α-helices. The specificity for ANK- and cerebellar dysfunction. In addition, and the death domain, which results in a repeat–protein interactions is likely to be ankyrin-B has been mapped to human predicted extended length of up to 0.5- conferred by non-conserved residues chromosome 4q25-27, the linkage site 0.6 µm. These giant isoforms have that flank each ankyrin repeat, located at for human type 4 long QT syndrome specialized functions in unmyelinated the tips of exposed loops. Ankyrin (Schott et al., 1995). This disorder axons (ankyrin-B) and in the targeting of MBDs with 24 ANK repeats are results in bradycardia and can cause voltage-dependent Na+ channels to axon multivalent and can accommodate cardiac arrhythmias leading to loss of initial segments and nodes of Ranvier multiple protein interactions; thus, they consciousness or sudden death. As (480 kDa and 270 kDa ankyrin-G also may assemble multiprotein complexes at ankyrin-B is highly expressed in cardiac contain a 40 kDa serine/threonine-rich specific cellular sites. tissue, further examination of both domain glycosylated with GlucNAc ankyrin-B+/– and ankyrin-B–/– mice may monosaccharide residues). Finally, small Physiological roles of ankyrin-G and reveal a similar cardiac phenotype. More ankyrin isoforms lacking large portions ankyrin-B in targeting proteins to generally, ankyrin mutations could result of canonical ankyrins are localized to specialized membrane domains have in improper localization and, therefore, specialized membrane sites, for been demonstrated by gene-knockout activity, of ion channels identified example, 119 kDa ankyrin-G (Golgi), studies in mice. Mice with cerebellar- with ankyrin-binding domains and thus 100/120 kDa ankyrin-G (lysosomes) and specific loss of ankyrin-G display result in a variety of functional 26 kDa ankyrin-R (sarcoplasmic coordinate loss of voltage-gated Na+ ‘channelopathies’. reticulum). channels, β-IV spectrin and neurofascin at the plasma membrane of axon initial Current views of ankyrin function are segments, decreased ability of Purkinje References based on co-localization and neurons to fire action potentials, and Bennett, V. and Baines, A. J. (2001). Spectrin and biochemical interactions of ankyrin with progressive ataxia (Jenkins and Bennett, ankyrin-based pathways: metazoan inventions for integrating cells into tissues. Physiol. Rev. 81, other proteins. Ankyrin associates with a 2001; Zhou et al., 1998). Ankyrin-B-null 1353-1392. variety of membrane proteins including mice die at birth with multisystem Jenkins, S. M. and Bennett, V. (2001). Ankyrin- ion channels (Na+/K+ ATPase, H+/K+ disorders including degeneration of long G coordinates assembly of the spectrin-based ATPase, anion exchangers AE 1-3, axon tracts, myopathy and degeneration membrane skeleton, voltage-gated sodium + channels, and L1 CAMs at Purkinje neuron initial voltage-sensitive Na channels, of the thymus (Tuvia et al., 1999). segments. J. Cell Biol. 155, 739-746. + 2+ –/– Na /Ca exchanger), calcium-release Ankyrin-B cardiomyocytes display Mohler, P. J., Gramolini, A. O. and Bennett, V. channels [ryanodine receptor, inositol downregulation and mis-sorting of (2002). The ankyrin-B C-terminal domain (1,4,5)-trisphosphate receptor], cell calcium-release channels [ryanodine and determines activity of ankyrin-B/G chimeras in adhesion molecules [CD44, L1CAMs inositol (1,4,5)-trisphosphate receptors] rescue of abnormal inositol 1,4,5-trisphosphate- and ryanodine receptor distribution in ankyrin-B (L1, NgCAM, neurofascin, LAD-1, within the endoplasmic reticulum in (–/–) neonatal cardiomyocytes. J. Biol. Chem. 7, 7. NrCAM, neuroglian)], as well as cardiomyocytes that can be rescued by Schott, J. J., Charpentier, F., Peltier, S., Foley, cytoplasmic proteins, including clathrin transfection with cDNA encoding P., Drouin, E., Bouhour, J. B., Donnelly, P., and tubulin (Bennett and Baines, 2001). ankyrin-B. Both ankyrin-G and ankyrin- Vergnaud, G., Bachner, L., Moisan, J. P. et al. (1995). Mapping of a gene for long QT syndrome Many of these interactions are mediated R are expressed in cardiomyoctes, but to chromosome 4q25-27. Am. J. Hum. Genet. 57, by ANK repeats within the MBD, cannot compensate for loss of ankyrin- 1114-1122. although the Na+/K+ ATPase and H+/K+ B. Rescue studies with ankyrin-B/G Sedgwick, S. G. and Smerdon, S. J. (1999). The ATPase associate at least in part with the chimeras have identified the C-terminal ankyrin repeat: a diversity of interactions on a spectrin-binding domain. Finally, domain of ankyrin-B as the defining common structural framework. Trends Biochem. Sci. 24, 311-316. ankyrin phosphorylation is important for domain in specifying ankyrin-B activity Tuvia, S., Buhusi, M., Davis, L., Reedy, M. and regulating the affinity of ankyrin for (Mohler et al., 2002). A working Bennett, V. (1999). Ankyrin-B is required for specific proteins, including spectrin. hypothesis to explain the cellular basis intracellular sorting of structurally diverse Ca2+ for these phenotypes is that ankyrins homeostasis proteins. J. Cell Biol. 147, 995-1008. Zhou, D., Lambert, S., Malen, P. L., Carpenter, ANK repeats are 33-residue motifs play roles as chaperones or guides that S., Boland, L. M. and Bennett, V. (1998). involved in protein recognition and are direct vesicle transport of a variety of ion AnkyrinG is required for clustering of voltage- found in more than 325 unrelated human channels to sites in the plasma gated Na channels at axon initial segments and for proteins [>5800 repeats in more than membrane as well as the endoplasmic normal action potential firing. J. Cell Biol. 143, 1400 predicted proteins in the non- reticulum. 1295-1304. redundant sequence database (http://smart.embl-heidelberg.de)], Ankyrins have been implicated in human including tankyrase, p53-binding protein disease (Bennett and Baines, 2001). (53BP2), transcriptional regulators Hereditary spherocytosis results from Cell Science at a Glance on the Web GABPβ and NF-κB inhibitory protein decreased expression and/or mutated Electronic copies of the poster insert are IkBα, and the TRP family of ion forms of ankyrin-R. A similar model available in the online version of this article channels (Sedgwick and Smerdon, (nb/nb) is observed in mice due to a near at jcs.biologists.org. JPEG and PDF files 1999). ANK repeats fold into stacks of loss of ankyrin-R (210 kDa) in multiple (see supplemental material) can be downloaded for printing or use as slides. antiparallel α-helices interconnected by cell types, which causes anemia,.