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2.  A Single Divergent Exon Inhibits Ankyrin-B Association with the Plasma Membrane 
The Journal of Biological Chemistry  2013;288(21):14769-14779.
Background: Ankyrin-B is intracellular, whereas ankyrin-G associates with plasma membranes.
Results: An ankyrin-B-specific linker between the ANK repeat domain and the ZU52-UPA module inhibits ankyrin-B binding to membrane protein partners and localization to plasma membranes of epithelial cells and neurons.
Conclusion: The linker region contributes to functional differences between ankyrins.
Significance: This work explains the distinct behavior of ankyrins regarding plasma membrane localization.
Vertebrate ankyrin-B and ankyrin-G exhibit divergent subcellular localization and function despite their high sequence and structural similarity and common origin from a single ancestral gene at the onset of chordate evolution. Previous studies of ankyrin family diversity have focused on the C-terminal regulatory domain. Here, we identify an ankyrin-B-specific linker peptide connecting the ankyrin repeat domain to the ZU52-UPA module that inhibits binding of ankyrin-B to membrane protein partners E-cadherin and neurofascin 186 and prevents association of ankyrin-B with epithelial lateral membranes as well as neuronal plasma membranes. The residues of the ankyrin-B linker required for autoinhibition are encoded by a small exon that is highly divergent between ankyrin family members but conserved in the ankyrin-B lineage. We show that the ankyrin-B linker suppresses activity of the ANK repeat domain through an intramolecular interaction, likely with a groove on the surface of the ANK repeat solenoid, thereby regulating the affinities between ankyrin-B and its binding partners. These results provide a simple evolutionary explanation for how ankyrin-B and ankyrin-G have acquired striking differences in their plasma membrane association while maintaining overall high levels of sequence similarity.
PMCID: PMC3663501  PMID: 23569209
Epithelial Cell; Evolution; Neurons; Plasma Membrane; Transfection; Ankyrin Diversity; Autoinhibition; Intramolecular Interaction
3.  E-cadherin Polarity Is Determined by a Multifunction Motif Mediating Lateral Membrane Retention through Ankyrin-G and Apical-lateral Transcytosis through Clathrin 
The Journal of Biological Chemistry  2013;288(20):14018-14031.
Background: E-cadherin targets to the epithelial lateral membrane.
Results: Ankyrin-G and clathrin cooperate to control E-cadherin localization.
Conclusion: A multifunction motif on E-cadherin interacts with multiple pathways.
Significance: This finding provides novel insights into how basolateral targeting motifs function.
We report a highly conserved motif in the E-cadherin juxtamembrane domain that determines apical-lateral polarity by conferring both restricted mobility at the lateral membrane and transcytosis of apically mis-sorted protein to the lateral membrane. Mutations causing either increased lateral membrane mobility or loss of apical-lateral transcytosis result in partial mis-sorting of E-cadherin in Madin-Darby canine kidney cells. However, loss of both activities results in complete loss of polarity. We present evidence that residues required for restricted mobility mediate retention at the lateral membrane through interaction with ankyrin-G, whereas dileucine residues conferring apical-lateral transcytosis act through a clathrin-dependent process and function in an editing pathway. Ankyrin-G interaction with E-cadherin is abolished by the same mutations resulting in increased E-cadherin mobility. Clathrin heavy chain knockdown and dileucine mutation of E-cadherin both cause the same partial loss of polarity of E-cadherin. Moreover, clathrin knockdown causes no further change in polarity of E-cadherin with dileucine mutation but does completely randomize E-cadherin mutants lacking ankyrin-binding. Dileucine mutation, but not loss of ankyrin binding, prevented transcytosis of apically mis-sorted E-cadherin to the lateral membrane. Finally, neurofascin, which binds ankyrin but lacks dileucine residues, exhibited partial apical-lateral polarity that was abolished by mutation of its ankyrin-binding site but was not affected by clathrin knockdown. The polarity motif thus integrates complementary activities of lateral membrane retention through ankyrin-G and apical-lateral transcytosis of mis-localized protein through clathrin. Together, the combination of retention and editing function to ensure a high fidelity steady state localization of E-cadherin at the lateral membrane.
PMCID: PMC3656260  PMID: 23530049
Cell Adhesion; Cell Biology; Cell Junctions; Cell Polarity; Molecular Cell Biology
4.  Cysteine 70 of Ankyrin-G Is S-Palmitoylated and Is Required for Function of Ankyrin-G in Membrane Domain Assembly 
The Journal of Biological Chemistry  2012;287(52):43995-44005.
Background: Ankyrin-G targets to specialized membrane domains in multiple cell types.
Results: Ankyrin-G is S-palmitoylated at a conserved cysteine 70 located in the first loop of the ankyrin repeat solenoid.
Conclusion: Cysteine 70 is required for function of ankyrin-G in membrane domain assembly.
Significance: This finding provides new insights into how the ankyrin proteins exert their functions in formation and maintenance of membrane domains.
Ankyrin-G (AnkG) coordinates protein composition of diverse membrane domains, including epithelial lateral membranes and neuronal axon initial segments. However, how AnkG itself localizes to these membrane domains is not understood. We report that AnkG remains on the plasma membrane in Madin-Darby canine kidney (MDCK) cells grown in low calcium, although these cells lack apical-basal polarity and exhibit loss of plasma membrane association of AnkG partners, E-cadherin and β2-spectrin. We subsequently demonstrate using mutagenesis and mass spectrometry that AnkG is S-palmitoylated exclusively at Cys-70, which is located in a loop of the first ankyrin repeat and is conserved in the vertebrate ankyrin family. Moreover, C70A mutation abolishes membrane association of 190-kDa AnkG in MDCK cells grown in low calcium. C70A 190-kDa AnkG fails to restore biogenesis of epithelial lateral membranes in MDCK cells depleted of endogenous AnkG. In addition, C70A 270-kDa AnkG fails to cluster at the axon initial segment of AnkG-depleted cultured hippocampal neurons and fails to recruit neurofascin as well as voltage-gated sodium channels. These effects of C70A mutation combined with evidence for its S-palmitoylation are consistent with a requirement of palmitoylation for targeting and function of AnkG in membrane domain biogenesis at epithelial lateral membranes and neuronal axon initial segments.
PMCID: PMC3527982  PMID: 23129772
Axon; Cell Biology; Confocal Microscopy; Membrane Biogenesis; Protein Palmitoylation
5.  Ank3-dependent SVZ niche assembly is required for the continued production of new neurons 
Neuron  2011;71(1):61-75.
The rodent subventricular/subependymal zone (SVZ/SEZ) houses neural stem cells (NSCs) that generate olfactory bulb interneurons. It is unclear how the SVZ environment sustains neuronal production into adulthood. We discovered that the adapter molecule Ankyrin-3 (Ank3) is specifically upregulated in radial glia destined to become SVZ ependymal niche cells, but not in NSCs, and is required for SVZ assembly through progenitor lateral adhesion. Furthermore, we found that Ank3 expression is controlled by Foxj1, a transcriptional regulator of multicilia formation, and genetic deletion of this pathway led to complete loss of SVZ niche structure. In its absence, radial glia continued to transition into postnatal NSCs. However, inducible ependymal deletion of Foxj1-Ank3 after SVZ niche assembly resulted in dramatic depletion of neurogenesis. Targeting a novel pathway regulating ependymal organization/assembly and showing its requirement for new neuron production, our results have important implications for environmental control of adult neurogenesis and harvesting NSCs for replacement therapy.
PMCID: PMC3134799  PMID: 21745638
6.  Mutation of Conserved Histidines Alters Tertiary Structure and Nanomechanics of Consensus Ankyrin Repeats* 
The Journal of Biological Chemistry  2012;287(23):19115-19121.
Background: The His → Arg mutation in a native ankyrin-R protein is responsible for converting RBCs to spherocytes, causing hereditary spherocytosis (HS).
Results: The mutant unfolds and refolds at lower forces compared with the wild type.
Conclusion: The His → Arg mutation weakens the mechanical stability of ankyrin repeats.
Significance: The His → Arg mutation in ankyrin-R may cause HS by decreasing the mechanical stability and affecting its structure recovery ability.
The conserved TPLH tetrapeptide motif of ankyrin repeats (ARs) plays an important role in stabilizing AR proteins, and histidine (TPLH)-to-arginine (TPLR) mutations in this motif have been associated with a hereditary human anemia, spherocytosis. Here, we used a combination of atomic force microscopy-based single-molecule force spectroscopy and molecular dynamics simulations to examine the mechanical effects of His → Arg substitutions in TPLH motifs in a model AR protein, NI6C. Our molecular dynamics results show that the mutant protein is less mechanically stable than the WT protein. Our atomic force microscopy results indicate that the mechanical energy input necessary to fully unfold the mutant protein is only half of that necessary to unfold the WT protein (53 versus 106 kcal/mol). In addition, the ability of the mutant to generate refolding forces is also reduced. Moreover, the mutant protein subjected to cyclic stretch-relax measurements displays mechanical fatigue, which is absent in the WT protein. Taken together, these results indicate that the His → Arg substitutions in TPLH motifs compromise mechanical properties of ARs and suggest that the origin of hereditary spherocytosis may be related to mechanical failure of ARs.
PMCID: PMC3365944  PMID: 22514283
Atomic Force Microscopy; Molecular Dynamics; Protein Folding; Protein Stability; Single-molecule Biophysics
7.  Membrane Domains Based on Ankyrin and Spectrin Associated with Cell–Cell Interactions 
Nodes of Ranvier and axon initial segments of myelinated nerves, sites of cell–cell contact in early embryos and epithelial cells, and neuromuscular junctions of skeletal muscle all perform physiological functions that depend on clustering of functionally related but structurally diverse ion transporters and cell adhesion molecules within microdomains of the plasma membrane. These specialized cell surface domains appeared at different times in metazoan evolution, involve a variety of cell types, and are populated by distinct membrane-spanning proteins. Nevertheless, recent work has shown that these domains all share on their cytoplasmic surfaces a membrane skeleton comprised of members of the ankyrin and spectrin families. This review will summarize basic features of ankyrins and spectrins, and will discuss emerging evidence that these proteins are key players in a conserved mechanism responsible for assembly and maintenance of physiologically important domains on the surfaces of diverse cells.
Ankyrins and spectrins form a “skeleton” beneath the plasma membrane, clustering ion channels and other proteins together to generate distinct microdomains.
PMCID: PMC2882121  PMID: 20457566
8.  Ankyrin-B Interactions with Spectrin and Dynactin-4 Are Required for Dystrophin-based Protection of Skeletal Muscle from Exercise Injury* 
The Journal of Biological Chemistry  2010;286(9):7370-7378.
Costameres are cellular sites of mechanotransduction in heart and skeletal muscle where dystrophin and its membrane-spanning partner dystroglycan distribute intracellular contractile forces into the surrounding extracellular matrix. Resolution of a functional costamere interactome is still limited but likely to be critical for understanding forms of muscular dystrophy and cardiomyopathy. Dystrophin binds a set of membrane-associated proteins (the dystrophin-glycoprotein complex) as well as γ-actin and microtubules and also is required to align sarcolemmal microtubules with costameres. Ankyrin-B binds to dystrophin, dynactin-4, and microtubules and is required for sarcolemmal association of these proteins as well as dystroglycan. We report here that ankyrin-B interactions with β2 spectrin and dynactin-4 are required for localization of dystrophin, dystroglycan, and microtubules at costameres as well as protection of muscle from exercise-induced injury. Knockdown of dynactin-4 in adult mouse skeletal muscle phenocopied depletion of ankyrin-B and resulted in loss of sarcolemmal dystrophin, dystroglycan, and microtubules. Moreover, mutations of ankyrin-B and of dynactin-4 that selectively impaired binary interactions between these proteins resulted in loss of their costamere-localizing activity and increased muscle fiber fragility as a result of loss of costamere-associated dystrophin and dystroglycan. In addition, costamere-association of dynactin-4 did not require dystrophin but did depend on β2 spectrin and ankyrin-B, whereas costamere association of ankyrin-B required β2 spectrin. Together, these results are consistent with a functional hierarchy beginning with β2 spectrin recruitment of ankyrin-B to costameres. Ankyrin-B then interacts with dynactin-4 and dystrophin, whereas dynactin-4 collaborates with dystrophin in coordinating costamere-aligned microtubules.
PMCID: PMC3044993  PMID: 21186323
Cytoskeleton; Microtubules; Muscular Dystrophy; Protein Targeting; Skeletal Muscle; Ankyrin; Dynactin; Dystrophin; Dystrophin-Glycoprotein Complex; Spectrin
9.  Cyclic Nucleotide-Gated Channels Require Ankyrin-G for Transport to the Sensory Cilium of Rod Photoreceptors 
Science (New York, N.Y.)  2009;323(5921):1614-1617.
Cyclic nucleotide-gated channels localize exclusively to the plasma membrane of photosensitive outer segments of rod photoreceptors where they generate the electrical response to light. Here we found that targeting of cyclic nucleotide-gated channels to the rod outer segment required their interaction with ankyrin-G. Ankyrin-G localized exclusively to rod outer segments, coimmunoprecipitated with the cyclic nucleotide-gated channel, and bound to the C-terminal domain of the β1-subunit. Ankyrin-G depletion in neonatal mouse retinas markedly reduced cyclic nucleotide-gated channel expression. Transgenic expression of cyclic nucleotide-gated channel β-subunit mutants in Xenopus rods showed that ankyrin-G binding was necessary and sufficient for targeting of the β1-subunit to outer segments. Thus ankyrin-G is required for transport of cyclic nucleotide-gated channels to the plasma membrane of rod outer segments.
PMCID: PMC2792576  PMID: 19299621
10.  Being there: cellular targeting of voltage-gated sodium channels in the heart 
The Journal of Cell Biology  2008;180(1):13-15.
Voltage-gated sodium (Nav) channels in cardiomyocytes are localized in specialized membrane domains that optimize their functions in propagating action potentials across cell junctions and in stimulating voltage-gated calcium channels located in T tubules. Mutation of the ankyrin-binding site of Nav1.5, the principal Nav channel in the heart, was previously known to cause cardiac arrhythmia and the retention of Nav1.5 in an intracellular compartment in cardiomyocytes. Conclusive evidence is now provided that direct interaction between Nav1.5 and ankyrin-G is necessary for the expression of Nav1.5 at the cardiomyocyte cell surface.
PMCID: PMC2213601  PMID: 18180365
11.  Adducin Promotes Micrometer-Scale Organization of β2-Spectrin in Lateral Membranes of Bronchial Epithelial Cells 
Molecular Biology of the Cell  2008;19(2):536-545.
Adducin promotes assembly of spectrin–actin complexes, and is a target for regulation by calmodulin, protein kinase C, and rho kinase. We demonstrate here that adducin is required to stabilize preformed lateral membranes of human bronchial epithelial (HBE) cells through interaction with β2-spectrin. We use a Tet-on regulated inducible small interfering RNA (siRNA) system to deplete α-adducin from confluent HBE cells. Depletion of α-adducin resulted in increased detergent solubility of spectrin after normal membrane biogenesis during mitosis. Conversely, depletion of β2-spectrin resulted in loss of adducin from the lateral membrane. siRNA–resistant α-adducin prevented loss of lateral membrane, but only if α-adducin retained the MARCKS domain that mediates spectrin–actin interactions. Phospho-mimetic versions of adducin with S/D substitutions at protein kinase C phosphorylation sites in the MARCKS domain were not active in rescue. We find that adducin modulates long-range organization of the lateral membrane based on several criteria. First, the lateral membrane of adducin-depleted cells exhibited reduced height, increased curvature, and expansion into the basal surface. Moreover, E-cadherin-GFP, which normally is restricted in lateral mobility, rapidly diffuses over distances up to 10 μm. We conclude that adducin acting through spectrin provides a novel mechanism to regulate global properties of the lateral membrane of bronchial epithelial cells.
PMCID: PMC2230604  PMID: 18003973
12.  Ankyrin-B Syndrome: Enhanced Cardiac Function Balanced by Risk of Cardiac Death and Premature Senescence 
PLoS ONE  2007;2(10):e1051.
Here we report the unexpected finding that specific human ANK2 variants represent a new example of balanced human variants. The prevalence of certain ANK2 (encodes ankyrin-B) variants range from 2 percent of European individuals to 8 percent in individuals from West Africa. Ankyrin-B variants associated with severe human arrhythmia phenotypes (eg E1425G, V1516D, R1788W) were rare in the general population. Variants associated with less severe clinical and in vitro phenotypes were unexpectedly common. Studies with the ankyrin-B+/− mouse reveal both benefits of enhanced cardiac contractility, as well as costs in earlier senescence and reduced lifespan. Together these findings suggest a constellation of traits that we term “ankyrin-B syndrome”, which may contribute to both aging-related disorders and enhanced cardiac function.
PMCID: PMC2013943  PMID: 17940615
13.  L1-dependent neuritogenesis involves ankyrinB that mediates L1-CAM coupling with retrograde actin flow 
The Journal of Cell Biology  2003;163(5):1077-1088.
The cell adhesion molecule L1 (L1-CAM) plays critical roles in neurite growth. Its cytoplasmic domain (L1CD) binds to ankyrins that associate with the spectrin–actin network. This paper demonstrates that L1-CAM interactions with ankyrinB (but not with ankyrinG) are involved in the initial formation of neurites. In the membranous protrusions surrounding the soma before neuritogenesis, filamentous actin (F-actin) and ankyrinB continuously move toward the soma (retrograde flow). Bead-tracking experiments show that ankyrinB mediates L1-CAM coupling with retrograde F-actin flow in these perisomatic structures. Ligation of the L1-CAM ectodomain by an immobile substrate induces L1CD–ankyrinB binding and the formation of stationary ankyrinB clusters. Neurite initiation preferentially occurs at the site of these clusters. In contrast, ankyrinB is involved neither in L1-CAM coupling with F-actin flow in growth cones nor in L1-based neurite elongation. Our results indicate that ankyrinB promotes neurite initiation by acting as a component of the clutch module that transmits traction force generated by F-actin flow to the extracellular substrate via L1-CAM.
PMCID: PMC2173603  PMID: 14657231
ankyrin; L1-CAM; adhesion; neurite; clutch
14.  α-Adducin dissociates from F-actin and spectrin during platelet activation 
The Journal of Cell Biology  2003;161(3):557-570.
Aspectrin-based skeleton uniformly underlies and supports the plasma membrane of the resting platelet, but remodels and centralizes in the activated platelet. α-Adducin, a phosphoprotein that forms a ternary complex with F-actin and spectrin, is dephosphorylated and mostly bound to spectrin in the membrane skeleton of the resting platelet at sites where actin filaments attach to the ends of spectrin molecules. Platelets activated through protease-activated receptor 1, FcγRIIA, or by treatment with PMA phosphorylate adducin at Ser726. Phosphoadducin releases from the membrane skeleton concomitant with its dissociation from spectrin and actin. Inhibition of PKC blunts adducin phosphorylation and release from spectrin and actin, preventing the centralization of spectrin that normally follows cell activation. We conclude that adducin targets actin filament ends to spectrin to complete the assembly of the resting membrane skeleton. Dissociation of phosphoadducin releases spectrin from actin, facilitating centralization of spectrin, and leads to the exposure of barbed actin filament ends that may then participate in converting the resting platelet's disc shape into its active form.
PMCID: PMC2172952  PMID: 12743105
spectrin; adducin; actin; platelet; cell motility
15.  Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments 
The Journal of Cell Biology  2001;155(5):739-746.
The axon initial segment is an excitable membrane highly enriched in voltage-gated sodium channels that integrates neuronal inputs and initiates action potentials. This study identifies Nav1.6 as the voltage-gated sodium channel isoform at mature Purkinje neuron initial segments and reports an essential role for ankyrin-G in coordinating the physiological assembly of Nav1.6, βIV spectrin, and the L1 cell adhesion molecules (L1 CAMs) neurofascin and NrCAM at initial segments of cerebellar Purkinje neurons. Ankyrin-G and βIV spectrin appear at axon initial segments by postnatal day 2, whereas L1 CAMs and Nav1.6 are not fully assembled at continuous high density along axon initial segments until postnatal day 9. L1 CAMs and Nav1.6 therefore do not initiate protein assembly at initial segments. βIV spectrin, Nav1.6, and L1 CAMs are not clustered in adult Purkinje neuron initial segments of mice lacking cerebellar ankyrin-G. These results support the conclusion that ankyrin-G coordinates the physiological assembly of a protein complex containing transmembrane adhesion molecules, voltage-gated sodium channels, and the spectrin membrane skeleton at axon initial segments.
PMCID: PMC2150881  PMID: 11724816
βIV spectrin; sodium channel Nav1.6; neurofascin; NrCAM; axon hillock
16.  LAD-1, the Caenorhabditis elegans L1CAM homologue, participates in embryonic and gonadal morphogenesis and is a substrate for fibroblast growth factor receptor pathway-dependent phosphotyrosine-based signaling 
The Journal of Cell Biology  2001;154(4):841-856.
This study shows that L1-like adhesion (LAD-1), the sole Caenorhabditis elegans homologue of the L1 family of neuronal adhesion molecules, is required for proper development of the germline and the early embryo and embryonic and gonadal morphogenesis. In addition, the ubiquitously expressed LAD-1, which binds to ankyrin-G, colocalizes with the C. elegans ankyrin, UNC-44, in multiple tissues at sites of cell–cell contact. Finally, we show that LAD-1 is phosphorylated in a fibroblast growth factor receptor (FGFR) pathway-dependent manner on a tyrosine residue in the highly conserved ankyrin-binding motif, FIGQY, which was shown previously to abolish the L1 family of cell adhesion molecule (L1CAM) binding to ankyrin in cultured cells. Immunofluorescence studies revealed that FIGQY-tyrosine–phosphorylated LAD-1 does not colocalize with nonphosphorylated LAD-1 or UNC-44 ankyrin but instead is localized to sites that undergo mechanical stress in polarized epithelia and axon–body wall muscle junctions. These findings suggest a novel ankyrin-independent role for LAD-1 related to FGFR signaling. Taken together, these results indicate that L1CAMs constitute a family of ubiquitous adhesion molecules, which participate in tissue morphogenesis and maintaining tissue integrity in metazoans.
PMCID: PMC2196473  PMID: 11502758
L1CAM; C. elegans; UNC-44 ankyrin; tyrosine phosphorylation; cell migration
17.  Caenorhabditis elegans β-G Spectrin Is Dispensable for Establishment of Epithelial Polarity, but Essential for Muscular and Neuronal Function 
The Journal of Cell Biology  2000;149(4):915-930.
The Caenorhabditis elegans genome encodes one α spectrin subunit, a β spectrin subunit (β-G), and a β-H spectrin subunit. Our experiments show that the phenotype resulting from the loss of the C. elegans α spectrin is reproduced by tandem depletion of both β-G and β-H spectrins. We propose that α spectrin combines with the β-G and β-H subunits to form α/β-G and α/β-H heteromers that perform the entire repertoire of spectrin function in the nematode. The expression patterns of nematode β-G spectrin and vertebrate β spectrins exhibit three striking parallels including: (1) β spectrins are associated with the sites of cell–cell contact in epithelial tissues; (2) the highest levels of β-G spectrin occur in the nervous system; and (3) β spec-trin-G in striated muscle is associated with points of attachment of the myofilament apparatus to adjacent cells. Nematode β-G spectrin associates with plasma membranes at sites of cell–cell contact, beginning at the two-cell stage, and with a dramatic increase in intensity after gastrulation when most cell proliferation has been completed. Strikingly, depletion of nematode β-G spectrin by RNA-mediated interference to undetectable levels does not affect the establishment of structural and functional polarity in epidermis and intestine. Contrary to recent speculation, β-G spectrin is not associated with internal membranes and depletion of β-G spectrin was not associated with any detectable defects in secretion. Instead β-G spectrin-deficient nematodes arrest as early larvae with progressive defects in the musculature and nervous system. Therefore, C. elegans β-G spectrin is required for normal muscle and neuron function, but is dispensable for embryonic elongation and establishment of early epithelial polarity. We hypothesize that heteromeric spectrin evolved in metazoans in response to the needs of cells in the context of mechanically integrated tissues that can withstand the rigors imposed by an active organism.
PMCID: PMC2174577  PMID: 10811831
membrane skeleton; unc-70; RNAi; cell–cell contact
18.  Ankyrin-B Is Required for Intracellular Sorting of Structurally Diverse Ca2+ Homeostasis Proteins 
The Journal of Cell Biology  1999;147(5):995-1008.
This report describes a congenital myopathy and major loss of thymic lymphocytes in ankyrin-B (−/−) mice as well as dramatic alterations in intracellular localization of key components of the Ca2+ homeostasis machinery in ankyrin-B (−/−) striated muscle and thymus. The sacoplasmic reticulum (SR) and SR/T-tubule junctions are apparently preserved in a normal distribution in ankyrin-B (−/−) skeletal muscle based on electron microscopy and the presence of a normal pattern of triadin and dihydropyridine receptor. Therefore, the abnormal localization of SR/ER Ca ATPase (SERCA) and ryanodine receptors represents a defect in intracellular sorting of these proteins in skeletal muscle. Extrapolation of these observations suggests defective targeting as the basis for abnormal localization of ryanodine receptors, IP3 receptors and SERCA in heart, and of IP3 receptors in the thymus of ankyrin-B (−/−) mice. Mis-sorting of SERCA 2 and ryanodine receptor 2 in ankyrin-B (−/−) cardiomyocytes is rescued by expression of 220-kD ankyrin-B, demonstrating that lack of the 220-kD ankyrin-B polypeptide is the primary defect in these cells. Ankyrin-B is associated with intracellular vesicles, but is not colocalized with the bulk of SERCA 1 or ryanodine receptor type 1 in skeletal muscle. These data provide the first evidence of a physiological requirement for ankyrin-B in intracellular targeting of the calcium homeostasis machinery of striated muscle and immune system, and moreover, support a catalytic role that does not involve permanent stoichiometric complexes between ankyrin-B and targeted proteins. Ankyrin-B is a member of a family of adapter proteins implicated in restriction of diverse proteins to specialized plasma membrane domains. Similar mechanisms involving ankyrins may be essential for segregation of functionally defined proteins within specialized regions of the plasma membrane and within the Ca2+ homeostasis compartment of the ER.
PMCID: PMC2169334  PMID: 10579720
calcium homeostasis; IP3 receptor; ankyrin; sarcoplasmic reticulum; gene knockout
19.  Phosphorylation of Adducin by Rho-Kinase Plays a Crucial Role in Cell Motility  
The Journal of Cell Biology  1999;145(2):347-361.
Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates α-adducin and thereby enhances the F-actin–binding activity of α-adducin in vitro. Here we identified the sites of phosphorylation of α-adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized α-adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated α-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated α-adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or α-adducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migra- tion in NRK49F cells. α-AdducinT445D,T480D (substi- tution of Thr445 and Thr480 by Asp), but not α-adducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates α-adducin downstream of Rho in vivo, and that the phosphorylation of adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.
PMCID: PMC2133101  PMID: 10209029
Rho; Rho-kinase; adducin; membrane ruffling; cell motility
20.  AnkyrinG Is Required for Clustering of Voltage-gated Na Channels at Axon Initial Segments and for Normal Action Potential Firing  
The Journal of Cell Biology  1998;143(5):1295-1304.
Voltage-gated sodium channels (NaCh) are colocalized with isoforms of the membrane-skeletal protein ankyrinG at axon initial segments, nodes of Ranvier, and postsynaptic folds of the mammalian neuromuscular junction. The role of ankyrinG in directing NaCh localization to axon initial segments was evaluated by region-specific knockout of ankyrinG in the mouse cerebellum. Mutant mice exhibited a progressive ataxia beginning around postnatal day P16 and subsequent loss of Purkinje neurons. In mutant mouse cerebella, NaCh were absent from axon initial segments of granule cell neurons, and Purkinje cells showed deficiencies in their ability to initiate action potentials and support rapid, repetitive firing. Neurofascin, a member of the L1CAM family of ankyrin-binding cell adhesion molecules, also exhibited impaired localization to initial segments of Purkinje cell neurons. These results demonstrate that ankyrinG is essential for clustering NaCh and neurofascin at axon initial segments and is required for physiological levels of sodium channel activity.
PMCID: PMC2133082  PMID: 9832557
ankyrinG; sodium channel; neurofascin; clustering; action potential
21.  Nervous System Defects of AnkyrinB (−/−) Mice Suggest Functional Overlap between the Cell Adhesion Molecule L1 and 440-kD AnkyrinB in Premyelinated Axons  
The Journal of Cell Biology  1998;143(5):1305-1315.
The L1 CAM family of cell adhesion molecules and the ankyrin family of spectrin-binding proteins are candidates to collaborate in transcellular complexes used in diverse contexts in nervous systems of vertebrates and invertebrates. This report presents evidence for functional coupling between L1 and 440-kD ankyrinB in premyelinated axons in the mouse nervous system. L1 and 440-kD ankyrinB are colocalized in premyelinated axon tracts in the developing nervous system and are both down-regulated after myelination. AnkyrinB (−/−) mice exhibit a phenotype similar to, but more severe, than L1 (−/−) mice and share features of human patients with L1 mutations. AnkyrinB (−/−) mice exhibit hypoplasia of the corpus callosum and pyramidal tracts, dilated ventricles, and extensive degeneration of the optic nerve, and they die by postnatal day 21. AnkyrinB (−/−) mice have reduced L1 in premyelinated axons of long fiber tracts, including the corpus callosum, fimbria, and internal capsule in the brain, and pyramidal tracts and lateral columns of the spinal cord. L1 was evident in the optic nerve at postnatal day 1 but disappeared by postnatal day 7 in mutant mice while NCAM was unchanged. Optic nerve axons of ankyrinB (−/−) mice become dilated with diameters up to eightfold greater than normal, and they degenerated by day 20. These findings provide the first evidence for a role of ankyrinB in the nervous system and support an interaction between 440-kD ankyrinB and L1 that is essential for maintenance of premyelinated axons in vivo.
PMCID: PMC2133070  PMID: 9832558
ankyrin; L1; cell adhesion molecule; mental retardation; gene knock-out
22.  Restriction of 480/270-kD Ankyrin G to Axon Proximal Segments Requires Multiple Ankyrin G-specific Domains  
The Journal of Cell Biology  1998;142(6):1571-1581.
AnkyrinG (−/−) neurons fail to concentrate voltage-sensitive sodium channels and neurofascin at their axon proximal segments, suggesting that ankyrinG is a key component of a structural pathway involved in assembly of specialized membrane domains at axon proximal segments and possibly nodes of Ranvier (Zhou, D., S. Lambert, D.L. Malen, S. Carpenter, L. Boland, and V. Bennett, manuscript submitted for publication). This paper addresses the mechanism for restriction of 270-kD ankyrinG to axon proximal segments by evaluation of localization of GFP-tagged ankyrinG constructs transfected into cultured dorsal root ganglion neurons, as well as measurements of fluorescence recovery after photobleaching of neurofascin– GFP-tagged ankyrinG complexes in nonneuronal cells. A conclusion is that multiple ankyrinG-specific domains, in addition to the conserved membrane-binding domain, contribute to restriction of ankyrinG to the axonal plasma membrane in dorsal root ganglion neurons. The ankyrinG-specific spectrin-binding and tail domains are capable of binding directly to sites on the plasma membrane of neuronal cell bodies and axon proximal segments, and presumably have yet to be identified docking sites. The serine-rich domain, which is present only in 480- and 270-kD ankyrinG polypeptides, contributes to restriction of ankyrinG to axon proximal segments as well as limiting lateral diffusion of ankyrinG–neurofascin complexes. The membrane-binding, spectrin-binding, and tail domains of ankyrinG also contribute to limiting the lateral mobility of ankyrinG–neurofascin complexes. AnkyrinG thus functions as an integrated mechanism involving cooperation among multiple domains heretofore regarded as modular units. This complex behavior explains ability of ankyrinB and ankyrinG to sort to distinct sites in neurons and the fact that these ankyrins do not compensate for each other in ankyrin gene knockouts in mice.
PMCID: PMC2141775  PMID: 9744885
ankyrin; axon initial segment; cell polarity; membrane dynamics; spectrin
23.  Adducin Is an In Vivo Substrate for Protein Kinase C: Phosphorylation in the MARCKS-related Domain Inhibits Activity in Promoting Spectrin–Actin Complexes and Occurs in Many Cells, Including Dendritic Spines of Neurons  
The Journal of Cell Biology  1998;142(2):485-497.
Adducin is a heteromeric protein with subunits containing a COOH-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that caps and preferentially recruits spectrin to the fast-growing ends of actin filaments. The basic MARCKS-related domain, present in α, β, and γ adducin subunits, binds calmodulin and contains the major phosphorylation site for protein kinase C (PKC). This report presents the first evidence that phosphorylation of the MARCKS-related domain modifies in vitro and in vivo activities of adducin involving actin and spectrin, and we demonstrate that adducin is a prominent in vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation of native and recombinant adducin inhibited actin capping measured using pyrene-actin polymerization and abolished activity of adducin in recruiting spectrin to ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS-serine, which is the major PKC phosphorylation site in the MARCKS-related domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increased twofold in rat hippocampal slices, eight- to ninefold in human embryonal kidney (HEK 293) cells, threefold in MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of adducin is an in vivo phosphorylation site for PKC or other PMA-activated kinases but not for cAMP-dependent protein kinase in a variety of cell types. Physiological consequences of the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type or PKC-unphosphorylatable S716A/S726A mutant α adducin. The mutant α adducin was no longer concentrated at the cell membrane at sites of cell–cell contact, and instead it was distributed as a cytoplasmic punctate pattern. Moreover, the cells expressing the mutant α adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant α adducin in a punctate pattern. Immunofluorescence with the phosphoadducin-specific antibody revealed the RTPS-serine phosphorylation of adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoadducin were detected in the dendritic spines of cultured hippocampal neurons. Spectrin also was a component of dendritic spines, although at distinct sites from the ones containing phosphoadducin. These data demonstrate that adducin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin occurs in dendritic spines that are believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures.
PMCID: PMC2133059  PMID: 9679146
membrane skeleton; cytoskeleton; actin binding protein; synapse; synaptic plasticity
24.  Tyrosine Phosphorylation at a Site Highly Conserved in the L1 Family of Cell Adhesion Molecules Abolishes Ankyrin Binding and Increases Lateral Mobility of Neurofascin 
The Journal of Cell Biology  1997;137(3):703-714.
This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.
PMCID: PMC2139872  PMID: 9151675
25.  Ankyrin-B Coordinates the Na/K ATPase, Na/Ca Exchanger, and InsP3 Receptor in a Cardiac T-Tubule/SR Microdomain 
PLoS Biology  2005;3(12):e423.
We report identification of an ankyrin-B-based macromolecular complex of Na/K ATPase (alpha 1 and alpha 2 isoforms), Na/Ca exchanger 1, and InsP3 receptor that is localized in cardiomyocyte T-tubules in discrete microdomains distinct from classic dihydropyridine receptor/ryanodine receptor “dyads.” E1425G mutation of ankyrin-B, which causes human cardiac arrhythmia, also blocks binding of ankyrin-B to all three components of the complex. The ankyrin-B complex is markedly reduced in adult ankyrin-B+/− cardiomyocytes, which may explain elevated [Ca2+]i transients in these cells. Thus, loss of the ankyrin-B complex provides a molecular basis for cardiac arrhythmia in humans and mice. T-tubule-associated ankyrin-B, Na/Ca exchanger, and Na/K ATPase are not present in skeletal muscle, where ankyrin-B is expressed at 10-fold lower levels than in heart. Ankyrin-B also is not abundantly expressed in smooth muscle. We propose that the ankyrin-B-based complex is a specialized adaptation of cardiomyocytes with a role for cytosolic Ca2+ modulation.
The authors describe an ankyrin-B-based macromolecular complex localized in cardiomyocyte T-tubules, which the authors propose is a specialized adaptation of these cells with a role for cytosolic Ca2+ modulation.
PMCID: PMC1287507  PMID: 16292983

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