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2005 | 52 | 2 | 469-476
Article title

Calcium-binding calmyrin forms stable covalent dimers in vitro, but in vivo is found in monomeric form.

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The EF-hand Ca^(2+)-binding protein calmyrin is expressed in many tissues and can interact with multiple effector proteins, probably as a sensor transferring Ca^(2+) signals. As oligomerization may represent one of Ca^(2+)-signal transduction mechanisms, we characterised recombinant calmyrin forms using non-reducing SDS/PAGE, analytical ultracentrifugation and gel filtration. We also aimed at identification of biologically active calmyrin forms. Non-reducing SDS/PAGE showed that in vitro apo- and Ca^(2+)-bound calmyrin oligomerizes forming stable intermolecular disulfide bridges. Ultracentrifugation indicated that at a 220 µM initial protein concentration apo-calmyrin existed in an equilibrium of a 21.9 kDa monomer and a 43.8 kDa dimer (trimeric or tetrameric species were not detected). The dimerization constant was calculated as Ka = 1.78 × 103 M^(-1) at 6oC. Gel filtration of apo- and Ca^(2+)-bound calmyrin at a 100 µM protein concentration confirmed an equilibrium of a monomer and a covalent dimer state. Importantly, both monomer and dimer underwent significant conformational changes in response to binding of Ca^(2+). However, when calmyrin forms were analyzed under non-reducing conditions in cell extracts by Western blotting, only monomeric calmyrin was detected in human platelets and lymphocytes, and in rat brain. Moreover, in contrast to recombinant calmyrin, crosslinking did not preserve any dimeric species of calmyrin regardless of Ca^(2+) concentrations. In summary, our data indicate that although calmyrin forms stable covalent dimers in vitro, it most probably functions as a monomer in vivo.
Physical description
  • Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, Warsaw, Poland
  • Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, Warsaw, Poland
  • Laboratory of Biochemistry, NHLBI/NIH, Bethesda, MD 20892-8012, USA
  • Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, Warsaw, Poland
  • Nencki Institute of Experimental Biology, Laboratory of Calcium-Binding Proteins, Warsaw, Poland
  • Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, Warsaw, Poland
  • Ames JB, Ishima R, Tanaka T, Gordon JI, Stryer L, Ikura M (1997) Molecular mechanics of calcium-myristoyl switches. Nature 389: 198-202.
  • Atkinson RA, Joseph C, Kelly G, Muskett FW, Frenkiel TA, Nietlispach D, Pastore A (2001) Ca2+-independent binding of an EF-hand domain to a novel motif in the alpha-actinin-titin complex. Nat Struct Biol 8: 853-857.
  • Barry WT, Boudignon-Proudhon C, Shock DD, McFadden A, Weiss JM, Sondek J, Parise LV (2002) Molecular basis of CIB binding to the integrin IIb cytoplasmic domain. J Biol Chem 277: 28877-28883.
  • Bernstein HG, Blazejczyk M, Rudka T, Gundelfinger ED, Dobrowolny H, Bogerts B, Kreutz MR, Kuznicki J, Wojda U (2005) The Alzheimer disease-related calcium-binding protein Calmyrin is present in human forebrain with an altered distribution in Alzheimer’s as compared to normal aging brains. Neuropathol Appl Neurobiol 31: 314-324.
  • Berridge MJ (1998) Neuronal calcium signaling. Neuron 21: 13-26.
  • Burgoyne RD, Weiss JL (2001) The neuronal calcium sensor family of Ca2+-binding proteins. Biochem J 353: 1-12.
  • Donato R (2001) S100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular functional roles. Int J Biochem Cell Biol 33: 637-668.
  • Gentry HR, Singer AU, Betts L, Yang C, Ferrara JD, Sondek J, Parise LV (2005) Structural and biochemical characterization of CIB1 delineates a new family of EF-hand containing proteins. J Biol Chem 280: 8407-8415.
  • Fang X, Chen C, Wang Q, Gu J, Chi C (2001) The interaction of the calcium- and integrin-binding protein (CIBP) with the coagulation factor VIII. Thromb Res 102: 177-185.
  • Haglid KG, Yang Q, Hamberger A, Berman S, Widerberg A, Danielsen N (1997) S100beta stimulates neurite outgrowth in the rat sciatic grafted with acellular nuscle transplants. Brain Res 753: 196-201.
  • Heizmann CW, Hunziker W (1991) Intracellular calcium-binding proteins: more sites than insights. Trends Biochem Sci 16: 98-103.
  • Henderson MJ, Russell AJ, Hird S, Munoz M, Clancy JL, Lehrbach GM, Calanni ST, Jans DA, Sutherland RL, Watts CK (2002) EDD, the human hyperplastic discs protein, has a role in progesterone receptor coactivation and potential involvement in DNA damage response. J Biol Chem 277: 26468-26478.
  • Hollenbach AD, McPherson CJ, Lagutina I, Grosveld G (2002) The EF-hand calcium-binding protein calmyrin inhibits the transcriptional and DNA-binding activity of Pax3. Biochim Biophys Acta 1574: 321-328.
  • Hwang PM, Vogel HJ (2000) Structures of the platelet calcium- and integrin-binding protein and the alphaIIb-integrin cytoplasmic domain suggest a mechanism for calcium regulated recognition; homology modelling and NMR studies. J Mol Recognit 13: 83-92.
  • Ikura M (1996) Calcium binding and conformational response in EF-hand proteins. Trends Biochem Sci 21: 14-17.
  • Ito A, Uehara T, Nomura Y (2000) Isolation of Ich-1S (caspase-2S)-binding protein that partially inhibits caspase activity. FEBS Lett 470: 360-364.
  • Kauselmann G, Weiler M, Wulff P, Jessberger S, Konietzko U, Scafidi J et al. (1999) The polo-like protein kinases Fnk and Snk associate with Ca2+- and integrin-binding protein and are regulated dynamically with synaptic plasticity. EMBO J 18: 5528-5539.
  • Koltzscher M, Neumann C, Konig S, Gerke V (2003) Ca2+-dependent binding and activation of dormant ezrin by dimeric S100P. Mol Biol Cell 14: 2372-2384.
  • Lee GE, Yu EY, Cho CH, Lee J, Muller MT, Chung IK (2004) DNA-protein kinase catalytic subunit-interacting protein KIP binds telomerase by interacting with human telomerase reverse transcriptase. J Biol Chem 279: 34750-34755.
  • Ma S, Liu MA, Yuan YL, Erikson RL (2003) The serum-inducible protein kinase Snk is a G1 phase polo-like kinase that is inhibited by the calcium- and integrin-binding protein CIB. Mol Cancer Res 1: 376-384.
  • Maler L, Sastry M, Chazin WJ (2002) A structural basis for S100 protein specificity derived from comparative analysis of apo and Ca(2+)-calcyclin. J Mol Biol 317: 279-290.
  • Meyer T, York JD (1999) Calcium-myristoyl switches turn on new lights. Nat Cell Biol 1: E93-E95.
  • Miller G (1974) The oncogenicity of Epstein-Barr virus. J Infect Dis 130: 187-205.
  • Moroz OV, Dodson GG, Wilson KS, Lukanidin E, Bronstein IB (2003) Structural states of S100A12: A key to its functional diversity. Microsc Res Tech 60: 581-592.
  • Nakayama S, Kretsinger RH (1994) Evolution of the EF-hand family of proteins. Annu Rev Biophys Biomol Struct 23: 473-507.
  • Naik MU, Naik UP (2003) Calcium- and integrin-binding protein regulates focal adhesion kinase activity during platelet spreading on immobilized fibrinogen. Blood 102: 3629-3636.
  • Naik UP, Patel PM, Parise LV (1997) Identification of a novel calcium-binding protein that interacts with the integrin alphaIIb cytoplasmic domain. J Biol Chem 272: 4651-4654.
  • Nosworthy NJ, Peterkofsky A, König S, Seok Y-J, Szczepanowski RH, Ginsburg A (1998) Phosphorylation destabilizes the amino-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system. Biochemistry 37: 6718-6726.
  • Olshevskaya EV, Ermilov AN, Dizhoor AM (1999) Dimerization of guanylyl cyclase-activating protein and a mechanism of photoreceptor guanylyl cyclase activation. J Biol Chem 274: 25583-25587.
  • Osawa M, Tong KI, Lilliehook C, Wasco W, Buxbaum JD, Cheng HY, Penninger JM, Ikura M, Ames JB (2001) Calcium-regulated DNA binding and oligomerization of the neuronal calcium-sensing protein, calsenilin/DREAM/KChIP3. J Biol Chem 276: 41005-41013.
  • Potts BC, Smith J, Akke M, Macke TJ, Okazaki K, Hidaka H, Case DA, Chazin WJ (1995) The structure of calcyclin reveals a novel homodimeric fold for S100 Ca(2+)-binding proteins. Nat Struct Biol 2: 790-796.
  • Seidenbecher CI, Landwehr M, Smalla KH, Kreutz M, Dieterich DC, Zuschratter W, Reissner C, Hammarback JA, Bockers TM, Gundelfinger ED, Kreutz MR (2004) Caldendrin but not calmodulin binds to light chain 3 of MAP1A/B: an association with the microtubule cytoskeleton highlighting exclusive binding partners for neuronal Ca2+-sensor proteins. J Mol Biol 336: 957-970.
  • Shock DD, Naik UP, Brittain JE, Alahari SK, Sondek J, Parise LV (1999) Calcium-dependent properties of CIB binding to the integrin alphaIIb cytoplasmic domain and translocation to the platelet cytoskeleton. Biochem J 342: 729-735.
  • Stabler SM, Ostrowski LL, Janicki SM, Monteiro MJ (1999) A myristoylated calcium-binding protein that preferentially interacts with the Alzheimer’s disease presenilin 2 protein. J Cell Biol 145: 1277-1292.
  • Vijay-Kumar S, Kumar VD (1999) Crystal structure of recombinant bovine neurocalcin. Nat Struct Biol 6: 80-88.
  • Wendland M, von Figura K, Pohlmann R (1991) Mutational analysis of disulfide bridges in the Mr 46,000 mannose 6-phosphate receptor. Localization and role for ligand binding. J Biol Chem 266: 7132-7136.
  • Winnigham-Major F, Staecker JL, Barger SW, Coats S, Van Eldik LJ (1989) Neurite extension and neuronal survival activities of recombinant S100 beta proteins that differ in the content and position of cysteine residues. J Cell Biol 109: 3063-3071.
  • Wojda U, Kuznicki J (1993a) Biochemical properties of calcyclin - a potential marker of some diseases. Acta Biochim Polon 40: 171-175.
  • Wojda U, Kuznicki J (1993b) Characterization of calcyclin fragments obtained by CNBr-cleavage. Int J Biochem 25: 999-1007.
  • Wojda U, Kuznicki J (1994) Calcyclin from mouse Ehrlich ascites tumour cells and rabbit lung form non-covalent dimers. Biochim Biophys Acta 1209: 248-252.
  • Wu X, Lieber MR (1997) Interaction between DNA-dependent protein kinase and a novel protein, KIP. Mutat Res 385: 13-20.
  • Whitehouse C, Chambers J, Howe K, Cobourne M, Sharpe P, Solomon E (2002) NBR1 interacts with fasciculation and elongation protein zeta-1 (FEZ1) and calcium and integrin binding protein (CIB) and shows developmentally restricted expression in the neural tube. Eur J Biochem 269: 538-545.
  • Yamniuk AP, Nguyen LT, Hoang TT, Vogel HJ (2004) Metal ion binding properties and conformational states of calcium- and integrin-binding protein. Biochemistry 43: 2558-2568.
  • Zamyatnin AA (1984) Amino acid, peptide and protein volume in solution. Annu Rev Biophys Bioeng 13: 145-165.
  • Zhou W, Qian Y, Kunjilwar K, Pfaffinger PJ, Choe S (2004) Structural insights into the functional interaction of KChIP1 with Shal-type K(+) channels. Neuron 41: 573-586.
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