- Research Article
- Published:
Non-erythroid beta spectrin interacting proteins and their effects on spectrin tetramerization
Cellular & Molecular Biology Letters volume 16, Article number: 595 (2011)
Abstract
With yeast two-hybrid methods, we used a C-terminal fragment (residues 1697–2145) of non-erythroid beta spectrin (βII-C), including the region involved in the association with alpha spectrin to form tetramers, as the bait to screen a human brain cDNA library to identify proteins interacting with βII-C. We applied stringent selection steps to eliminate false positives and identified 17 proteins that interacted with βII-C (IPβII-C s). The proteins include a fragment (residues 38–284) of “THAP domain containing, apoptosis associated protein 3, isoform CRA g”, “glioma tumor suppressor candidate region gene 2” (residues 1-478), a fragment (residues 74–442) of septin 8 isoform c, a fragment (residues 704–953) of “coatomer protein complex, subunit beta 1, a fragment (residues 146–614) of zinc-finger protein 251, and a fragment (residues 284–435) of syntaxin binding protein 1. We used yeast three-hybrid system to determine the effects of these βII-C interacting proteins as well as of 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IPαII-N s) [1] on spectrin tetramer formation. The results showed that 3 IPβII-C s were able to bind βII-C even in the presence of αII-N, and 4 IPαII-N s were able to bind αII-N in the presence of βII-C. We also found that the syntaxin binding protein 1 fragment abolished αII-N and βII-C interaction, suggesting that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.
Abbreviations
- αII:
-
non-erythroid (brain) alpha spectrin
- αII-N:
-
a recombinant protein consisting of the N-terminal region 359 residues of αII
- AD:
-
activation domain of GAL4
- βII:
-
non-erythroid (brain) beta spectrin
- βII-C:
-
a recombinant protein consisting of residues 1697-2145 at the C-terminus of βII
- BD:
-
binding domain of GAL4
- IPαII-N :
-
proteins interacting with αII-N
- IPβII-C :
-
proteins interacting with βII-C
- pAD:
-
yeast twohybrid cloning vector pGADT7
- pBD:
-
yeast two-hybrid cloning vector pGBKT7
- pBR:
-
yeast three-hybrid cloning vector pBridge
- QDO:
-
quadruple drop-out
- SD:
-
synthetic defined
- TDO:
-
triple drop-out
- X-α-gal:
-
5-bromo-4-chloro-3-indolyl-α-galactopyranoside
- YPDA:
-
yeast growth medium with yeast extract, peptone, dextrose and adenine
References
Oh, Y. and Fung, L.W.-M. Brain proteins interacting with the tetramerization region of non-erythroid alpha spectrin. Cell. Mol. Biol. Lett. 12 (2007) 604–620.
Marchesi, V.T. and Steers, E. Selective solubilization of a protein component of the red cell membrane. Science 159 (1968) 203–204.
Hiller, G. and Weber, K. Spectrin is absent in various tissue culture cells. Nature 299 (1977) 181–183.
Levine, J. and Willard, M. Axonally transported polypeptides associated with the internal periphery of many cells. J. Cell Biol. 90 (1981) 631–643.
Lee, J.K., Coyne, R.S., Dubreuil, R.R., Goldstein, L.S.B. and Branton, D. Cell shape and interaction defects in α-spectrin mutants of Drosophila Melanogaster. J. Cell Biol. 123 (1993) 1797–1809.
Pinder, J.C. and Baines, A.J. A protein accumulator. Nature 406 (2000) 253–254.
Djinovic-Carugo, K., Gautel, M., Ylanne, J. and Young, P. The spectrin repeat: a structural platform for cytoskeletal protein assemblies. FEBS Lett. 513 (2002) 119–123.
Gascard, P. and Mohandas, N. New insights into functions of erythroid proteins in nonerythroid cells. Curr. Opin. Hematol. 7 (2000) 123–129.
Sridharan, D.M., McMahon, L.W. and Lambert, M. W. αII-spectrin interacts with five groups of functionally important proteins in the nucleus. Cell Biol. Int. 30 (2006) 866–878.
Kanda, K., Tanaka, T. and Sobue, K. Calspectin (fodrin or nonerythroid spectrin)-actin interaction: a possible involvement of 4,1-related protein. Biochem. Biophys. Res. Commun. 140 (1986) 1051–1058.
Tsukita, S., Tsukita, S., Ishikawa, H., Kurokawa, M., Morimoto, K., Sobue, K. and Kakiuchi, S. Binding sited of calmodulin and actin on the brain spectrin, calspectin. J. Cell Biol. 97 (1983) 574–578.
Sobue, K., Kanda, K. and Kakiuchi, S. Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin: a spectrin-like calmodulin-binding protein (fodrin). FEBS Lett. 150 (1982) 185–190.
Riederer, B.M., Lopresti, L.L., Krebs, K.E., Zagon, I.S. and Goodman, S.R. Brain spectrin (240/235) and brain spectrin (240/235E): conservation of structure and location within mammalian neural tissue. Brain Res. Bull. 21 (1988) 607–616.
Ohara, O., Ohara, R., Yamakawa, H., Nakajima, D. and Nakayama, M. Characterization of a new β-spectrin gene which is predominantly expressed in brain. Mol. Brain Res. 57 (1998) 181–192.
Tang, Y., Katuri, V., Iqbal, S., Narayan, T., Wang, Z., Lu, R.S., Mishra, L. and Mishra, B. ELF a beta-spectrin is a neuronal precursor cell marker in developing mammalian brain; structure and organization of the elf/beta-G spectrin gene. Oncogene 21 (2002) 5255–5267.
Lambert, S. and Bennett, V. Postmitotic expression of ankyrinR and beta R-spectrin in discrete neuronal populations of the rat brain. J. Neurosci. 13 (1993) 3725–3735.
Tang, Y., Katuri, V., Dillner, A., Mishra, B., Deng, C.-X. and Mishra, L. Disruption of transforming growth factor-β signaling in ELF β-spectrindeficient mice. Science 299 (2003) 574–577.
Bennett, V. and Baines, A.J. Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues. Physiol. Rev. 81 (2001) 1353–1392.
Norman, K.R. and Moerman, D.G. Alpha spectrin is essential for morphogenesis and body wall muscle formation in Caenorhabditis elegant. J. Cell. Biol. 157 (2002) 665–677.
McMahon, K.R., Zhang, P., Sridharan, D.M., Lefferts, J.A. and Lambert, M.W. Knockdown of alpha II spectrin in normal human cells by siRNA leads to chromosomal instability and decreased DNA interstrand cross-link repair. Biochem. Biophys. Res. Commun. 381 (2009) 288–293.
DeSilva, T.M., Peng, K.-C., Speicher, K.D. and Speicher, D.W. Analysis of human red cell spectrin tetramer (head-to-head) assembly using complementary univalent peptides. Biochemistry 31 (1992) 10872–10878.
Bignone, P.A., King, M.D., Pinder, J.C. and Baines, A.J. Phosphorylation of a threonine unique to the short C-terminal isoform of betaII-spectrin links regulation of alpha-spectrin interaction to neuritogenesis. J. Biol. Chem. 232 (2007) 888–896.
Speicher, D., DeSilva, T., Speicher, K., Ursitti, J., Hembach, P. and Weglarz, L. Location of the human red cell spectrin tetramer binding site and detection of a related “closed” hairpin loop dimer using proteolytic footprinting. J. Biol. Chem. 268 (1993) 4227–4235.
Mehboob, S., Luo, B.-H., Fu, W., Johnson, M.E. and Fung, L.W.-M. Conformational studies of the tetramerization site of human erythroid spectrin by cysteine-scanning spin-labeling EPR methods. Biochemistry 44 (2005) 15898–15905.
Ipsaro, J.J., Harper, S.L., Messick, T.E., Marmorstein, R., Mondragon, A. and Speicher, D.W. Crystal structure and functional interpretation of the erythrocyte spectrin tetramerization domain complex. Blood 115 (2010) 4843–4852.
Song, Y., Antoniou, C., Memic, A., Kay, B.K. and Fung, L.W.-M. Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs. Protein Sci. 20 (2011) 867–879.
Antoniou, A., Lam, V.Q. and Fung, L.W.-M. Conformational changes at the tetramerization site of erythroid α-spectrin upon binding β-spectrin: a spin label EPR study. Biochemistry 47 (2008) 10765–10772.
Song, Y., Pipala, N.H. and Fung, L.W.-M. The L49F mutation in alpha erythroid spectrin induces local disorder in the tetramer association region: fluorescence and molecular dynamics studies of free and bound alpha spectrin. Protein Sci. 18 (2009) 1916–1925.
Mehboob, S., Song, Y., Witek, M., Long, F., Santarsiero, B.D., Johnson, M.E. and Fung, L.W.-M. Crystal structure of the nonerythroid α-spectrin tetramerization site reveals differences between erythroid and nonerythroid spectrin tetramer formation. J. Biol. Chem. 285 (2010) 14572–14587.
Mehboob, S., Luo, B.-H., Patel, B.M. and Fung, L.W.-M. αβ spectrin coiled coil association at the tetramerization site. Biochemistry 40 (2001) 12457–12464.
Mehboob, S., Jacob, J., May, M., Kotula, L., Thiyagarajan, P., Johnson, M.E. and Fung, L.W.-M. Structural analysis of the αN-terminal region of erythroid and nonerythroid spectrins by small-angle X-ray scattering. Biochemistry 42 (2003) 14702–14710.
Begg, G.E., Morris, M.B. and Ralston G.B. Comparison of the saltdependent self-association of brain and erythroid spectrin. Biochemistry 36 (1997) 6977–6985.
Sumandea, C.A. and Fung, L.W.-M. Mutational effects at the tetramerization site of nonerythroid alpha spectrin. Mol. Brain Res. 136 (2005) 81–90.
Marchler-Bauer, A., Lu, S., Anderson, J.B., Chitsaz, F., Derbyshire M.K., DeWeese-Scott, C., Fong, J.H., Geer, L.Y., Geer, R.C., Gonzales, N.R., Gwadz, M., Hurwitz, D.I., Jackson, J.D., Ke, Z., Lanczycki, C.J., Lu, F., Marchler, G.H., Mullokandov, M., Omelchenko, M.V., Robertson, C.L., Song, J.S., Thanki, N., Yamashita, R.A., Zhang, D., Zhang, N., Zheng, C. and Bryant, S.H. CDD: a conserved domain database for the functional annotation of proteins. Nucleic Acids Res. 39 (2011) D225–D229.
Roussigne, M., Kossida, S., Lavigne, A.-C., Clouaire, T., Ecochard, V., Glories, A., Amalric, F. and Girard, J.-P. The THAP domain: a novel protein motif with similarity to the DNA-binding domain of P lelement transposase. Trends Biochem. Sci. 28 (2003) 66–69.
Macara, I.G., Baldarelli, R., Field, C.M., Glotzer, M., Hayashi, Y., Hsu, S.C., Kennedy, M.B., Kinoshita, M., Longtine, M., Low, C., Maltais, L.J., McKenzie, L., Mitchison, T.J., Nishikawa, T., Noda, M., Petty, E.M., Peifer, M., Pringle, J.R., Robinson, P.J., Roth, D., Russel, S., Stuhlmann, H., Tanaka, M., Tanaka, R., Trimble, W., Ware, J., Zeleznik-Le, N.J. and Zieger, B. Mammalian septins nomenclature. Mol. Biol. Cell 13 (2002) 4141–4143.
Peterson, E.A. and Petty, E.M. Conquering the complex world of human septins: implications for health and disease. Clin. Genet. 77 (2010) 511–524.
Han, G.A., Malintan, N.T., Collins, B.M., Meunier, F.A. and Sugita, S. Munc18-1 as a key regulator of neurosecretion. J. Neurochem. 115 (2010) 1–10.
David, Y., Ziv, T., Admon, A. and Navon, A. The E2 ubiquitin-conjugating enzymes direct polyubiquitination to preferred lysines. J. Biol. Chem. 285 (2010) 8595–8604.
Ardley, H.C., Moynihan, T.P. Markham, A.F. and Robinson, P.A. Promoter analysis of the human ubiquitin-conjugating enzyme gene family UBE2L1-4, including UBE2L3 which encodes UbcH7. Biochim. Biophys. Acta 1491 (2000) 57–64.
Good, M.C., Zalatan, J.G. and Lim, W.A. Scaffold proteins: hubs for controlling the flow of cellular information. Science 332 (2011) 680–686.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Sevinc, A., Fung, L.W.M. Non-erythroid beta spectrin interacting proteins and their effects on spectrin tetramerization. Cell Mol Biol Lett 16, 595 (2011). https://doi.org/10.2478/s11658-011-0025-9
Received:
Accepted:
Published:
DOI: https://doi.org/10.2478/s11658-011-0025-9