Skip to main content
PMC home page
Cellular & Molecular Biology Letters logoLink to Cellular & Molecular Biology Letters
. 2007 Jul 3;12(4):604–620. doi: 10.2478/s11658-007-0028-8

Brain proteins interacting with the tetramerization region of non-erythroid alpha spectrin

Younsang Oh 1, Leslie W -M Fung 1,✉
PMCID: PMC6275721  PMID: 17607528

Abstract

The N-terminal region of non-erythroid alpha spectrin (SpαII) is responsible for interacting with its binding partner, beta spectrin, to form functional spectrin tetramers. We used a yeast-two-hybrid system, with an N-terminal segment of alpha spectrin representing the functional tetramerization site, as a bait to screen human brain c-DNA library for proteins that interact with the alpha spectrin segment. In addition to several beta spectrin isoforms, we identified 14 proteins that interact with SpαII. Seven of the 14 were matched to 6 known proteins: Duo protein, Lysyl-tRNA synthetase, TBP associated factor 1, two isoforms (b and c) of a protein kinase A interacting protein and Zinc finger protein 333 (2 different segments). Four of the 6 proteins are located primarily in the nucleus, suggesting that spectrin plays important roles in nuclear functions. The remaining 7 proteins were unknown to the protein data base. Structural predictions show that many of the 14 proteins consist of a large portion of unstructured regions, suggesting that many of these proteins fold into a rather flexible conformation. It is interesting to note that all but 3 of the 14 proteins are predicted to consist of one to four coiled coils (amphiphilic helices). A mutation in SpαII, V22D, which interferes with the coiled coil bundling of SpαII with beta spectrin, also affects SpαII interaction with Duo protein, TBP associated factor 1 and Lysyl-tRNA synthetase, suggesting that they may compete with beta spectrin for interaction with SpαII. Future structural and functional studies of these proteins to provide interaction mechanisms will no doubt lead to a better understanding of brain physiology and pathophysiology.

Key words: Spectrin, Tetramerization site, Protein-protein interaction, Yeast-two hybrid system, Brain protein, Spectrin mutation

Full Text

The Full Text of this article is available as a PDF (463.2 KB).

Abbreviations used

KRAB

Krueppel-associated box

QDO

quadruple drop-out

PKA

protein kinase A

SH3

src homology 3

SpαII

non-erythroid alpha spectrin

SpI

erythroid spectrin

SpβII

spectrin beta II

TBP

TATA box binding protein

TDO

triple drop-out

References

  • 1.Yu J., Fischman D.A., Steck T.L. Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. J. Supramol. Struct. 1973;1:233–248. doi: 10.1002/jss.400010308. [DOI] [PubMed] [Google Scholar]
  • 2.Mohandas N., Chasis J.A., Shohet S.B. The influence of membrane skeleton on red cell deformability, membrane material properties, and shape. Semin. Hematol. 1983;20:225–242. [PubMed] [Google Scholar]
  • 3.Park S., Caffrey M.S., Johnson M.E., Fung L.W. Solution structural studies on human erythrocyte alpha-spectrin tetramerization site. J. Biol. Chem. 2003;278:21837–21844. doi: 10.1074/jbc.M300617200. [DOI] [PubMed] [Google Scholar]
  • 4.Yan Y., Winograd E., Viel A., Cronin T., Harrison S.C., Branton D. Crystal structure of the repetitive segments of spectrin. Science. 1993;262:2027–2030. doi: 10.1126/science.8266097. [DOI] [PubMed] [Google Scholar]
  • 5.Pascual J., Pfuhl M., Rivas G., Pastore A., Saraste M. The spectrin repeat folds into a three-helix bundle in solution. FEBS Lett. 1996;383:201–207. doi: 10.1016/0014-5793(96)00251-7. [DOI] [PubMed] [Google Scholar]
  • 6.Mehboob S., Luo B.H., Johnson M.E., Fung L.W.-M. Conformational studies of the tetramerization site of the human erythroid spectrin by cysteine-scanning spin-labeling EPR methods. Biochemistry. 2005;44:15898–15905. doi: 10.1021/bi051009m. [DOI] [PubMed] [Google Scholar]
  • 7.Mehboob S., Luo B.H., Fung L.W.-M. αβ spectrin association: A model system to mimic helical bundling at the tetramerization site. Biochemistry. 2001;40:12457–12464. doi: 10.1021/bi010984k. [DOI] [PubMed] [Google Scholar]
  • 8.Hiller G., Weber K. Spectrin is absent in various tissue culture cells. Nature. 1977;266:181–183. doi: 10.1038/266181a0. [DOI] [PubMed] [Google Scholar]
  • 9.Beck K.A., Nelson W.J. A spectrin membrane skeleton of the Golgi complex. Biochim. Biophys. Acta. 1998;1404:153–160. doi: 10.1016/S0167-4889(98)00054-8. [DOI] [PubMed] [Google Scholar]
  • 10.Goodman S.R. Discovery of nonerythroid spectrin to the demonstration of its key role in synaptic transmission. Brain Res. Bull. 1999;50:345–346. doi: 10.1016/S0361-9230(99)00098-2. [DOI] [PubMed] [Google Scholar]
  • 11.De Matteis M.A., Morrow J.S. Spectrin tethers and mesh in the biosynthetic pathway. J. Cell Sci. 2000;113:2331–2343. doi: 10.1242/jcs.113.13.2331. [DOI] [PubMed] [Google Scholar]
  • 12.Gascard P., Mohandas N. New insights into functions of erythroid proteins in nonerythroid cells. Curr. Opin. Hematol. 2000;7:123–129. doi: 10.1097/00062752-200003000-00009. [DOI] [PubMed] [Google Scholar]
  • 13.Kordeli E. The spectrin-based skeleton at the postsynaptic membrane of the neuromuscular junction. Microsc. Res. Tech. 2000;49:101–107. doi: 10.1002/(SICI)1097-0029(20000401)49:1<101::AID-JEMT11>3.0.CO;2-U. [DOI] [PubMed] [Google Scholar]
  • 14.Bennett V., Baines A.J. Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues. Physiol. Rev. 2001;81:1353–1392. doi: 10.1152/physrev.2001.81.3.1353. [DOI] [PubMed] [Google Scholar]
  • 15.Giorgi M., Cianci C., Gallagher P., Morrow J.S. Spectrin oligomerization is cooperatively coupled to membrane assembly: A linkage targeted by many hereditary hemolytic anemias. Exp. Mol. Pathol. 2001;70:215–230. doi: 10.1006/exmp.2001.2377. [DOI] [PubMed] [Google Scholar]
  • 16.Djinovic-Carugo K., Gautel M., Ylanne J., Young P. The spectrin repeat: a structural platform for cytoskeletal protein assemblies. FEBS Lett. 2002;513:119–123. doi: 10.1016/S0014-5793(01)03304-X. [DOI] [PubMed] [Google Scholar]
  • 17.Lee J.K., Coyne R.S., Dubreuil R.R., Goldstein L.S., Branton D. Cell shape and interaction defects in alpha-spectrin mutants of Drosophila melanogaster. J. Cell Biol. 1993;123:1797–1809. doi: 10.1083/jcb.123.6.1797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Pinder J.C., Baines A.J. A protein accumulator. Nature. 2000;406:253–254. doi: 10.1038/35018679. [DOI] [PubMed] [Google Scholar]
  • 19.McMahon L.W., Sangerman J., Goodman S.R., Kumaresan K., Lambert M.W. Human alpha spectrin II and the FANCA, FANCC and FANCG proteins bind to DNA containing psoralen interstrand cross-links. Biochemistry. 2001;40:7025–7034. doi: 10.1021/bi002917g. [DOI] [PubMed] [Google Scholar]
  • 20.Sridharan D.M., McMahon L.W., Lambert M.W. alphaII-Spectrin interacts with five groups of functionally important proteins in the nucleus. Cell Biol. Int. 2006;30:866–878. doi: 10.1016/j.cellbi.2006.06.005. [DOI] [PubMed] [Google Scholar]
  • 21.Goodman S.R., Zimmer W.E., Clark M.B., Zagon I.S., Barker J.E., Bloom M.L. Brain spectrin: of mice and men. Brain Res. Bull. 1995;36:593–606. doi: 10.1016/0361-9230(94)00264-2. [DOI] [PubMed] [Google Scholar]
  • 22.Kanda K., Tanaka T., Sobue K. Calspectin (fodrin or nonerythroid spectrin)-actin interaction: a possible involvement of 4.1-related protein. Biochem. Biophys. Res. Commun. 1986;140:1051–1058. doi: 10.1016/0006-291X(86)90741-2. [DOI] [PubMed] [Google Scholar]
  • 23.Tsukita S., Tsukita S., Ishikawa H., Kurokawa M., Morimoto K., Sobue K., Kakiuchi S. Binding sites of calmodulin and actin on the brain spectrin, calspectin. J. Cell Biol. 1983;97:574–578. doi: 10.1083/jcb.97.2.574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Sobue K., Kanda K., 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. 1982;150:185–190. doi: 10.1016/0014-5793(82)81331-8. [DOI] [PubMed] [Google Scholar]
  • 25.Riederer B.M., Lopresti L.L., Krebs K.E., Zagon I.S., Goodman S.R. Brain spectrin(240/235) and brain spectrin(240/235E): conservation of structure and location within mammalian neural tissue. Brain Res Bull. 1988;21:607–616. doi: 10.1016/0361-9230(88)90200-6. [DOI] [PubMed] [Google Scholar]
  • 26.Ohara O., Ohara R., Yamakawa H., Nakajima D., Nakayama M. Characterization of a new beta-spectrin gene which is predominantly expressed in brain. Brain Res. Mol. Brain Res. 1998;57:181–192. doi: 10.1016/S0169-328X(98)00068-0. [DOI] [PubMed] [Google Scholar]
  • 27.Stankewich M.C., Tse W.T., Peters L.L., Ch’ng Y., John K.M., Stabach P.R., Devarajan P., Morrow J.S., Lux S.E. A widely expressed betaIII spectrin associated with Golgi and cytoplasmic vesicles. Proc. Natl. Acad. Sci. U. S. A. 1998;95:14158–14163. doi: 10.1073/pnas.95.24.14158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Levine J., Willard M. Fodrin: axonally transported polypeptides associated with the internal periphery of many cells. J. Cell. Biol. 1981;90:631–642. doi: 10.1083/jcb.90.3.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Burridge K., Kelly T., Mangeat P. Nonerythrocyte spectrins: actinmembrane attachment proteins occurring in many cell types. J. Cell. Biol. 1982;95:478–486. doi: 10.1083/jcb.95.2.478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Bachs O., Lanini L., Serratosa J., Coll M.J., Bastos R., Aligue R., Rius E., Carafoli E. Calmodulin-binding proteins in the nuclei of quiescent and proliferatively activated rat liver cells. J. Biol. Chem. 1990;265:18595–18600. [PubMed] [Google Scholar]
  • 31.Vendrell M., Aligue R., Bachs O., Seratosa J. Presence of calmodulin and calmodulin-binding proteins in the nuclei of brain cells. J. Neurochem. 1991;57:622–628. doi: 10.1111/j.1471-4159.1991.tb03793.x. [DOI] [PubMed] [Google Scholar]
  • 32.Sumandea C.A., Fung L.W.-M. Mutational Effects at the Tetramerization Site of Nonerythroid Alpha Spectrin. Mol. Brain Res. 2005;136:81–90. doi: 10.1016/j.molbrainres.2005.01.003. [DOI] [PubMed] [Google Scholar]
  • 33.Tse W.T., Tang J., Jin O., Korsgren C., John K.M., Kung A.L., Gwynn B., Peters L.L., Lux S.E. A new spectrin, beta IV, has a major truncated isoform that associates with promyelocytic leukemia protein nuclear bodies and the nuclear matrix. J. Biol. Chem. 2001;276:23974–23985. doi: 10.1074/jbc.M009307200. [DOI] [PubMed] [Google Scholar]
  • 34.Nagase T., Kikuno R., Hattori A., Kondo Y., Okumura K., Ohara O. Prediction of the coding sequences of unidentified human genes. XIX. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res. 2000;7:347–355. doi: 10.1093/dnares/7.6.347. [DOI] [PubMed] [Google Scholar]
  • 35.Colomer V., Engelender S., Sharp A.H., Duan K., Cooper J.K., Lanahan A., Lyford G., Worley P., Ross C.A. Huntingtin-associated protein 1 (HAP1) binds to a Trio-like polypeptide, with a rac1 guanine nucleotide exchange factor domain. Hum. Mol. Genet. 1997;6:1519–1525. doi: 10.1093/hmg/6.9.1519. [DOI] [PubMed] [Google Scholar]
  • 36.Freist W., Gauss D.H. Lysyl-tRNA synthetase. Biol. Chem. Hoppe. Seyler. 1995;376:451–472. doi: 10.1515/bchm3.1995.376.8.451. [DOI] [PubMed] [Google Scholar]
  • 37.Tolkunova E., Park H., Xia J., King M.P., Davidson E. The human lysyltRNA synthetase gene encodes both the cytoplasmic and mitochondrial enzymes by means of an unusual alternative splicing of the primary transcript. J. Biol. Chem. 2000;275:35063–35069. doi: 10.1074/jbc.M006265200. [DOI] [PubMed] [Google Scholar]
  • 38.Hisatake K., Hasegawa S., Takada R., Nakatani Y., Horikoshi M., Roeder R.G. The p250 subunit of native TATA box-binding factor TFIID is the cellcycle regulatory protein CCG1. Nature. 1993;362:179–181. doi: 10.1038/362179a0. [DOI] [PubMed] [Google Scholar]
  • 39.Jacobson R.H., Ladurner A.G., King D.S., Tjian R. Structure and function of a human TAFII250 double bromodomain module. Science. 2000;288:1422–1425. doi: 10.1126/science.288.5470.1422. [DOI] [PubMed] [Google Scholar]
  • 40.Maile T., Kwoczynski S., Katzenberger R.J., Wassarman D.A., Sauer F. TAF1 activates transcription by phosphorylation of serine 33 in histone H2B. Science. 2004;304:1010–1014. doi: 10.1126/science.1095001. [DOI] [PubMed] [Google Scholar]
  • 41.Tian Y., Breedveld G.J., Huang S., Oostra B.A., Heutink P., Lo W.H. Characterization of ZNF333, a novel double KRAB domain containing zinc finger gene on human chromosome 19p13.1. Biochim. Biophys. Acta. 2002;1577:121–125. doi: 10.1016/s0167-4781(02)00397-4. [DOI] [PubMed] [Google Scholar]
  • 42.Jing Z., Liu Y., Dong M., Hu S., Huang S. Identification of the DNA binding element of the human ZNF333 protein. J. Biochem. Mol. Biol. 2004;37:663–670. doi: 10.5483/bmbrep.2004.37.6.663. [DOI] [PubMed] [Google Scholar]
  • 43.Sastri M., Barraclough D.M., Carmichael P.T., Taylor S.S. A-kinaseinteracting protein localizes protein kinase A in the nucleus. Proc. Natl. Acad. Sci. U. S. A. 2005;102:349–354. doi: 10.1073/pnas.0408608102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Narayan V.A., Kriwacki R.W., Caradonna J.P. Structures of zinc finger domains from transcription factor Sp1. Insights into sequence-specific protein-DNA recognition. J Biol. Chem. 1997;272:7801–7809. doi: 10.1074/jbc.272.49.30619. [DOI] [PubMed] [Google Scholar]
  • 45.Yang M., Wu Z., Field S. Protein-protein interactions analyzed with the yeast two-hybrid system. Nucleic Acid Res. 1995;23:1152–1156. doi: 10.1093/nar/23.7.1152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Mehboob S., Jacob J., May M., Kotula L., Thiyagarajan P., Johnson M.E., Fung L.W.-M. Structural analysis of the alpha N-terminal region of erythroid and nonerythroid spectrins by small-angle X-ray scattering. Biochemistry. 2003;42:14702–14710. doi: 10.1021/bi0353833. [DOI] [PubMed] [Google Scholar]
  • 47.Adamson J.G., Zhou N.E., Hodges R.S. Structure, function and application of the coiled-coil protein folding motif. Curr. Opin. Biotechnol. 1993;4:428–437. doi: 10.1016/0958-1669(93)90008-K. [DOI] [PubMed] [Google Scholar]
  • 48.McMahon L.W., Walsh C.E., Lambert M.W. Human alpha spectrin II and the Fanconi anemia proteins FANCA and FANCC interact to form a nuclear complex. J. Biol. Chem. 1999;274:32904–32908. doi: 10.1074/jbc.274.46.32904. [DOI] [PubMed] [Google Scholar]
  • 49.Sridharan D., Brown M., Lambert W.C., McMahon L.W., Lambert M.W. Nonerythroid alphaII spectrin is required for recruitment of FANCA and XPF to nuclear foci induced by DNA interstrand cross-links. J. Cell. Sci. 2003;116:823–835. doi: 10.1242/jcs.00294. [DOI] [PubMed] [Google Scholar]
  • 50.Lallena M.J., Correas I. Transcription-dependent redistribution of nuclear protein 4.1 to SC35-enriched nuclear domains. J. Cell Sci. 1997;110:239–247. doi: 10.1242/jcs.110.2.239. [DOI] [PubMed] [Google Scholar]
  • 51.Lallena M.J., Martinez C., Valcarcel J., Correas I. Functional association of nuclear protein 4.1 with pre-mRNA splicing factors. J. Cell Sci. 1998;111:1963–1971. doi: 10.1242/jcs.111.14.1963. [DOI] [PubMed] [Google Scholar]
  • 52.Mattagajasingh S.N., Huang S.C., Hartenstein J.S., Snyder M., Marchesi V.T., Benz E.J. A nonerythroid isoform of protein 4.1R interacts with the nuclear mitotic apparatus (NuMA) protein. J. Cell Biol. 1999;5:29–43. doi: 10.1083/jcb.145.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Ye K., Compton D.A., Lai M.M., Walensky L.D., Snyder S.H. Protein 4.1N binding to nuclear mitotic apparatus protein in PC12 cells mediates the antiproliferative actions of nerve growth factor. J. Neurosci. 1999;19:10747–10756. doi: 10.1523/JNEUROSCI.19-24-10747.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Carmo-Fonseca M. The contribution of nuclear compartmentalization to gene regulation. Cell. 2002;108:513–521. doi: 10.1016/S0092-8674(02)00650-5. [DOI] [PubMed] [Google Scholar]
  • 55.Chubb J.R., Bickmore W.A. Considering nuclear compartmentalization in the light of nuclear dynamics. Cell. 2003;112:403–406. doi: 10.1016/S0092-8674(03)00078-3. [DOI] [PubMed] [Google Scholar]
  • 56.Palstra R.J., Tolhuis B., Splinter E., Nijmeijer R., Grosveld F., de Laat W. The beta-globin nuclear compartment in development and erythroid differentiation. Nat. Genet. 2003;35:190–194. doi: 10.1038/ng1244. [DOI] [PubMed] [Google Scholar]

Articles from Cellular & Molecular Biology Letters are provided here courtesy of BMC

RESOURCES