Isolation and Characterization of a cDNA Encoding Chick a-Actinin

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Sep 12, 1986 - Michael D. Baron, Matthew D. Davison, Peter Jones, Bipin Patel, and David R. CritchleyS. From the Department of Biochemistry, University of ...
THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Vol. 262, No.6, Issue of February 25, pp. 2558-2561, 1987 Printed in U.S.A.

Isolation and Characterizationof a cDNA Encoding a Chick a-Actinin* (Received for publication, September 12,1986)

Michael D.Baron, Matthew D. Davison, Peter Jones, Bipin Patel, and David R. CritchleyS From the Department of Biochemistry, University of Leicester, University Road, Leicester LEI 7RH, Great Britain

W e have isolated and sequenced a 2.1-kilobase cDNA encoding 86%of the sequence of a-actinin. The cDNA clone was isolated from a chick embryo fibroblast cDNA library constructed in the expression vector Xgtll. Identification of this sequence as a-actinin was confirmed by immunological methods and by comparing the deduced protein sequence with the sequence of several CNBr fragments obtained from adult chicken smooth muscle (gizzard) a-actinin. The deduced protein sequence shows two distinct domains, one of which consists of four repeats of approximately 120 amino acids. This region corresponds to a previously identified 50-kDa tryptic peptide involved in formation of the a-actinin dimer. The last 19 residues of C-terminal sequence display an homology with the so-called E-F hand of Ca2*-bindingproteins. Hybridization analysis reveals only one size of mRNA (approximately 3.5 kilobases) in fibroblasts, but multiple bands in genomic cDNA.

different, peptide maps, suggesting that they are the product of more than one gene. In addition, muscle and non-muscle a-actinins differ in that most of the latter are Ca2+-sensitive for actin binding (8, 9, 16). As yet, however, there is no information as to the actual sequence differences between different isoforms or the degree of genetic complexity of aactinin. We report here the isolation and characterization of a 2.1-kilobase a-actinin cDNA. Analysis of the deduced protein sequence has revealed an internal repeat structure in the domain thought to be involved in formation of the a-actinin dimer. Hybridization of this cDNA to genomicDNA has provided evidence consistent with the existence of more than one a-actinin gene.

* This work was supported by Medical Research Council and the Cancer Research Campaign Grants (toD. R. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. has been submitted The nucleotide sequence(s) reported in this paper to the GenBankTM/EMBLData Bank withaccessionnumber(s) J 02666. 4 To whom correspondence should be addressed.

Portions of this paper (including “Materials and Methods” and Figs. 1, 3, and 4) are presented in miniprint at the endof this paper. The abbreviations used are: SDS, sodium dodecyl sulfate; DTT, dithiothreitol. Miniprint is easily read with the aid of a magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-3165, cite the authors, and include a check or money order for $3.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

MATERIALS AND METHODS’ RESULTS AND DISCUSSION

The “small insert” Xgtll cDNA library was screened with affinity-purified anti-a-actinin, and two positive clones were plaque-purified. The fusion proteins encoded by these recombinants were expressed in Escherichia coli strain C600 and a-Actinin was first isolated as a minor component of rabbit analyzed by sodium dodecyl sulfate-polyacrylamide gel elecskeletal muscle (1).It has subsequently been found in cardiac trophoresis and Western blotting. The two fusion proteins and smooth muscle (2,3) and in a variety of non-muscle cells were of similar size, and both were labeled by anti-a-actinin (4-9). The native molecule consists of two subunits of ap- (Fig. 1).The antibody did not label any equivalent protein in proximately 97 kDa, and is thought to be a homodimer. C600 or in Agtll-infected C600 cells. However, since the aElectron microscopy reveals a molecule with a high axial actinin used to affinity purify the antibody was only 95% ratio, dimensions 3-4 nm by30-40 nm (lo), whereas CD pure, it was possible that some minor component of the spectra (11)predict a high a-helical content. It appears to be antiserum was binding to the fusion protein. This possibility an integral part of actin-containing structures. It is found in was tested by eluting antibody from fusion protein that had the Z-disk in striated muscle (12) and in functionally similar been bound to nitrocellulose filters. This anti-fusion protein dense bodies and dense plaques in smooth muscle (13). In antibody could be used to label pure a-actinin (97-kDa band) non-muscle cells, a-actinin is distributed periodically along in Western blots (not shown), confirming that the fusion microfilament bundles and at their ends, where they appear protein and a-actinin sharedat least one common epitope. One cDNA clone (C5) was subcloned into M13mp18, and to be attached to the membrane in adherens-type junctions recombinants were selected with the insert in each orienta(adhesion plaques, macula adherens) (4, 14). Little is knownabout the function of a-actinin. Theprotein tion. This allowed the entire sequence of the 176-base pair binds to F-actin although probably only at the ends of fila- insert to be determined on both strands without further ments under in uiuo conditions of temperature and pH (9,15, subcloning. The deduced peptide sequence from C5 matched 16). Given its wide distribution in different cell types, it may or overlapped the sequences of three CNBr peptides obtained be involved in theanchorage of F-actin filaments to a variety from smooth muscle a-actinin (Fig. 2), thus confirming that of intracellular structures. Distinctisoforms of a-actinin have this cDNA had been derived from mRNA coding for an abeen isolated from different tissues and even from within the actinin. C5 was then used as a probe to isolate clones from the same tissue (8, 16-20). These different isoforms show small differences in molecular mass (3-5 kDa) andhave similar, but “large insert” cDNA library. Ten positive clones were purified,

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FIG. 2. The sequence of cDNA clones C5 and C18, the corresponding deduced protein sequence, and the sequence of a-actinin peptides. The numbering applies to clone C18. The sequence of C5 is also shown, with the two single-base differences underlined. The peptide sequence determined from purified a-actinin is shown by overlining the corresponding deduced sequence, a broken line indicating that thepeptide amino acid was not uniquely identified at thatpoint. Where peptide sequence and deduced sequence diverge, both sequences are given (boned).The startof the 50-kDa tryptic peptide is marked with a dot.

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Chick &-ActinincDNA Sequence

and the size of the inserts determined. The largest of these (ClS) was approximately 2.1 kilobases. This was also subcloned into M13mp18 and sequenced. The full sequence of this 2128-base pair cDNA and the translation into protein is shown in Fig. 2.99% of the sequence was determined on both strands. The sequence of C5 was entirely contained within this sequence, beginning at base 1649, although we did find two (silent) single-base differences between these two clones, the simplest explanation for which is allelic variation at this locus. The sequence of C18 was checked against the EMBL and GenBank DNA sequence data bases, but no significant homologies werefound. C18 contained a single open reading frame which extended the entire length of the sequence, encoding 708 amino acids. Although there is a methionine at amino acid 6, the sequence around it does not correspond to the consensus sequence for initiation of translation (40), so it is probable that we will need to obtain further sequence a t both 5’ and 3‘ ends to complete the sequence of the mRNA. The deduced protein sequence represents just over 86% of the sequence of the full a-actinin monomer. Of seven peptides sequenced, six completely matched the predicted sequence; the seventh, located at theC-terminalend of the predicted sequence, showed divergence over its last 5residues (Fig. 2). Given the different origins of the protein (smooth muscle) and cDNA (embryo fibroblasts) that we have sequenced, it is actually surprising that so few differences were observed.This suggests that there is strong conservation of protein structure in a-actinin, despite the different locations in which a-actinin is found in these two cell types. Interestingly, the last 19 C-terminal residues of the deduced amino acid sequence showclose homology with the consensus sequence for the “E-F hand” Ca2+-bindingsite (41). The divergent sequence found in the smooth muscle a-actinin disrupts thishomology. It is tempting to speculate that this may reflect the known difference in Ca2+sensitivity between muscle and non-muscle a-actinins. We are currently attempting to clone a-actinin cDNAs containing the sequence coding for the rest of this putative Ca2+binding site in order to clarify this point. The pTotein sequence deduced from C18 has a composition very similar to those previously published for a-actinin (17). Although, like several other cytoskeletal proteins (e.g. myosin, tropomyosin), a-actinin is a dimer and an extended molecule with a high axial ratio, we could find no trace of the heptad repeating unit common to these proteins which has been interpreted (42) in terms of an a-helical coiled coil. However, when the protein sequence of a-actinin was compared with itself, using the program DIAGON (37), two distinct domains were found (Fig. 3). The more C-terminal of these, comprising roughly residues 210-690, contains four repeats of approximately 120 amino acids. The natureof this repeat remains to be determined, but it is interesting to note that thebeginning of the repeats coincides with the beginning of a 50-kDa tryptic peptide of a-actinin which we have isolated, and for which we have determined the N-terminal sequence (Fig. 2). It has been shown (43) that this50-kDa tryptic peptide is assembled into dimers, whereas a 32-kDa N-terminal peptide, which binds to actin, is not. The four repeats we have seen here have a theoretical molecular mass of approximately 55 kDa, and it is possible, therefore, that this structure is involved in the formation of a-actinin dimers? Northern blot analysis of chick fibroblast mRNA revealed only a single band of approximately 3.5 kilobases (not shown), although this does not preclude the presence of more than

* Detailed analysis of the homologies of the repeat structure using sequence and structural criteria will be published separately.

one species of similar size. Southern blots of chick genomic DNA probed with either C18 DNA (2128 base pairs) (Fig. 4, lune a ) or C5DNA (178 pb) (data not shown) revealed a relatively complex picture. The apparent genetic complexity was also observed whenC18DNAwas hybridized to rat genomicDNA(Fig. 4, lune b). The marked difference in peptide maps of certain a-actinin isoforms (16-20) has provided evidence for the existence of more than one functional a-actinin gene, a conclusion with which these current observations are compatible. However, our results do not exclude the possible existence of one or more non-functional genes. The hybridization of C18 to rat genomic DNA under conditions of high stringency suggests that a-actininsequences are conserved at the DNA level across phylogenetic boundaries. Acknowledgments-We are grateful to Prof. W. J. Brammar, Dr. I. C. Eperon, and Dr. D. Pappin for much helpful advice and discussion and to James Turner for oligonucleotide synthesis. REFERENCES 1. Ebashi, S., and Ebashi, F. (1965) J. Biochern. (Tokyo) 58,7-12 2. Robson, R. M., and Zeece, M. G. (1973) Biochirn. Biophys. Acta 295,208-224 3. Singh, I., Goll, D. E., Robson, R. M., and Stromer, M. H. (1977) Biochim. Biophys. Acta4 9 1 , 29-45 4. Lazarides, E., and Burridge, K. (1975) Cell 6,,289-298 5. Jockush, B. M., Burger, M. M., Da Prada, M., Richards, J. G., Chaponnier, C., and Gabbiani, G. (1977) Nature 270,628-629 6. Craig, S.W., and Pardo, J. V. (1979) J. Cell Biol. 80, 203-210 7. Hoessli, D., Ruyger-Brandle, E., Jockush, B. M., and Gabbiani, G. (1980) J. Cell Bwl. 8 4 , 305-314 8. Burridge, K., and Feramisco, J. R. (1981) Nature 294, 565-567 9. Bennett, J. P., Zaner, K. S., and Stossel, T. P. (1984) Biochemistry 23,5081-5086 10. Podlubnaya, Z. A., Tskhovrebova, L. A., Zaalishvili, M. M., and Stefanenko, G. A. (1975) J. Mol. Biol. 92,357-359 11. Suzuki, A., Goll, D. E.,Singh, I., Allen, R. E., Robson, R. M., and Stromer, M. H. (1976) J. Bwl. Chem. 2 5 1 , 6860-6870 12. Masaki, T., Endo, M., and Ebashi, S. (1967) J. Biochem. (Tokyo) 62,630-632 13. Kay, F. S., Fujiwara, K., Rees, D. D., and Fogarty, K. E. (1982) J. Cell Biol. 9 6 , 783-795 14. Lazarides, E. (1976) J . Cell Biol. 68,202-219 15. Goll, D. E., Suzuki, A., Temple, J., and Holmes, G. R. (1972) J. Mol. Biol. 6 7 , 469-488 16. Landon, F., Gache, Y., Toniton, H., and Olomucki, A. (1985) Eur. J. Biochem. 1 5 3 , 231-237 17. Bretscher, A., Vandekerckhove, J., and Weber, K. (1979) Eur. J. Biochem. 100,237-243 18. Endo, T., and Masaki, T. (1982) J. Biochern. (Tokyo) 9 2 , 14571468 19. Kobayashi, R., Itoh, H., and Tashima, Y. (1983) Eur. J. Biochem. 133,607-611 20. Schachat, F. H., Canine, A. C., Briggs, M. M., and Fkedy, M. C. (1985) J. Cell Biol. 101,1001-1008 21. Tamkun, J. W., DeSimone, D. W., Fonda, D., Patel, R. S., Buck, C., Horwitz, A. F., and Hynes, R. 0.(1986) Cell 4 6 , 271-282 22. Huynh, T.V., Young, R. A., and Davis, R. W. (1985) in DNA Cloning, a Practical Approach (Glover, D. M., ed) Vol. 1, pp. 49-78, IRL Press Limited, Oxford 23. Kellie, S., Patel, B., Pierce, E. J., and Critchley, D. R. (1983) J. Cell Biol. 97,447-454 24. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,NY 25. Tautz, D., and Renz, M. (1983) Anal. Biochem. 132, 14-19 26. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 1 3 2 , 6-13 27. Feinberg, A. P., and Vogelstein, B. (1984) Anal. Biochem. 137, 266-267 28. Laemmli, U. K. (1970) Nature 227,680-685 29. Towbin, H., Staehelin, T.,and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,4350-4354 30. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Nutl. Acad. Sci. U. S. A. 74,5463-5467

cDNA a-Actinin Chick 31. Sanger, F., Coulson, A. R., Barell, B. G., Smith, A. J. A., and Roe, B. A. (1980) J. Mol. Biol. 143, 161-178 32. Brenner, D. G., and Shaw, W. V. (1985) EMBO J. 4,561-568 33.Feramisco, J. R., and Burridge,K. (1980) J. Biol. Chem. 2 5 5 , 1194-1199 34. Brett, M., and Findlay, J. B. C. (1983) Biochem. J. 211,661-670 35. Pappin, D. J. C., and Findlay, J. B. C. (1984) Biochem. J. 217, 605-613 36. Staden, R. (1982) Nucleic Acids Res. 10,4731-4751

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37. Staden, R. (1982) Nucleic Acids Res. 10,2951-2961 38. Staden, R. (1984) Nucleic Acids Res. 12,521-538 39. Bishop, M. J., and Thompson, E. (1984) Nucleic Acids Res. 12, 5471-5474 40. Kozak, M. (1984) Nucleic Acids Res. 12,857-872 41. Tufty, R. M., and Kretsinger, R. H.(1975) Science 185,167-169 42. McLachlan, A. D., and Karn, J. (1983) J. Mol. Biol. 164, 605626 43. Arakawa, N., Goll, D. E., Robson, R. M., and Kleese, W. C.(1985) J. Cell. Biochem. Suppl. 9 (Part B), 7

Lysates of C600 cultures ( b . 0 , or cultures that had been infected with wild type hgtll (d,h) or recanbinmt phages C1 (c,g) or CS @.e) were analyred m an SD5-7.51 polyacrylamide gel. and the pmteim transferred to nitmcellulose filren. Filters were stained for pmtein, and the p s i t l o n of the 6-galactosidase pmduced by hgtll (d) or the fmionpmteins pmduced the recmbinm~s(a&) were marked w l t h dats before destaining (see &th&). Filters were then pmbedwith affiniry-purlfied rabbit anrio-acrinin ( 2 u p m l )(a-d) or p r e - m e senm (l/lm)(e-h), andbomd antibody detected U S ~ Ea wmxidase system as described I" k h & .

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Figure 3.

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A canparison of the deduced o-actinin amino acid sequence (U8)with itself was generated using the pmgrm DIAm' (371, w i t h a span length of 29 and a cut-off score of 314. 'The axes are calxbrated in amino acid

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