Rat embryonic fibroblast tropomyosin 1. cDNA and complete primary amino acid sequence

A cDNA expression library of approximately 80,000 members was prepared from rat embryonic fibroblast mRNA using the plasmid expression vectors pUC8 and pUC9. Using an immunological screening procedure and 32P-labeled cDNA probes, clones encoding rat embryonic fibroblast tropomyosin 1 (TM-1) were identified and isolated. DNA sequence analysis was carried out to determine the amino acid sequence of the protein. Rat embryonic fibroblast TM-1 was found to contain 284 amino acids and is most homologous to smooth muscle alpha-tropomyosin compared with skeletal muscle alpha- and beta-tropomyosins and platelet beta-tropomyosin. Among the various tropomyosins, two regions where the greatest sequence divergence is evident are between amino acids 185 and 216 and amino acids 258 and 284. Rat embryonic fibroblast TM-1 and chicken smooth muscle alpha-tropomyosin are most closely related from amino acids 185 and 216 compared with skeletal muscle and platelet tropomyosins. In contrast, rat embryonic fibroblast TM-1, smooth muscle alpha-tropomyosin, and platelet tropomyosin are most homologous from amino acids 258 and 284 compared with skeletal muscle tropomyosins. These differences in sequences at the carboxyl-terminal region of the various tropomyosins are discussed in relation to differences in their binding to skeletal muscle troponin and its T1 fragment.     

Molecular properties and functions in vitro of chicken smooth-muscle alpha-actinin in comparison with those of striated-muscle alpha-actinins

alpha-Actinin purified from chicken gizzard smooth muscle was characterized in comparison with alpha-actinins from chicken striated muscles, or fast-skeletal muscle, slow-skeletal muscle, and cardiac muscle. The gizzard alpha-actinin molecule consisted of two apparently identical subunits with a molecular weight of 100,000 on SDS-polyacrylamide gel electrophoresis, as do striated-muscle alpha-actinins. Its isoelectric points in the presence of urea were similar to the striated-muscle counterparts. Despite these similarities, distinctive amino acid sequences between smooth-muscle alpha-actinin and striated-muscle alpha-actinins were revealed by peptide mapping using limited proteolysis in SDS. Gizzard alpha-actinin was immunologically distinguished from striated-muscle alpha-actinins. Gizzard alpha-actinin formed bundles of gizzard F-actin as well as of skeletal-muscle F-actin, but could not form any cross-bridges between adjacent actin filaments under conditions where skeletal-muscle alpha-actinin could. Temperature-dependent competition between gizzard alpha-actinin and tropomyosin on binding to gizzard thin filaments was demonstrated by electron microscopic observations. Gizzard alpha-actinin promoted Mg2+-ATPase activity of reconstituted skeletal actomyosin, gizzard acto-skeletal myosin, and gizzard actomyosin. This promoting effect was depressed by the addition of gizzard tropomyosin. These findings imply that, despite structural differences between gizzard and striated-muscle alpha-actinin molecules, they function similarly in vitro, and that gizzard alpha-actinin can interact not only with smooth-muscle actin (gamma- and beta-actin) but also with skeletal-muscle actin (alpha-actin).     

Amino acid sequence of chicken gizzard gamma-tropomyosin

Chicken gizzard muscle tropomyosin has been fractionated into its two major components, beta and gamma and the amino acid sequence of the gamma component established by the isolation and sequence analysis of fragments derived from cyanogen bromide cleavage and tryptic digestions. Despite its much slower mobility on sodium dodecyl sulfate-polyacrylamide electrophoretic gels, it has the same polypeptide chain length (284 residues) as the alpha and beta components of rabbit skeletal muscle. Evidence for microheterogeneity of the chicken gizzard component was detected both on electrophoretic gels and in the sequence analysis. The gamma component is more closely related to rabbit skeletal alpha-tropomyosin than to the beta component. While the protein is highly homologous to the rabbit skeletal tropomyosins, significant sequence differences are observed in two regions; between residues 42-83 and 258-284. In the latter region (COOH-terminal) the alterations in sequence are very similar to those seen in platelet tropomyosin when compared with the skeletal proteins.     

Isolation and characterization of related cDNA clones encoding skeletal muscle beta-tropomyosin and a low-molecular-weight nonmuscle tropomyosin isoform.

We have isolated and characterized cDNA clones from chicken cDNA libraries derived from skeletal muscle, body wall, and cultured fibroblasts. A clone isolated from a skeletal muscle cDNA library contains the complete protein-coding sequence of the 284-amino-acid skeletal muscle beta-tropomyosin together with 72 bases of 5' untranslated sequence and nearly the entire 3' untranslated region (about 660 bases), lacking only the last 4 bases and the poly(A) tail. A second clone, isolated from the fibroblast cDNA library, contains the complete protein-coding sequence of a 248-amino-acid fibroblast tropomyosin together with 77 bases of 5' untranslated sequence and 235 bases of 3' untranslated sequence through the poly(A) tract. The derived amino acid sequence from this clone exhibits only 82% homology with rat fibroblast tropomyosin 4 and 80% homology with human fibroblast tropomyosin TM30nm, indicating that this clone encodes a third 248-amino-acid tropomyosin isoform class. The protein product of this mRNA is fibroblast tropomyosin 3b, one of two low-molecular-weight isoforms expressed in chicken fibroblast cultures. Comparing the sequences of the skeletal muscle and fibroblast cDNAs with a previously characterized clone which encodes the smooth muscle alpha-tropomyosin reveals two regions of absolute homology, suggesting that these three clones were derived from the same gene by alternative RNA splicing.     

Chemical and immunochemical characteristics of tropomyosins from striated and smooth muscle

1. On electrophoresis in dissociating conditions the tropomyosins isolated from skeletal muscles of mammalian, avian and amphibian species migrated as two components. These were comparable with the alpha and beta subunits of tropomyosin present in rabbit skeletal muscle. 2. The alpha and beta components of all skeletal-muscle tropomyosins contained 1 and 2 residues of cysteine per 34000g respectively. 3. The ratio of the amounts of alpha and beta subunit present in skeletal muscle tropomyosins was characteristic for the muscle type. Muscle consisting of slow red fibres contained a greater proportion of beta-tropomyosin than muscles consisting predominantly of white fast fibres. 4. Mammalian and avian cardiac muscle tropomyosins consisted of alpha-tropomyosin only. 5. Mammalian and avian smooth-muscle tropomyosins differed both chemically and immunologically from striated-muscle tropomyosins. 6. Antibody raised against rabbit skeletal alpha-tropomyosin was species non-specific, reacting with all other striated muscle alpha-tropomyosin subunits tested. 7. Antibody raised against rabbit skeletal beta-tropomyosin subunit was species-specific.     

Amino acid sequence of chicken gizzard beta-tropomyosin. Comparison of the chicken gizzard, rabbit skeletal, and equine platelet tropomyosins

Chicken gizzard beta-tropomyosin has the same chain length (284 residues) as other muscle tropomyosins, and is most closely related to the beta component of rabbit skeletal muscle. The majority of the amino acid substitutions are restricted to two regions of the structure, residues 185-216 and 258-284. The altered sequences at the COOH-terminal ends (residue 258-284) of the two gizzard components are very similar to each other and to those in platelet tropomyosin and can be correlated with the reduced affinity of interaction of all three tropomyosins with skeletal troponin T and its T1 fragment. The virtually identical NH2-terminal sequences of all four muscle tropomyosin chains indicates that the gizzard proteins' greater ability to polymerize head-to-tail is due to the sequence changes at its COOH terminus. On the other hand, the weaker head-to-tail aggregation of the platelet protein must be due to its NH2-terminal sequence alterations. Examination of the distribution of amino acids and the frequency of their substitution in the a to g positions of the repeating pseudoheptapeptide for all five tropomyosin sequences (four muscle and one platelet) emphasizes the importance of Glu residues at position e. Examination of those features of the muscle sequences implicated in the stabilization of their coiled-coil structures and in their interactions with F-actin suggest only marginal differences among them, with the possible exception of the chicken gizzard gamma component.     

In vitro phosphorylation of tropomyosin by a kinase from chicken embryo

A tropomyosin kinase has been partially purified from the leg muscle of 11-day-old chick embryos by ammonium sulfate precipitation and DEAE and phosphocellulose chromatography. The tropomyosin kinase requires Mg2+ for its activity, but Ca2+ and cyclic AMP are not needed. Increase in KC1 concentration decreased the tropomyosin kinase activity with over 90% inhibition at 0.2 M KC1. The alpha-tropomyosin subunit from rabbit and chicken skeletal muscle was phosphorylated about five times faster than the beta-tropomyosin subunit. Smooth muscle tropomyosin from chicken gizzard was not phosphorylated. The in vitro phosphorylation site in rabbit and chicken skeletal tropomyosins is a single serine residue close to the COOH terminus, a region intimately engaged in the head to tail polymerization of tropomyosin. Since the amino acid sequences of rabbit alpha- and beta-tropomyosin and chicken alpha-tropomyosin in this region are known, their phosphorylation sites can be unambiguously assigned as the penultimate residue, serine 283.     

The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin

Analysis of two recombinant variants of chicken striated muscle alpha-tropomyosin has shown that the structure of the amino terminus is crucial for most aspects of tropomyosin function: affinity to actin, promotion of binding to actin by troponin, and regulation of the actomyosin MgATPase. Initial characterization of variants expressed and isolated from Escherichia coli has been published (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Fusion tropomyosin contains 80 amino acids of a nonstructural influenza virus protein (NS1) on the amino terminus. Nonfusion tropomyosin is a variant because the amino-terminal methionine is not acetylated (unacetylated tropomyosin). The affinity of tropomyosin labeled at Cys190 with N-[14C]ethylmaleimide for actin was measured by cosedimentation in a Beckman Airfuge. Fusion tropomyosin binds to actin with an affinity slightly greater than that of chicken striated muscle alpha-tropomyosin (Kapp = 1-2 X 10(7) versus 0.5-1 X 10(7) M-1) and more strongly than unacetylated tropomyosin (Kapp = 3 X 10(5) M-1). Both variants bind cooperatively to actin. Troponin increases the affinity of unacetylated tropomyosin for actin (+Ca2+, Kapp = 6 X 10(6) M-1; +EGTA, Kapp = 2 X 10(7) M-1), but the affinity is still lower than that of muscle tropomyosin for actin in the presence of troponin (Kapp much greater than 10(8) M-1). Troponin has no effect on the affinity of fusion tropomyosin for actin indicating that binding of troponin T to the over-lap region of the adjacent tropomyosin, presumably sterically prevented by the fusion peptide in fusion tropomyosin, is required for troponin to promote the binding of tropomyosin to actin. The role of troponin T in regulation and the mechanisms of cooperative binding of tropomyosin to actin have been discussed in relation to this work.     

Phosphorylation of alpha alpha- and beta beta-tropomyosin and synthetic peptide analogues

A tropomyosin kinase partially purified from chicken embryos was used to study the phosphorylation mechanism of alpha alpha- and beta beta-tropomyosin and synthetic peptides containing the site of phosphorylation at Ser-283 and corresponding to residues 264-284 of the tropomyosin isoforms. The apparent Km is 47 microM for alpha alpha- and 265 microM for beta beta-tropomyosin, whereas the Vmax values are similar. The alpha [264-284] and beta [264-284] peptides have apparent Km values of 500 microM and 650 microM, respectively, and Vmax values similar to that of the intact tropomyosin. This indicates that the conformation of the phosphorylation site at the COOH-terminal end of tropomyosin contributes significantly to the phosphorylation of the substrate. Furthermore, the marginal difference in the Km values of the alpha- and beta-peptide cannot account for the 5-fold difference in the Km of the native alpha alpha and beta beta isoforms, suggesting that the conformations of alpha alpha- and beta beta-tropomyosin at the phosphorylation sites are significantly different. Phosphorylation of beta-peptide analogues, each with a single substitution corresponding to the alpha sequence, indicates that His-276 and Ile-284 have negative influences on the phosphorylation of the beta-peptide, whereas Met-281 improves it. Direct analyses of the time courses of phosphorylation of alpha alpha-tropomyosin at 37 degrees C, where head-to-tail polymerization is minimized, show that a single exponential can fit the data satisfactorily. This indicates a random phosphorylation of two identical chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chicken cardiac tropomyosin and a low-molecular-weight nonmuscle tropomyosin are related by alternative splicing

We have isolated and characterized complementary DNAs (cDNAs) encoding chicken cardiac muscle tropomyosin and a low-molecular-weight nonmuscle tropomyosin. The cardiac muscle cDNA (pCHT-4) encodes a 284-amino acid protein that differs from chicken skeletal muscle alpha- and beta-tropomyosins throughout its length. The nonmuscle cDNA (pFT-C) encodes a 248-amino acid protein that is most similar (93-94%) to the tropomyosin class including rat fibroblast TM-4, equine platelet tropomyosin, and human fibroblast TM30pl. The nucleotide sequences of the cardiac and nonmuscle cDNAs are identical from the position encoding cardiac amino acid 81 (nonmuscle amino acid 45) through cardiac amino acid 257 (nonmuscle amino acid 221). The sequences differ both 5' and 3' of this region of identity. These comparisons suggest that the chicken cardiac tropomyosin and low-molecular-weight "platelet-like" tropomyosin are derived from the same genomic locus by alternative splicing. S1 analysis suggests that this locus encodes at least one other tropomyosin isoform.     

Isolation and characterization of tropomyosin kinase from chicken embryo

Tropomyosin kinase is partially purified from 14-day-old chicken embryos using DEAE-cellulose, cellulose phosphate and gel filtration chromatography. The purest enzyme preparation consists of two major bands of Mr = 76,000 and 43,000 on SDS-polyacrylamide gel electrophoresis. The molecular weight of the enzyme is 250,000 determined by gel filtration chromatography. It phosphorylates casein and skeletal tropomyosin equally well but histone and phosvitin at a much slower rate. Smooth muscle myosin light chain, tropomyosin from platelet, erythrocyte and smooth muscle are not phosphorylated. The apparent Km for skeletal alpha-tropomyosin and ATP is 50 microM and 200 microM, respectively. Vmax varies between 100-300 nmol/min per mg depending on the purity of the preparation. Mg2+ and dithiothreitol are essential for activity but Ca+, calmodulin and cAMP are not required. The optimum temperature is 37 degrees C and optimum pH is about 7.5. Heparin, a potent inhibitor of casein kinase II, has no inhibitory effect on the enzyme. Similar tropomyosin kinase activity is not detected in skeletal muscle in adult rabbit and chicken. The tropomyosin kinase described here represents a hitherto uncharacterized kinase responsible for phosphorylation of tropomyosin in the chicken embryo.     

Four fibroblast tropomyosin isoforms are expressed from the rat alpha-tropomyosin gene via alternative RNA splicing and the use of two promoters

cDNA clones encoding four rat tropomyosin isoforms, termed TM-2, TM-3, TM-5a, and TM-5b, were isolated and characterized. All are derived from the alpha-tropomyosin gene via alternative RNA processing and the use of two alternate promoters. The cDNA sequences predict that TM-2 and TM-3 both contain 284 amino acids and differ from each other only at an internal region of the protein from amino acids 189 through 213, due to alternative splicing of exons 6a and 6b. TM-5a and TM-5b both contain 248 amino acids and differ from each other only at an internal exon encoding amino acids 153 through 177, also due to alternative splicing of exons 6a and 6b. The differences in the amino acid sequence encoded by these alternate exons affects the theoretical actin-binding pattern of the tropomyosins, such that TM-5b is expected to bind actin with greater affinity than TM-5a. TM-2 and TM-3 are transcribed from the upstream promoter, and TM-5a and TM-5b are transcribed from an internal promoter. In addition, all four isoforms contain the identical COOH-terminal coding region. RNA protection analyses revealed that the mRNA for each isoform is expressed in a number of different tissues and cell types, although the expression of some isoforms is restricted to particular cell types. Furthermore, the expression of mRNA encoding these isoforms was found to be altered in a number of different virally transformed cell lines. The changes in the expression of tropomyosin mRNAs in transformed cells reflect changes in the relative use of the two promoters, as well as the relative use of alternatively spliced exons 6a and 6b.     

Alpha-tropomyosin gene organization. Alternative splicing of duplicated isotype-specific exons accounts for the production of smooth and striated muscle isoforms

We have previously isolated and characterized cloned complementary DNAs (cDNAs) for striated and smooth muscle alpha-tropomyosin. The sequences of these cDNA clones suggested that these two isoforms were encoded by the same gene. Here, we have determined the complete structure of the alpha-tropomyosin (alpha-TM) gene, establishing that a single gene, with a sequence complexity of 28 kilobase pairs, is split into 12 exons and produces the smooth and striated muscle alpha-TM mRNA isoforms by alternative splicing of a minimum of five exchangeable isotype-specific exons. The elucidation of the intron/exon organization of alpha-TM suggests that this gene evolved from an ancestral gene encoding a 21-aa protein that might represent the primordial actin binding domain. Sequence comparison between the pairs of exons coding for the "isotype switch regions" and among the corresponding regions of tropomyosin genes in a variety of species ranging from insects to mammals, suggests that the alternatively spliced exons are very old and might have arisen before the radiation of the arthropods, more than 600 million years ago. Additionally, the examination of the intronic sequences has uncovered potential alternative intramolecular secondary structures (hairpin-loop structures) which might be involved in the tissue-specific expression of the duplicated and mutually exclusive alpha-TM isotype-specific exons.