Novel form of non-muscle tropomyosin in human fibroblasts

130

We have found that cytoskeletal extracts of cultured chicken embryo fibroblasts contain at least seven distinct polypeptides (two major and five minor) which cross-react with antiserum to chicken smooth muscle tropomyosin. These polypeptides range in apparent molecular weight from 31,000 to 47,000, and each is encoded by mRNAs which specifically hybridize to cloned muscle tropomyosin cDNAs. These nonmuscle tropomyosin species and their respective mRNAs are electrophoretically distinct from those of chicken skeletal muscle and appear by genomic DNA blotting to comprise a part of a multigene tropomyosin family. In Rous sarcoma virus-transformed chicken embryo fibroblasts, synthesis of the tropomyosins is differentially repressed such that the synthesis of the major species (cp35 and cp33, cytoskeletal proteins of molecular weight 35,000 and 33,000, respectively) and three minor species is drastically reduced, whereas the synthesis of two of the minor species (cp32 and cp3l) remains essentially unchanged. Analysis of cellular mRNA and runoff nuclear transcription experiments indicate that the repression of tropomyosin synthesis by Rous sarcoma virus transformation occurs at the level of transcription. This repression of tropomyosin synthesis is partially mimicked in normal chicken embryo fibroblasts during incubation in high-NaCI medium, a condition in which chicken embryo fibroblasts acquire many characteristics of transformed cells.Tropomyosin (TM) is a protein which has been best studied in skeletal muscle, where it is associated with actin in thin filaments and, together with the troponin complex, is responsible for mediating the effect of calcium on the contraction-generating interaction of actin and myosin (11,24). In nonmuscle cells (which do not contain troponin), TM is associated with actin in microfilaments (28,36,50) and, although the function of TM in these filaments is not known, there is evidence suggesting that one function resides in its ability to stabilize F-actin polymers (3,13). TMs from a variety of nonmuscle sources have been characterized (9,10,14,15,17), and a distinctive feature of these nonmuscle species has been their apparent molecular weight of 30,000, significantly less than that of skeletal muscle TM, which has a molecular weight range of 34,000 to 36,000. However, two forms of nonmuscle TM with significantly larger apparent molecular weights of 35,000 and 33,000 were identified in the cytoskeleton of cultured chicken embryo fibroblasts (25). More recently, multiple forms of TM having a broad range of molecular weights have been identified in the cytoskeleton of human fibroblasts (44) and in microfilaments of a variety of other cultured mammalian cells (35,36).It is commonly believed that microfilaments play a major role in cell motility and cell shape changes (7,18,27). A general feature of oncogenic transformation is the morphological alteration of the cell (for reviews, see references 23 and 45). Correlated with this change in cell morphology is a dramatic change in the organizatio...

Section: Identificationmentioning

confidence: 99%

mentioning

confidence: 99%

Multiple tropomyosin polypeptides in chicken embryo fibroblasts: differential repression of transcription by Rous sarcoma virus transformation.

Hendricks¹,

Weintraub²

1984

130

“…Multiple forms of tropomyosin have been detected in many cultures of nonmuscle cells (33,34,38,50,51,59,60,85,92). For example, rat embryonic fibroblasts contain three major tropomyosins termed 1, 2, and 4 (apparent molecular weights of 40,000, 36,500, and 32,400, respectively) and two relatively minor tropomyosins termed 3 and 5 (apparent molecular weights of 35,000 and 32,000, respectively) (59,60).…”

mentioning

confidence: 99%

“…For example, the molecular bases for the occurrence of the multiple forms of tropomyosin in fibroblasts are not fully understood. In vitro translation of mRNA isolated from fibroblasts indicates that each form is encoded by a separate mRNA rather than formed by posttranslational modifications of the proteins (39,50,59,92). The mRNA for each species of tropomyosin could be encoded by a separate gene.…”

mentioning

confidence: 99%

Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation.

Helfman¹,

Cheley²,

Kuismanen³

et al. 1986

175

122

The molecular basis for the expression of rat embryonic fibroblast tropomyosin 1 and skeletal muscle beta-tropomyosin was determined. cDNA clones encoding these tropomyosin isoforms exhibit complete identity except for two carboxy-proximal regions (amino acids 189 to 213 and 258 to 284) and different 3'-untranslated sequences. The isoform-specific regions delineate the troponin T-binding domains of skeletal muscle tropomyosin. Analysis of genomic clones indicates that there are two separate loci in the rat genome that contain sequences complementary to these mRNAs. One locus is a pseudogene. The other locus contains a single gene made up of 11 exons and spans approximately 10 kilobases. Sequences common to all mRNAs were found in exons 1 through 5 (amino acids 1 to 188) and exons 8 and 9 (amino acids 214 to 257). Exons 6 and 11 are specific for fibroblast mRNA (amino acids 189 to 213 and 258 to 284, respectively), while exons 7 and 10 are specific for skeletal muscle mRNA (amino acids 189 to 213 and 258 to 284, respectively). In addition, exons 10 and 11 each contain the entire 3'-untranslated sequences of the respective mRNAs including the polyadenylation site. Although the gene is also expressed in smooth muscle (stomach, uterus, and vas deferens), only the fibroblast-type splice products can be detected in these tissues. S1 and primer extension analyses indicate that all mRNAs expressed from this gene are transcribed from a single promoter. The promoter was found to contain G-C-rich sequences, a TATA-like sequence TTTTA, no identifiable CCAAT box, and two putative Sp1-binding sites.

“…The poly(A)-containing RNA was denatured with formaldehyde, sub-jected to electrophoresis in a 1.2% agarose gel and transferred to nitrocellulose by the procedure described previously (33). Probes were prepared by exonuclease III digestion, followed by "filling in" with DNA polymerase (Klenow fragment) in the presence of [32P]dCTP (Amersham, >3000 Ci/mmol) as described previously (34). Probes unique for the skaTM.…”

Section: Methodsmentioning

confidence: 99%

Human hTM alpha gene: expression in muscle and nonmuscle tissue.

MacLeod¹,

Gooding²

1988

We have isolated a cDNA clone from a human skeletal muscle library which contains the complete protein-coding sequence of a skeletal muscle a-tropomyosin. This cDNA sequence defines a fourth human tropomyosin gene, the hTMa gene, which is distinct from the hTMnm gene encoding a closely related isoform of skeletal muscle a-tropomyosin. In cultured human fibroblasts, the hTMa gene encodes both skeletal-muscleand smooth-muscle-type a-tropomyosins by using an alternative mRNA-splicing mechanism.