Eur.J.Biochem.224,1-10(1994)
FEBS. 1994
Expression, cytoskeletal utilization and dimer formation
of tropomyosin derived from retroviral-mediated cDNA transfer
Metabolism of tropomyosin from transduced cDNA
Gaddamanugu L.PRASAD,Rebecca A.FULDNER,RichardBRAVERMAN,ElwoodMCDUFFIEand HerbertL.COOPER
Cell and Molecular Physiology Section, Laboratory of Tumor Immunology and Biology,National Cancer Institute, Bethesda,USA
(Received February 21,1994)-EJB 940250/1
Expression of the tropomyosin-1 isoform was enhanced by cDNA transfer in non-transformed
murine 3T3 fibroblasts and also in v-Ki-ras transformed fibroblasts in which native tropomyosin-1
expression had been reduced and tropomyosin-2 synthesis virtually eliminated by action of the
oncogene. The level of synthesis of insert-derived tropomyosin-1 was similar in normal and trans-
expression resulted in a considerable increase in tropomyosin-1 utilization in the cytoskeleton of
transformed cells, but this expression still did not reach normal levels, suggesting an oncogene-
lelated inhibition of tropomyosin utilization. A large proportion of newly synthesized native tropo-
myosin-1 in normal, unmodified fibroblasts appeared in homodimers which,upon prolonged incuba-
tion, were largely converted to the heterodimers. Excess tropomyosin-1 derived from the inserted
cDNA also appeared largely as the homodimer in both normal and transformed cells. This homodi-
merwas utilized effectively in the formation of cytoskeletal structures but was partially converted
to heterodimer by chain exchange. Under steady-state conditions, approximately 33% of the cy-
normal fibroblasts. The results show that the increased amounf of tropomyosin-1 homodimer enter-
ing the cytoskeleton under conditions of tropomyosin-1 excess,results in an atypical microfilament
composition.The effect of this excess of tropomyosin-1 homodimers on stability or function of
microfilament fibers remains to be determined.Theresults also confirm that themechanisms of
rapid homodimer formation with conversion to heterodimers by chain exchange,known from in
vitro studies,also occur in vivo.
ity, and interaction with extracellular supporting elements
The synthesis of several microfilament-associated pro-
(Pollard et al.,1976; Coté, 1983; Stossel et al.,1985).The
teins, including tropomyosin (Cooper et al., 1985), vinculin
terminology of tropomyosins used in this study is one in
(Raz and Geiger, 1982, Fernandez et ai., 1992), α-actinin
general use for tropomyosins expressed in fibroblasts (Lea-
(Gluck et al.,1993),and gelsolin (Vandekerckhove et al.,
vitt et al.,1986).Tropomyosins-1-3 correspond,in increas-
1990) have been reported to be suppressed in neoplastically
ing order of electrophoretic mobility, to the high-M, tropo-
transformed fibroblasts. Among these, tropomyosin has been
myosins (284 amino acids), which are homologous to tropo-
extensively studied.
myosins expressed in muscle celis, while tropomyosin-4 and
Tropomyosins are a family of ubiquitous actin-binding
tropomyosin-5 correspond to the low-M, tropomyosins (247
proteins with extensive a-helical structure that play a well-
or 248 amino acids) characteristic of non-muscle cells.The
defined role in muscle contraction (Leavis and Gergely,
reader is also referred to footnote 2 of Lehrer and Qian
1984; Payne and Rudnick, 1985; Lees-Miller and Helfman,
(1990) regarding the terminology of smooth muscle tropo-
1991).In non-muscle cells, the role of tuopomyosins is less
myosins.A number of studies have shown that down-regula-
clearly understood,and it is generally thought that they are
tion of expression of the high-M, tropomyosin isoforms con-
involved in stabilization of actin microfilaments which,in
sistently accompanies neoplastic transformation ofmurine
tum, play an important role in maintaining cell shape, motil-
and avian fibroblasts by a variety of retroviral oncogenes,
Correspondence to G.L.Prasad, Building 10,Room 8BO7,
chemical mutagens and transforming growth factors (Hen-
Laboratory of Tumor Immunology and biology, National Institutes
dricks and Weintraub,1981; Le0nardi et al.,1982; Matsu-
ofHealth,Bethesda,MD 20892,USA
mura et al., 1983; Hendricks and Weintraub, 1984; Cooper
Fax:+13014962756.
et al., 1985, 1987; Leavitt et al., 1986). These findings have
Abbreviations.Nbs,5,5'-dithiobis-2-nitrobenzoate;PhMeSOF,
led to the proposal that deficiency of tropomyosin expression
phenylmethylsulfonyl fluoride; TMe1, cDNA encoding human.
maybe a causal biochemical event in neoplastic transforma-
tropomyosin-1.
