(Received for publication, August 18, 1995; and in revised form, November 2, 1995)
From the
Rad, a prototypic member of a subfamily of Ras-related GTPases,
is overexpressed in skeletal muscle of type II diabetic humans. By
expression screening of mouse embryo and human skeletal muscle cDNA
libraries, we found that Rad interacted with skeletal muscle
-tropomyosin. In the mouse skeletal muscle cell line
C
C
, this interaction was significantly
increased by the calcium ionophore A23187. A23187 also caused a time-
and concentration-dependent decrease in total cellular Rad with
increased interaction between tropomyosin and Rad in the
detergent-soluble fraction and the appearance of Rad in the
cytoskeleton. In C
C
cells stably
overexpressing a putative dominant negative mutant of Rad (S105N),
there was an increase in the amount of tropomyosin in Rad
immunoprecipitates. In cells overexpressing wild type Rad, much of Rad
was associated with the cytoskeleton and was no longer responsive to
A23187. In far-Western blotting and guanine nucleotide saturation
studies, GDP-Rad bound to tropomyosin far better than GTP-Rad. We
conclude that Rad interacts with skeletal muscle
-tropomyosin and
the cytoskeleton in a guanine nucleotide-dependent manner. These data
suggest that Rad may be involved in skeletal muscle motor function and
cytoskeletal organization.
Rad is the prototype of a growing subfamily of Ras-related
GTPases with distinct amino and carboxyl termini. It was initially
identified by subtractive cloning as a gene product overexpressed in
the skeletal muscle of humans with type II diabetes(1) . In
normal humans, it is highly expressed in skeletal muscle, lung, and
heart. It has a unique magnesium dependence with regard to guanine
nucleotide binding and is a target of a cellular serine/threonine
protein kinase(2) . Evidence also suggests the existence of
Rad-specific GTPase-activating protein (GAP). ()Another
Ras-related protein, Gem, shares significant sequence homology with Rad
and is a mitogen-induced immediate early gene product in T
lymphocytes(3) . Kir is probably an alternatively spliced form
of Gem, and its expression is inducible by oncogenic tyrosine kinases
in pre-B cells(4) .
Evidence suggests that many members of the Ras GTPase superfamily play very diversified roles within the cells. Some of them are involved in normal cell functions such as cell signaling(5) , growth and differentiation(6) , vesicular transport(7) , and nucleoprotein import (8) . Recently, it has also been suggested that some play a role in cytoskeleton arrangement(9) . For example, Rho has been shown to regulate the formation of actin stress fibers(10) . Very recently, a direct interaction between Rho family members (Rac1, RhoA, and CDC42) and a novel myosin, myr 5, has been described in vitro(11) . Although this has not yet been demonstrated in vivo, myr 5 appears to promote GTP hydrolysis by these Rho-related GTPases.
Since the cloning of rad, we have
begun a series of studies in an attempt to determine the role of Rad in
normal cell function. Rad is readily phosphorylated by the catalytic
subunit of protein kinase A at the carboxyl terminus(2) ,
enabling us to use phosphorylated Rad as a probe to screen expression
libraries for potential Rad-associated proteins, similar to the
approach used to identify the Ran-associated protein,
RanBP1(12) . By screening both mouse embryo and human skeletal
muscle expression libraries, we found that Rad interacted with skeletal
muscle -tropomyosin. This study reports the results of that
screening and demonstrates that this interaction occurs in intact
cells, is guanine nucleotide-dependent, and may be regulated by changes
in calcium flux.
The cDNA was cloned into the EXlox vector
(Novagen). The cDNA and EcoR I/HindIII directional
linkers were phosphorylated with T4 polynucleotide kinase. The linkers
were then ligated to the cDNA via blunt end ligation. The HindIII and EcoR I sites were then cleaved with their
respective restriction enzymes. The library was
phenol/chloroform-extracted and size-fractionated using Sephacryl
S-400, which isolates double-stranded DNA of
300 bp and larger.
After an ethanol precipitation, the cDNA was resuspended in 20 ml of
TE. An aliquot of 3 ml of the cDNA was ligated to 0.5 mg of
EXlox
vector arms, and the DNA was packaged into virus using Novagen's
packaging extract. This yielded a library with a titer of
2.0
10
plaque-forming units/ml.
The BOSC23
packaging cell line, a gift from D. Baltimore, was maintained in DMEM
containing 10% FBS at 37 °C in a humidified atmosphere of 10%
CO, as described previously (15) . Cells were
transfected by calcium phosphate co-precipitation with 10 µg each
of the plasmids. Transiently produced viral supernatants were used to
infect C
C
cells in the presence of 4 µg/ml
Polybrene. After elimination of uninfected cells by puromycin, stable
cell lines were maintained in DMEM containing 10% FBS. The expression
levels were determined by immunoblotting with an antibody (JD68)
generated against a fusion protein of GST with amino acid residues
40-308 of Rad (starting from the second in-frame ATG of rad). Expression of wild type Rad and mutant Rad were
30-40-fold and 20-30-fold, respectively, over that of
endogenous Rad.
Figure 1:
Rad recognizes
skeletal muscle -tropomyosin in the expression libraries. A, GST-Rad was phosphorylated by protein kinase A and used to
probe a 16-day mouse embryo cDNA library and a human skeletal muscle
cDNA library. Protein expression was induced by incubation of plates
with nitrocellulose filters impregnated in 10 mM
isopropyl-
-D-thiogalactoside. Filters were washed,
blocked, probed with [
P]Rad-GST at 4 °C
overnight, and washed again. Autoradiograms of the primary, secondary,
and tertiary filter lifts of one of the positive plaques in mouse
embryo library are shown. B, schematic representation of mouse
skeletal muscle
-tropomyosin cDNA sequences isolated from the
mouse embryo library.
Figure 2:
Tropomyosin co-immunoprecipitates with
Rad. CC
cells were grown to confluence. Cell
differentiation into myotubes was induced by culture in DMEM containing
low serum. Cells were serum-starved overnight and treated with
different agents, washed with cold PBS, and lysed in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM
dithiothreitol, 1 mM MgCl
, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 10
µg/ml leupeptin, 1% Nonidet P-40, and 0.1% SDS. After 1 h
solubilization at 4 °C, detergent-soluble and insoluble fractions
were separated by centrifugation in a microcentrifuge for 15 min. The
supernatant was divided and subjected to immunoprecipitation with
anti-Rad or anti-tropomyosin antibodies. Immunoprecipitates were washed
and further divided, resolved by 10% SDS-PAGE, and blotted with either
of the antibodies. A, cells were treated with the
Me
SO vehicle, 10% FBS plus Me
SO for 5 min, or
10 µM A23187 for 18 min at 37 °C. For direct blotting,
an aliquot of 45 µl of the detergent-soluble supernatant (out of 1
ml) was withdrawn and resolved by 10% SDS-PAGE for Western blotting. B, myoblasts were frozen in the lysis buffer at -70
°C just before confluence and processed together with myotubes when
the latter were available. Protein concentrations were normalized.
Cells were either treated with Me
SO or 10 µM A23187 for 18 min at 37 °C. Samples were processed as in A. C, cells were seeded on day 0 at a density of 6
10
/10-cm dish, allowed to grow, and taken daily to
prepare cell lysates as stated in Fig. 2. Protein concentrations
were normalized and directly subjected to Western blotting. The
positions of immunoglobulin (Ig), Rad, and tropomyosin are
indicated.
Expression of Rad and its association
with tropomyosin was also dependent on the state of differentiation of
the cells. Thus, Rad was virtually undetectable by immunoprecipitation
in CC
myoblasts in which tropomyosin was
already expressed in appreciable quantities (Fig. 2B).
Only in myotubes did association of tropomyosin with Rad occur, and
this interaction was increased by A23187 treatment. Direct
immunoblotting of C
C
cells at different stages
of differentiation showed no detectable Rad in myoblasts, but Rad
expression was elevated beginning on day 1 of differentiation (Fig. 2C). Therefore, it appeared that the expression
of Rad increased with the extent of cell differentiation and that
increased calcium influx caused a decrease in cellular Rad and an
increase in the association between tropomyosin and Rad.
Figure 3:
A23187 causes translocation of Rad to the
cytoskeleton, a decrease in total cellular Rad content, and an increase
in association of Rad with tropomyosin in a time- and dose-dependent
manner. CC
myotubes were treated with
Me
SO or 10 µM A23187 for indicated times (panel A) or 18 min with various A23187 concentrations (panel B) at 37 °C. Cells were lysed and samples were
subjected to immunoprecipitation or direct blotting as described in Fig. 2. The positions of immunoglobulin (Ig), Rad, and
tropomyosin are indicated.
Figure 4:
GDP-Rad associates with detergent-soluble
tropomyosin and GTP-Rad interacts with cytoskeleton. Wild type Rad and
the potential dominant negative mutant of Rad (S105N) were
overexpressed in CC
cells by retroviral
infection as described under ''Experimental Procedures.``
Myotubes were treated with Me
SO or 10 µM A23187 for 18 min, and samples were processed as described in Fig. 2.
Figure 5:
GDP-Rad binds tropomyosin more strongly
than GTP-Rad on far-Western blotting. Increasing amount of purified
rabbit skeletal muscle tropomyosin were resolved in 10% SDS-PAGE,
transferred to nitrocellulose filters, blocked, and probed with
phosphorylated Rad as described for library screening under
''Experimental Procedures`` except that the probe
([P]Rad, 1.8 µM) was equilibrated
with either 0.5 mM GDP or 0.5 mM GTP
S at room
temperature for 10 min before addition to the filters. After incubation
at 4 °C for 2 h, the filters were washed as described under
''Experimental Procedures`` and exposed to x-ray
films.
Co-immunoprecipitation of tropomyosin with Rad was also obtained
with human skeletal muscle (Fig. 6). Preincubation with GDP
caused an increase in co-immunoprecipitation of tropomyosin with Rad,
whereas preincubation with GTPS failed to do so. Therefore, it is
clear that in order for a strong interaction to occur between
tropomyosin and Rad, the GDP- rather than GTP-bound state of Rad is
preferred.
Figure 6:
Tropomyosin binds GDP-Rad, but not
GTP-Rad, in human skeletal muscle. Skeletal muscle from type I and type
II diabetic humans was homogenized with a Polytron homogenizer in 50
mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, 1 mM MgCl, and various
protease inhibitors. Nonidet P-40 and SDS were added to final
concentrations of 1% and 0.1%, respectively. After incubation at 4
°C for 1 h, samples were centrifuged in a microcentrifuge for 15
min. The supernatant (0.5 mg of protein) was incubated with 0.5 mM GDP or GTP at room temperature for 15 min. Antibodies to Rad and
tropomyosin were added for immunoprecipitation. Samples were processed
as described in Fig. 2.
Rad was the first member of a 35-39-kDa class of novel
Ras-related GTPases, which includes Gem, Kir, and possibly other
uncharacterized members. All of these GTPases possess highly conserved
NH-terminal extensions and COOH termini, which lack typical
prenylation sites. They contain unique sequence variations in the G1
and G3 domains as compared to Ras, and novel G2 (effector)
domains(2) . Each was also identified in a specific context;
Rad is increased in skeletal muscle of type II diabetes, whereas Gem
and Kir are increased in activated T-lymphocytes and pre-B cells,
respectively. The exact functions of these GTPases, however, remain
unknown.
In order to understand the function of Rad in cells, we
have searched for the proteins that interact with Rad by screening
expression libraries. Using this approach, we find that Rad directly
interacts with skeletal muscle -tropomyosin. This interaction
occurs using both intact cells and cDNA libraries from rodents and
humans. Ninety-six amino acid residues of the COOH terminus of skeletal
muscle
-tropomyosin are sufficient to mediate the interaction with
Rad. Tropomyosin is a classical molecule of
-helical coiled-coil
with seven so-called
-repeats throughout its
sequence(16) . The COOH terminus of tropomyosin is important in
mediating its binding to F-actin(17) .
In the original
report on the cloning of rad, we have shown that Rad is
overexpressed in the skeletal muscle of type II diabetic humans, and
that the highest expression in the normal humans is in skeletal muscle,
heart, and lung(1) . In rodents, heart and lung remain the
highest expressors of rad mRNA, while skeletal muscle has
somewhat lower level of expression. ()Thus, it is
conceivable that skeletal and cardiac muscle are two of the major
tissues in which Rad exerts its functions. Furthermore, although Rad is
below the detection level in C
C
myoblasts, its
expression dramatically increases as the cells differentiate to
myotubes, which suggests the regulation of Rad expression during
myogenesis. Since the duration and extent of C
C
cell differentiation are not altered by overexpression of either
wild type or a putative dominant negative mutant of Rad, it is likely
that Rad is not involved in the control of myogenesis, but rather that
its expression is controlled along with or by myogenesis. Likewise,
overexpression of Rad does not affect insulin- or serum-stimulated DNA
synthesis or MAP kinase pathway, although it does reduce
insulin-stimulated glucose uptake(18) .
In light of the recent observation that a novel member of the myosin family, myr 5, possesses a GAP activity toward the Rho subfamily of Ras-like small GTPases(11) , we have tested the effect of skeletal muscle tropomyosin on GTP binding to Rad, GDP dissociation from Rad, and the GTPase activity of Rad in vitro. No significant changes were observed; thus, under the conditions employed, skeletal muscle tropomyosin does not seem to have Rad GAP-like activity, nor does it act as a guanine nucleotide exchange factor for Rad (not shown). However, it is still possible that in vivo, additional molecules are needed to elaborate these specific effects.
The
interaction between Rad and tropomyosin is highly regulated. In the
basal state, a small fraction of Rad interacts with tropomyosin. This
interaction is unaffected by insulin or serum but is increased by an
elevated intracellular Ca concentration induced by
the calcium ionophore A23187. This is perhaps not surprising when we
consider that skeletal muscle tropomyosin is involved in
calcium-mediated muscle contraction. A23187 treatment also results in
translocation of Rad to a detergent-insoluble fraction. This fraction
is considered a cytoskeletal fraction(19) . It is not known by
which means and to which molecule Rad might attach; however, this
interaction is extremely tight since it can withstand repeated washes
in 0.5 M NaCl with 1% Nonidet P-40 and 0.1% SDS (data not
shown). In platelets, Rap2B is translocated to the detergent-insoluble
cytoskeleton upon thrombin stimulation, and it is therefore
hypothesized that Rap2B may be involved in platelet
activation(19) . Similarly, translocation of Rad to
cytoskeleton upon A23187 stimulation suggests a regulatory role for Rad
in muscle contraction and/or cytoskeleton arrangement.
The second
effect of A23187 treatment is a dramatic decrease in the cellular
content of Rad in a relatively short period of time (estimated t
10 min). This rapid decrease appears to
be due to a rapid protein degradation mediated by calcium-sensitive
proteases rather than relocation. The third effect of A23187 treatment
is an increase in the association of Rad with detergent-soluble
tropomyosin, despite the significant decrease in total Rad. The time
course and dose dependence of this association correlates with those of
Rad translocation and degradation.
In order to further understand
the association of Rad with tropomyosin, we expressed wild type and a
putative dominant negative mutant of Rad in CC
cells. This mutant Rad (S105N) is analogous to the dominant
negative mutant of Ras (S17N) and only binds GDP but not GTP.
Interestingly, a large amount of tropomyosin is co-immunoprecipitated
with Rad in cells overexpressing mutant Rad, whereas translocation of
Rad to the cytoskeleton upon A23187 stimulation remains the same as in
the parental cells. This strongly suggests that the detergent-soluble
tropomyosin is associated with Rad when the latter is in a GDP-bound
state. On the other hand, significant amounts of Rad are found in the
detergent-insoluble cytoskeleton in cells overexpressing wild type Rad,
and this association cannot be further stimulated with A23187. These
observations have two implications. First, A23187 increases the number
of Rad molecules in the GTP-bound state and translocation to
cytoskeleton occurs. Second, there are a limited number of binding
sites in the cytoskeleton for Rad such that, once they are occupied due
to overexpressed Rad, Ca
influx is no longer able to
stimulate further binding.
Further evidence for the dependence of Rad-tropomyosin interaction in the state of guanine nucleotide binding is provided by far-Western blotting and by immunoprecipitation of guanine nucleotide-saturated human skeletal muscle detergent extracts. It is clear that GDP-bound Rad associates with tropomyosin much more strongly than GTP-bound form. This difference is even more significant in human skeletal muscle when both Rad and tropomyosin are at their native conformations.
Thus, it is conceivable that skeletal muscle cells may have dual regulatory mechanisms with regard to Rad in response to calcium signal; a portion of Rad is translocated to cytoskeleton, where Rad may exert some as of yet unidentified function, e.g. involving the rearrangement of cell structure. Simultaneously, a proteolytic process is initiated, which may quickly reduce the effect of Rad in the cells. A well established mechanism by which activity of Ras-related small GTPases is terminated is their regulation by specific GAPs(20) . The latter promote hydrolysis of GTP to GDP by GTPases, thus ``switching-off'' the GTPases. Besides this classical mode of regulation, rapid degradation may also serve as a ``switching-off'' mechanism. The finding that GDP-Rad binds to detergent-soluble tropomyosin upon A23187 stimulation suggests that tropomyosin can serve to sequester and compartmentalize Rad in its inactive GDP-bound form, thus providing a third possible means of ``switching-off'' the protein. The latter is similar to the reversible interaction between c-Jun and tropomyosin, which acts to sequester c-Jun in the cytoplasm (21) , and to the Rab GDI that forms a complex with GDP-Rab, thus inhibiting the latter from binding to membranes(22) . Alternatively, binding of GDP-Rad to tropomyosin may somehow regulate the functions of tropomyosin. The effect of Rad at different guanine nucleotide binding states on tropomyosin function is currently under investigation.