(Received for publication, December 11, 1995; and in revised form, January 24, 1996)
From the
We have previously reported that Ras protein is a potent
cysteine proteinase inhibitor. In order to examine whether the cysteine
proteinase-inhibitory activity of Ras is involved in carcinogenesis,
the effects of the following probes were investigated. Cystatin
is a cysteine proteinase-specific inhibitor and has some amino acid
sequence homology with Ras. Ras has a CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid) at the
carboxyl terminus, which is indispensable for the biological activity.
Thus, cystatin
carrying a CAAX motif (cystatin
-CVLS) was examined. A v-Ha-Ras deletion mutant,
Ras
42-49, has undetectable GTP binding activity, yet it
retains a similar protease inhibitory activity to that of wild-type
v-Ras. These genes were inserted into a eukaryotic inducible expression
vector and transfected into NIH3T3 cells. The expression was
effectively induced by treatment with a glucocorticoid hormone,
dexamethasone. The expression of cystatin
-CVLS or
Ras
42-49 alone induced neither transformation nor
morphological changes. However, when their expression was induced in
the presence of a tumor-promoting phorbol ester, a remarkable increase
in the anchorage-independent growth was observed in cystatin
-CVLS- and Ras
42-49-transfected clones. These results
suggest that cysteine proteinase inhibitors and a tumor promoter
synergistically transformed NIH3T3 cells. It is thus possible that the
cysteine proteinase-inhibitory activity of Ras might play a key role in
the early stage of carcinogenesis.
A number of studies have been made on the action mechanism of ras oncogene product (Ras), and multiple proteins have been proposed as the effectors of Ras protein. These are rasGAP, protein kinase C, c-Raf-1, ralGDS, and phosphatidylinositol 3-kinase(1, 2, 3, 4, 5) . Although Ras associates with these proteins in a GTP-dependent manner, the direct effect of Ras remains to be proved. It has been suggested that Ras does not directly activate the kinase activity of Raf but translocates it from cytosol to membrane (6) . We have previously reported that Ras is a potent protease inhibitor(7, 8, 9) . The inhibitory activity of Ras is specific for cysteine proteinases such as cathepsins B and L. Consistently, the amino acid sequence of Ras is partly homologous to those of the cysteine proteinase inhibitor family designated ``cystatin superfamily''(7, 10) . If the cysteine proteinase-inhibitory activity of Ras is involved in the transformation, cystatins might have a similar transforming activity. The present results suggest that inhibition of cysteine proteinases is sufficient for the conversion from a normal cell to an initiated one.
Binding between Ras proteins and GST-c-Raf was
investigated as described previously(13) . Purified GST or
GST-c-Raf protein (1 µg) was first incubated at 4 °C for 30 min
with glutathione-Sepharose in binding buffer (100 mM KCl, 6.33
mM MgCl, 20 mM Tris-HCl (pH 7.4), 1.0 mg
of bovine serum albumin/ml, 25 µM ZnCl
), and
the unbound proteins were removed by washing with binding buffer.
Guanine nucleotides prebound to Ras were exchanged by incubation of Ras
at 30 °C for 15 min in loading buffer (50 mM HEPES (pH
7.4), 5 mM EDTA, 1 mM dithiothreitol, 75 µg of
bovine serum albumin/ml, and 1 mM GDP or GMP-PNP. GST or
GST-c-Raf immobilized on glutathione-Sepharose was then incubated with
Ras (1 µg) at 4 °C for 30 min in binding buffer and washed 4
times with wash buffer (100 mM KCl, 6.33 mM MgCl
, 20 mM Tris-HCl (pH 7.4) and 0.25%
Triton X-100). The bound materials were analyzed by Western blot using
an anti-Ras monoclonal antibody, NCC-RAS-001(14) .
The cysteine proteinase-inhibitory activity of cystatin
was more potent than that of Ras(7, 19) . However,
cystatin
possesses neither guanine nucleotide binding activity
nor a CAAX motif. A CAAX motif at the
carboxyl-terminal end of Ras is necessary not only for
post-translational farnesylation but also for the biological
activity(20) . Thus the biological activity of cystatin
carrying the CAAX motif (cystatin
-CVLS) was also
investigated.
Other probes used were two deletion mutants of
v-Ha-Ras, Ras43-45 and Ras
42-49. The most
conserved amino acid sequence among the cystatin superfamily is
Gln-Val-Val(10) , which is also found in Ras at amino acid
positions between 43 and 45(7) . These Ras proteins were
bacterially expressed and purified. In spite of the deletion of the
conserved amino acid sequence, Ras
43-45 and
Ras
42-49 showed similar protease-inhibitory activities as
that of v-Ras(WT)(17) . For example, the K
values of v-Ras(WT), Ras
42-49, and Ras
43-45
were 48, 102, and 40 nM, respectively, toward bovine cathepsin
B and 14, 11, and 8 nM, respectively, toward papain. These
results are consistent with the previous report that point mutants in
the Gln-Val-Val region did not significantly affected the
protease-inhibitory activity of cystatin A (21) . Thus, the
Gln-Val-Val sequence might be important but not essential for the
interaction with cysteine proteinases.
Guanine nucleotide binding
activities of these two deletion mutants were also compared with that
of v-Ras(WT). Fig. 1shows the results of GTP binding examined
by the filter overlay assay using
[-
P]GTP(11) . v-Ras(WT) showed a
potent GTP binding activity while Ras
42-49 and
Ras
43-45 showed no detectable GTP binding activity. The GTP
binding activity was also examined by rapid filtration according to the
method of Gibbs et al.(22) , and the results showed
both Ras
42-49 and Ras
43-45 had less than 1%
guanine nucleotide binding activity of that of v-Ras(WT)(17) .
Figure 1:
GTP binding activity of v-Ras(WT),
Ras43-45, and Ras
42-49. GTP binding activity was
investigated by the filter overlay method. v-Ras(WT) (lane 1),
Ras
43-45 (lane 2), and Ras
42-49 (lane 3) were electrophoresed and blotted on a nitrocellulose
filter, which was then incubated with
[
-
P]GTP and washed extensively. The
autoradiogram is shown. An arrow indicates the position of
v-Ras(WT).
We have also investigated the in vitro association of Ras
and c-Raf-1. c-Ha-Ras bound to GST-c-Raf only in the presence of
GMP-PNP, a non-hydrolyzable GTP analogue (Fig. 2). No
significant binding was observed toward GST. Under the same conditions,
v-Ras(WT) bound to GST-c-Raf irrespective of the GDP- or GMP-PNP-bound
form. Guanine nucleotides sometimes do not affect the association
between Ras and c-Raf under certain experimental conditions as reported
previously(23, 24) . Ras42-49 showed
similar results as those of v-Ras(WT), suggesting that the region
between amino acid positions 42 and 49 is not necessarily required for
Raf binding. However, Ras
43-45 failed to bind to GST-c-Raf
probably because the deletion caused some additional alteration in the
three-dimensional conformation of Ras.
Figure 2:
Binding of Ras proteins and c-Raf-1. Ras
proteins, which had been loaded with GDP or GMP-PNP, were mixed with
GST-c-Raf or GST immobilized to glutathione-Sepharose. The bound
proteins were examined by immunoblot analysis using anti-Ras monoclonal
antibody. The lowest bands correspond to Ras monomers while the upper
bands represent Ras dimers(31) . The mobility of
Ras42-49 in the gel was higher than that theoretically
expected, and thus trimer-like complexes were also observed. The
mobility of Ras
43-49 was almost the same as that of
v-Ras(WT), yet there was no detectable
signal.
DNAs for cystatin ,
cystatin
-CVLS, v-Ras(WT), Ras
42-49, and
Ras
43-45 were inserted into a downstream region of mouse
mammary tumor virus-long terminal repeat of a eukaryotic expression
vector, pMSG, and transfected into NIH3T3 cells. Western blot analysis
demonstrated that the transfected genes were effectively induced after
treatment with Dex(9, 16, 17) . Induction of
cystatin
, cystatin
-CVLS, Ras
42-49, and
Ras
43-45 did not cause any significant morphological changes
whereas induction of v-Ras(WT) resulted in drastic morphological
changes as well as the appearance of dense transformed foci (not
shown).
Because anchorage-independent growth is one of the specific
phenotypes observed in malignant cells(18) , growth of
transfected clones in soft agar medium was investigated.
Vector-transfected clones, NV1, NV2, and NV3, as well as the parent
NIH3T3 cells did not grow in soft agar medium while
v-ras(WT)-transfected clones, NR24 and NR37, showed high
colony-forming efficiencies in the presence of Dex (Fig. 3).
These clones grew in soft agar medium even in the absence of Dex
probably due to the leaked expression of Ras in the absence of
Dex(9, 16) . On the other hand, induction of cystatin
, cystatin
-CVLS, Ras
42-49, and
Ras
43-45 by Dex did not result in any discernible change in
the colony-forming ability. This implies that induction of cystatin
, cystatin
-CVLS, Ras
42-49, or Ras
43-45
alone is insufficient to induce anchorage-independent transformation.
Figure 3:
Colony-forming efficiency of transfected
clones in the presence or absence of Dex and/or TPA.
Anchorage-independent growth in soft agar medium was investigated for
vector-transfected control clones (NV-1, -2, and -3), cystatin
-transfected clones (NY-9, -21, and -23), cystatin
-CVLS-transfected clones (NS-13 and -18),
v-ras(WT)-transfected clones (NR-24 and -37), ras
42-49-transfected clones (NII-2 and -3), and ras
43-45-transfected clones (NIII-6 and -7). Column
values and error bars represent the average percentage ± S.E.,
respectively, over three independent
experiments.
These proteins might play a role at a certain stage in multistage
carcinogenesis. Two-stage carcinogenesis, which consists of initiation
and promotion, was first proposed by Berenblum(25) . Initiated
cells produced by irradiation or treatment with chemical carcinogens
are easily transformed in the presence of tumor promoter phorbol ester,
TPA, and the transformation can be evaluated by focus-forming assay (26, 27) . Some of the chemical carcinogens that
induce initiation have been shown to induce specific mutations in ras protooncogenes, and thus the involvement of Ras in the
early stage of carcinogenesis was
suggested(28, 29, 30) . Consequently,
cystatin might have a role at the stage of initiation and
cooperate with a tumor promoter to induce full transformation. Thus,
the anchorage-independent growth in the presence of TPA was also
investigated (Fig. 3). The culture of cystatin
-CVLS- and
Ras
42-49-transfected clones in the presence of both Dex and
TPA resulted in a remarkable increase in colony-forming efficiency,
which was significantly higher than that in the absence of either TPA
or Dex (p < 0.01). The presence of TPA alone was not
sufficient to induce the anchorage-independent transformation in these
transfectants. Vector-transfected control clones and NIH3T3 cells
showed no significant increase in colony numbers. Induction of cystatin
showed the intermediate results and the colony-forming
efficiencies of NY clones in the presence of Dex and TPA were between
10 and 30% (0.01 < p < 0.1) versus that in the
absence of TPA. This suggests that the expression of cystatin
partly induces the changes corresponding to initiation.
The transformed colonies formed by NY, NS, and NII clones were similar to those formed by NR clones. However, the sizes of the former were smaller than those of the latter (Fig. 4), i.e. the diameters of most colonies of NY, NS, and NII clones were between 0.05 and 0.2 mm while those of NR clones were larger than 0.2 mm. The colony-forming efficiency of NR clones could be underestimated because some colonies were overlapped.
Figure 4:
Phase morphology of transformed colonies.
Colonies formed by NY-21 (A), NS-13 (B), NII-3 (C), and NR-37 (D) in soft agar in the presence of
both Dex and TPA are shown. Original magnification was 60. The bar in C represents 0.2
mm.
The results obtained in the present
study are summarized in Table 1. Although cystatin -CVLS
alone cannot induce full transformation, it induced
anchorage-independent transformation in the presence of a tumor
promoter. Ras
42-49 also induced anchorage-independent growth
at a high efficiency in the presence of TPA in spite of the drastic
decrease in the GTP binding activity. This mutant retains the cysteine
proteinase-inhibitory activity, which is similar to that of v-Ras(WT).
Consequently, suppression of cysteine proteinases may be sufficient for
the conversion of a normal cell into an initiated one. The specific
role of cysteine protease inhibitors at the stage of initiation is
consistent with the possible involvement of ras oncogenes in
the stage of initiation(28, 29, 30) .
Cystatin -CVLS also induced initiation without any GTP binding
activity. The expression of Ras
43-45 resulted in a slight
increase in the colony formation even in the presence of TPA. Since
this mutant cannot bind to c-Raf-1, association with Raf protein might
be necessary for the transformation. Cystatin
-CVLS induced
similar transformation without binding to c-Raf-1, possibly because the
protease-inhibitory activity of cystatin
is higher than that of
v-Ras(WT)(7, 19) .
Even if the suppression of
cysteine proteinases induces initiation, the present results cannot
identify the target protease of Ras and cystatin . Higher
colony-forming efficiencies of NS clones as compared with those of NY
clones suggest that the target protease is localized at a membrane
fraction. In order to identify the substrates of the target protease,
we have investigated the intracellular contents of Ras-binding proteins
in the transfected cells before or after treatment with Dex. They were
c-Raf-1, A-Raf, MEK, MEK2, protein kinase C, rasGAP, and
phosphatidylinositol 3-kinase p85. However, none of them increased
reproducibly after induction of Ras or cystatin
(data not shown).
It is thus unlikely that Ras and cystatin
protect these proteins
from degradation. Further study is necessary to determine the target
protease and its substrates, which are involved in the induction of
initiation.
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