(Received for publication, August 14, 1995; and in revised form, October 3, 1995)
From the
R-Ras, belonging to the Ras small GTP-binding protein
superfamily, has been implicated in regulation of various cell
functions such as gene expression, cell proliferation, and apoptotic
cell death. In the present study, we purified an R-Ras-interacting
protein with molecular mass of about 98 kDa (p98) from bovine brain
cytosol by glutathione S-transferase (GST)-R-Ras affinity
column chromatography. This protein bound to GTPS (guanosine
5`-(3-O-thio)triphosphate, a nonhydrolyzable GTP
analog)
R-Ras but not to GDP
R-Ras, GTP
S
R-Ras with
a mutation in the effector domain (R-Ras
),
GTP
S
Ha-Ras, or GTP
S
RalA. We obtained a cDNA
encoding p98 on the basis of its partial amino acid sequences. The
predicted protein consists of 834 amino acids whose calculated mass,
95,384 Da, is close to the apparent molecular mass of p98. The amino
acid sequence shows a high degree of sequence similarity to the entire
sequence of Gap1
, one of the GTPase-activating proteins
(GAP) for Ha-Ras. A recombinant protein consisting of the GAP-related
domain of p98 fused to maltose-binding protein stimulated GTPase
activity of R-Ras, and showed a weak effect on that of Ha-Ras but not
that of Rap1 or Rho. These results clearly indicate that p98 is a novel
GAP for R-Ras. Thus, we designated this protein as R-Ras GAP.
Accumulating evidence indicates that Ras (Ha-Ras, Ki-Ras, N-Ras)
serve as downstream molecules for tyrosine kinase-type receptors such
as epidermal growth factor receptor, as well as Src family members (for
reviews, see (1) and (2) ). Ras appears to transmit
its signal to influence expression of genes that control cell cycle,
proliferation, and differentiation(1, 2) . Ras has a
GDP-bound inactive form and GTP-bound active form, the latter of which
recognizes target proteins including c-Raf-1. The GDP-bound form is
converted to the GTP-bound form by GDP/GTP exchange reaction, which is
regulated by GDP/GTP exchange proteins such as Smg GDS, mSos, and
mCdc25(3, 4, 5, 6) . The GTP-bound
form is converted to the GDP-bound form by intrinsic GTPase activity,
which is regulated by GTPase-activating proteins such as p120 GAP, ()NF1, and
Gap1
(7, 8, 9) .
R-Ras was
originally identified as the gene product of a Ras homologue (10) . R-Ras has been reported to physically associate with
Bcl-2, which is known to be a blocker of apoptotic cell
death(11, 12) . Recently, the activated R-Ras that presumably remains mostly in the GTP-bound form due to
impaired GTPase activity has been reported to enhance the apoptotic
cell death in cytokine-deprived 32D.3 cells and serum-deprived NIH/3T3
cells(13) . Bcl-2 abrogates most of the effects of
R-Ras
, indicating that R-Ras promotes apoptosis caused by
growth factor deprivation via a Bcl-2- suppressible mechanism. In
NIH/3T3 cells, R-Ras
confers the ability to proliferate
under low serum conditions, to form colonies in soft agar, and to form
tumors in nude mice, although its ability is weaker than that of the
activated Ha-Ras(14) . R-Ras as well as Ha-Ras have been shown
to interact with c-Raf-1, p120 GAP, and NF1, to induce MAP kinase
activation, and to stimulate Ras response elements in certain
cells(15, 16) . On the other hand, R-Ras does not
induce maturation of Xenopus oocytes or differentiation of
PC12 cells. Taken together, although Ras and R-Ras share some
biochemical and cellular functions, these proteins seem to play
different biological roles.
To understand the specific functions of R-Ras, we attempted to identify proteins that specifically interact with R-Ras in the present study, and have purified an R-Ras-interacting protein with molecular mass of about 98 kDa by GST-R-Ras affinity column chromatography, cloned its cDNA, determined its primary structure, and identified it as a novel R-Ras GAP.
Figure 2: Deduced amino acid sequence of p98. Amino acid sequences determined from native p98 are indicated by single underlines. Amino acid sequences used for amplification of p98 cDNA are indicated by double underlines.
Figure 1:
Purification of
R-Ras-interacting molecule. A, detection of R-Ras-interacting
molecule by GST-R-Ras affinity column chromatography. Bovine brain
cytosolic fraction was loaded onto GST-small G protein affinity
columns. The proteins bound to the affinity columns were eluted by
addition of 200 mM NaCl. Aliquots (40 µl) of the second
fraction of the 200 mM NaCl eluates were subjected to SDS-PAGE
and silver-stained. Lane 1, GST; lane 2,
GDPGST-R-Ras; lane 3, GTP
S
GST-R-Ras; lane
4, GTP
S
GST-R-Ras
. B, specific
interaction of p98 with GTP
S
GST-R-Ras. Affinity column
chromatography using various small G proteins was carried out as
described in the legend for panel A. Lane 1, GST; lane 2, GDP
GST-R-Ras; lane 3,
GTP
S
GST-R-Ras; lane 4, GDP
GST-Ha-Ras; lane 5, GTP
S
GST-Ha-Ras; lane 6,
GDP
GST-RalA; lane 7, GTP
S
GST-RalA. An arrow denotes the position of p98. The results shown are
representative of three independent
experiments.
Figure 3:
Schematic representation of p98 and rat
Gap1. The numbers indicate the amino acid sequence
identities in each domain. C2, C2 domain; GRD,
GAP-related domain; PH, pleckstrin homology
domain.
Figure 4:
Interaction of in vitro translated R-Ras GAP with GTPS
GST-R-Ras. In vitro translated R-Ras GAP was mixed with GST-small G proteins
immobilized to glutathione-Sepharose 4B beads. The interacting proteins
were eluted with GST-small G proteins by addition of glutathione.
Aliquots (40 µl) were subjected to SDS-PAGE and vacuum-dried
followed by autoradiography. Lane 1, in vitro translated R-Ras GAP; lane 2, GST; lane 3,
GDP
GST-R-Ras; lane 4, GTP
S
GST-R-Ras; lane
5, GTP
S
GST-R-Ras
; lane 6,
GDP
GST-Ha-Ras; lane 7, GTP
S
GST-Ha-Ras; lane 8, GDP
GST-RalA; lane 9,
GTP
S
GST-RalA. An arrow denotes the position of
R-Ras GAP. The results shown are representative of three independent
experiments.
Figure 5:
Time course for the GAP activity of R-Ras
GAP. [-
P]GTP
GST-small G proteins (100
nM each) were incubated at 30 °C for the indicated periods
of time with respective GRDs (30 nM).
,
, with
[
-
P]GTP
GST-Ha-Ras;
,
,
with [
-
P]GTP
GST-R-Ras;
,
, with
[
-
P]GTP
GST-R-Ras
. A,
,
,
, in the absence of GST-NF1-GRD;
,
,
, in the presence of GST-NF1-GRD. B,
,
,
, in the absence of MBP-R-Ras GAP-GRD;
,
,
, in the presence of MBP-R-Ras GAP-GRD. The values shown
are means ± S.E. of three independent
experiments.
Figure 6:
Dose-dependent effect of R-Ras GAP on
GTPase activity. [-
P]GTP
GST-small G
proteins (100 nM each) were incubated for 3 min at 30 °C
with indicated amounts of respective GRDs.
, with
[
-
P]GTP
GST-Ha-Ras;
, with
[
-
P]GTP
GST-R-Ras. A,
GST-NF1-GRD. B, MBP-R-Ras GAP-GRD. The values shown are means
± S.E. of three independent
experiments.
Figure 7:
RT-PCR analysis of R-Ras GAP expression in
various rat tissues. Total RNAs (1 µg of each) were subjected to
RT-PCR using rat R-Ras GAP or -actin primers as indicated. The PCR
products were electrophoresed on a 6% polyacrylamide gel. Lane
1, cerebrum; lane 2, cerebellum; lane 3, heart; lane 4, skeletal muscle; lane 5, spleen; lane
6, thymus; lane 7, lung; lane 8, liver; lane
9, kidney; lane 10, pancreas; lane 11, small
intestine; lane 12, adrenal gland; lane 13, testis.
The results shown are representative of three independent
experiments.
In the present study, we purified an R-Ras-interacting
protein, p98, by GST-R-Ras affinity column chromatography. p98
interacts with GTPS
R-Ras but not with GDP
R-Ras,
GTP
S
R-Ras
, GTP
S
Ha-Ras, or
GTP
S
RalA. We determined partial amino acid sequences of
peptides derived from p98, cloned its cDNA, and determined its primary
structure. p98 shows a high degree of amino acid sequence similarity to
Gap1
, and recombinant GRD of p98 showed GAP activity toward
R-Ras higher than that toward Ha-Ras. Taken together, these results
clearly indicate that p98 serves as GAP for R-Ras. Since GAP specific
for R-Ras was identified here for the first time, we designated p98 as
R-Ras GAP.
Among the small G protein-interacting proteins, both target proteins and GAP appear to interact with small G proteins in a GTP-dependent fashion, and not to interact with their effector mutants. Since R-Ras GAP is the first molecule that specifically interacts with R-Ras in a GTP-dependent fashion, we speculate that R-Ras GAP may serve as a downstream target for R-Ras rather than GAP. However, this possibility seems unlikely, because genetic evidence indicates that Gap1, which shows a high degree of amino acid sequence similarity with R-Ras GAP, functions as a GAP rather than a downstream target for Ras in Drosophila(26) .
The GAP activity for R-Ras was first detected in human spleen(30) . This protein has been partially purified and shown to be the same as p120 Ras GAP. NF1 was also reported to exhibit GAP activity toward R-Ras(15) . We have shown here that R-Ras GAP exhibits higher GAP activity toward R-Ras than toward Ha-Ras. Although we cannot rule out the possibility that p120 Ras GAP and NF1 serve as GAP for R-Ras as well as for Ha-Ras, it is more likely that p120 Ras GAP and NF1 primarily serve as GAP for Ha-Ras, and that R-Ras GAP primarily serves as GAP for R-Ras in vivo. Further studies are necessary to estimate how much R-Ras GAP contributes to the regulation of R-Ras in vivo.
RT-PCR experiments indicate that R-Ras GAP is highly expressed in cerebrum and cerebellum, moderately in heart, spleen, thymus, lung, liver, kidney, and pancreas and hardly in skeletal muscle, small intestine, adrenal grand, and testis, suggesting that R-Ras GAP plays important roles in brain. On the other hand, R-Ras is expressed in most tissues including skeletal muscle, small intestine, adrenal grand, and testis (data not shown). From these observations, it is conceivable that isoforms or different types of R-Ras GAP are expressed in the tissues where R-Ras GAP is hardly expressed.
R-Ras GAP has unique structural features
such as C2 domains and PH domain which are also observed in Gap1 and
Gap1. This suggests that Gap1
and R-Ras GAP may
share some functions or be regulated in a similar way in vivo.
The C2 domain, which is observed in protein kinase C, synaptotagmin,
and Rabphilin-3A, is believed to be involved in the binding to
Ca
and
phospholipid(27, 31, 32) . It is possible
that R-Ras GAP is recruited to membranes via the C2 domains upon influx
of Ca
into cells. The PH domain is assumed to be
involved in the binding to phosphatidylinositol-4,5-bisphosphate or
subunits of trimeric G proteins(33, 34) .
It is speculated that R-Ras GAP associates with these molecules through
the PH domain in vivo. Further studies may provide insight
into roles of the C2 and PH domains of R-Ras GAP, leading to better
understanding of modes of action and activation of R-Ras GAP.
R-Ras
has been reported to interact with Bcl-2(12) . Furthermore, it
has been shown that R-Ras increases the rate of apoptotic
cell death in the setting of growth factor withdrawal, and that Bcl-2
completely abrogates this effect of R-Ras(13) . R-Ras
has been shown to interact with c-Raf-1, to activate MAP kinase
cascade, and to induce transformation of NIH/3T3
cells(14, 15, 16) . However, it is not yet
clear how R-Ras
accelerates apoptotic cell death in
growth factor-deprived cells and promotes transformation of some types
of cells such as NIH/3T3 cells, and how R-Ras is activated presumably
downstream of receptors for some extracellular signals. Several groups
have demonstrated that Ras is involved in regulation of a variety of
cell functions including cell transformation, proliferation, and
differentiation by utilizing p120 Ras GAP as a
probe(35, 36) . Overexpression of p120 Ras GAP
suppresses growth factor- and Ras-mediated responses leading to cell
transformation, proliferation, and differentiation. Similarly to p120
Ras GAP, R-Ras GAP will enable us to dissect how R-Ras regulates
various cell functions and how R-Ras is regulated during actions of
certain extracellular signals.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U30857[GenBank].