(Received for publication, June 7, 1995; and in revised form, October 2, 1995)
From the
RalGDS is a GDP/GTP exchange protein for ral p24, a
member of small GTP-binding protein superfamily. We have recently shown
that RalGDS interacts directly with the GTP-bound active form of ras p21 through the effector loop of ras p21 in
vitro, in insect cells and in the yeast two-hybrid system. These
results suggest that RalGDS functions as an effector protein of ras p21. Here, we report that RalGDS interacts with ras p21
in mammalian cells in response to an extracellular signal. Epidermal
growth factor (EGF) induced the interaction of c-ras p21 and
RalGDS in COS cells expressing both proteins, but not in the cells
expressing RalGDS and c-ras p21, which is an
effector loop mutant of ras p21. We also found that cyclic
AMP-dependent protein kinase (protein kinase A) regulated the
selectivity of ras p21-binding to either RalGDS or Raf-1.
Protein kinase A phosphorylated RalGDS as well as (1-149)Raf
(amino acid residues 1-149). Although the phosphorylated
(1-149)Raf had a lower affinity for ras p21 than the
unphosphorylated (1-149)Raf, both the phosphorylated and
unphosphorylated RalGDS had the similar affinities for ras p21. The phosphorylation of RalGDS did not affect its activity to
stimulate the GDP/GTP exchange of ral p24. Pretreatment of COS
cells with forskolin further stimulated the interaction of ras p21 and RalGDS induced by EGF under the conditions that
EGF-dependent Raf-1 activity was inhibited. These results indicate that ras p21 distinguishes between RalGDS and Raf-1 by their
phosphorylation by protein kinase A.
ras p21 is a member of the small GTP-binding protein
superfamily and plays a pivotal role in cell growth and differentiation (1, 2) . ras p21 has GDP/GTP-binding and
GTPase activity and cycles between the GDP-bound inactive and GTP-bound
active forms. Recent studies have shown that growth factor receptors
that have tyrosine kinase activity regulate the GDP/GTP exchange
reaction and modulate the activity of ras p21(3, 4) . Growth factors such as EGF ()and platelet-derived growth factor induce
autophosphorylation of their receptors and create specific binding
sites for Src homology 2-containing proteins such as Grb2,
phospholipase C
, and the p85 subunit of PI
3-kinase(3, 5) . Grb2 is in a complex with Sos, a
GDP/GTP exchange protein for ras p21, in cytosol in the
absence of growth factors(6, 7, 8) . After a
growth factor induces the autophosphorylation of its receptor, Grb2-Sos
complex translocates from cytosol and associates with the receptor in
membranes, thereby placing it in the vicinity of ras p21. Sos
stimulates the conversion of the GDP-bound inactive form of ras p21 to the GTP-bound active form. The GTP-bound active form of ras p21 then transduces a signal(s) to downstream effector
protein(s).
One identified effector protein is Raf-1, a serine/threonine kinase (9, 10, 11, 12, 13, 14, 15, 16) . ras p21 interacts directly with Raf-1 and activates Raf-1, although the detailed mechanism of activation is not known. Then Raf-1 activates mitogen-activated protein kinase kinase, which in turn activates extracellular signal-regulated kinase, and Raf-1 exerts its function through this protein kinase cascade(9, 10, 17, 18, 19) . These data are consistent with previous observations that Raf-1 acts downstream of ras p21 in signaling pathways that mediate both the growth and differentiation responses to receptor tyrosine kinases(20, 21, 22) . However, it is possible that ras p21 has effector proteins other than Raf-1, since ras p21 exerts multiple functions(1, 2) . The first possible effector protein of ras p21 was GAP(23) . GAP interacts with only the GTP-bound form of ras p21 and fails to interact with the effector loop mutant of ras p21. Although it has been reported that GAP has an influence downstream of ras p21 in various signaling pathways(23, 24, 25) , it is not clear whether GAP is a real effector protein because GAP acts as a negative regulator of ras p21 by stimulating the GTPase activity of ras p21(23) . Another possible effector protein of ras p21 is PI 3-kinase(26) . PI 3-kinase consists of two subunits, p85 and p110. p110 associates with the GTP-bound form of ras p21. v-ras p21 elevates phosphorylated phosphoinositide levels, products of PI 3-kinase, in COS cells, and a dominant negative mutant of ras p21 inhibits nerve growth factor-induced phosphorylated phosphoinositide production in PC12 cells. However, we have reported that platelet-derived growth factor receptor mutant, which lacks the ability to bind to PI 3-kinase, is not able to stimulate GDP/GTP exchange of ras p21 in Chinese hamster ovary cells and epithelial murine mammary gland cells and that a constitutively active form of PI 3-kinase stimulates the GDP/GTP exchange of ras p21 in Xenopus oocytes (27, 28) . These results suggest that PI 3-kinase acts upstream of ras p21. Therefore, whether GAP and PI 3-kinase are effector proteins of ras p21 might be dependent on cell types.
We have recently shown that RalGDS is a potential effector protein of ras p21(29) . RalGDS has been originally isolated by polymerase chain reaction using regions conserved between CDC25 and ste6 proteins, two GDP/GTP exchange proteins known to regulate ras p21 in Saccharomyces cerevisiae and Saccharomyces pombe, respectively(30) . RalGDS is a 115-kDa protein that shares a high homology with the region of CDC25, which is important to stimulate the GDP/GTP exchange of ras p21. However, RalGDS does not affect the GDP/GTP exchange of ras p21. Among 13 different small G proteins, RalGDS stimulates the GDP/GTP exchange only of ralA p24 and ralB p24. ral p24 has been originally isolated by probing with an oligonucleotide corresponding to one of the GTP-binding domain of ras p21 (31) . Although the function of ral p24 has not yet been understood, RalGDS has been implicated in the regulation of the GTP state of ral p24(30) . We have found that RalGDS interacts with the GTP-bound active form of ras p21 but not with the GDP-bound inactive form, that the interaction of ras p21 and RalGDS requires the effector loop of ras p21, and that RalGDS inhibits the interaction of ras p21 with Raf-1 and GAP(29) . Thus, RalGDS fulfills the criteria expected of ras p21-effector protein interactions. Two other groups have reported similar results(32, 33) .
However, we have not yet demonstrated the interaction of ras p21 and RalGDS in intact mammalian cells in response to an extracellular signal. It has not been clarified how ras p21 distinguishes these possible effector proteins. Here we demonstrate that when COS cells are treated with EGF, RalGDS can be immunoprecipitated with ras p21. Furthermore, we show that protein kinase A regulates the selectivity of ras p21-binding to either RalGDS or Raf-1.
Figure 1:
Interaction of ras p21 and
RalGDS in COS-7 cells. A, coexpression of ras p21 and
RalGDS in COS cells. Aliquots (10 µl each) of lysates expressing no
protein (lane 1), RalGDS alone (lane 2), both
v-ras p21 and RalGDS (lane 3), or both ras p21 and RalGDS (lane 4) were probed with
the anti-HA and ras p21 antibodies. B, interaction of ras p21 and RalGDS in COS cells. COS cells expressing RalGDS
alone (lane 1), v-ras p21 alone (lane 2),
both v-ras p21 and RalGDS (lanes 3 and 5),
and both ras p21
and RalGDS (lane 4)
were lysed, and the proteins of the lysates were immunoprecipitated
with the anti-ras p21 antibody (Y13-238) (lanes
1-4) or nonimmune rat immunoglobulin (lane 5). The
precipitates were probed with the anti-HA and ras p21
antibodies. C, inability of Y13-259 to immunoprecipitate a ras p21-RalGDS complex. COS cells expressing both v-ras p21 and RalGDS were lysed, and the proteins of the lysates were
immunoprecipitated with Y13-238 (lane 1) or Y13-259 (lane
2). The precipitates were probed with the anti-HA and ras p21 antibodies. An arrowhead and an arrow indicate the positions of RalGDS and ras p21,
respectively. IP, immunoprecipitation; Ig,
immunoglobulin. The results shown are representative of three
independent experiments.
To characterize the
interaction of ras p21 and RalGDS further, we examined the
ability of RalGDS to interact with a ras p21 mutant, ras p21. ras p21
is well known
as a dominant negative mutant, which has a higher affinity for GDP than
GTP and strongly interacts with upstream molecules but not with
downstream molecules(1, 2, 39) . The
expression level of ras p21
was similar to that
of v-ras p21 (Fig. 1A, lanes 3 and 4). When the lysates coexpressing ras p21
and RalGDS were immunoprecipitated with the anti-ras p21
antibody, RalGDS was not coprecipitated with ras p21
under the same conditions that were used to coprecipitate
v-ras p21 and RalGDS (Fig. 1B, lanes 3 and 4). We used Y13-238 as the anti-ras p21
antibody to immunoprecipitate ras p21 for these experiments.
Another antibody, Y13-259, was tested for its ability to
immunoprecipitate a ras p21-RalGDS complex. Y13-259 is known
to be the neutralizing antibody(1, 40) . In contrast
to Y13-238, Y13-259 could not immunoprecipitate the ras p21-RalGDS complex from the lysate coexpressing v-ras p21
and RalGDS (Fig. 1C, lanes 1 and 2).
Y13-238 and Y13-259 immunoprecipitated the similar amounts of ras p21 from the lysates (Fig. 1C, lanes 1 and 2). These results indicate that RalGDS makes a
complex with v-ras p21 but not with ras p21
in COS cells, and the interaction of ras p21 and RalGDS
requires the effector loop of ras p21. These results in COS
cells are consistent with our previous observations in Sf9
cells(29) .
Figure 2:
Interaction of ras p21 and RalGDS
in COS-7 cells by stimulation with EGF. A, coexpression of
c-ras p21 or ras p21 and RalGDS in COS
cells. Aliquots (10 µl each) of the lysates expressing no protein (lane 1), both c-ras p21 and RalGDS (lane
2), or both ras p21
and RalGDS (lane
3) were probed with the anti-HA and ras p21 antibodies.
An arrowhead and an arrow indicate the positions of
RalGDS and ras p21, respectively. B, dose dependence.
COS cells expressing both c-ras p21 and RalGDS (
) or
both ras p21
and RalGDS (
) were stimulated
with the indicated concentrations of EGF for 10 min. After stimulation,
the cells were lysed, and the proteins of the lysates were
immunoprecipitated with the anti-ras p21 antibody. The
precipitates were probed with the anti-HA antibody. ECL system was used
for detection (left panel), and the developed bands were
quantified by personal densitometer (right panel). An arrowhead indicates the positions of RalGDS. Ig,
immunoglobulin. C, time course. COS cells expressing both
c-ras p21 and RalGDS (
) or both ras p21
and RalGDS (
) were stimulated with 100
ng/ml of EGF for the indicated time. After stimulation, the cells were
lysed, and the proteins of the lysates were immunoprecipitated with the
anti-ras p21 antibody. The precipitates were probed with the
anti-HA antibody. ECL system was used for detection and the developed
bands were quantified by personal densitometer. The results shown are
representative of three independent
experiments.
Figure 3:
Phosphorylation of RalGDS by protein
kinase A. A, protein staining of c-ras p21,
GST-(1-149)Raf, and RalGDS. The purified c-ras p21 (lane 1), GST-(1-149)Raf (lane 2), and RalGDS (lane 3) (0.5 µg of protein each) were subjected to
SDS-PAGE (12% polyacrylamide gel) and stained with Coomassie Brilliant
Blue. A big arrow, a small arrow, and an arrowhead indicate the positions of ras p21,
GST-(1-149)Raf, and RalGDS, respectively. B,
phosphorylation of c-ras p21, GST-(1-149)Raf, and RalGDS
by protein kinase A. c-ras p21 (lanes 1 and 2), GST-(1-149)Raf (lanes 3 and 4),
and RalGDS (lanes 5 and 6) (2 pmol each) were
incubated in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6)
of the catalytic subunit of protein kinase A for 20 min at 30 °C.
The reaction was stopped by the addition of Laemmli's buffer. The
samples were subjected to SDS-PAGE, followed by autoradiography. A big arrow, a small arrow, and an arrowhead indicate the positions of ras p21, GST-(1-149)Raf,
and RalGDS, respectively. C, effect of the phosphorylation of
GST-(1-149)Raf on its binding to ras p21. The
unphosphorylated () or phosphorylated (
) form of
GST-(1-149)Raf (20 pmol each) was incubated with the indicated
concentrations of the GTP
S-bound form of ras p21 for 30
min at 4 °C. The mixtures were immunoprecipitated with the
anti-ras p21 antibody, and the precipitates were probed with
the anti-GST antibody. The developed bands were quantified by personal
densitometer. D, effect of the phosphorylation of RalGDS on
its binding to ras p21. The unphosphorylated (
) or
phosphorylated (
) form of RalGDS (20 pmol each) was incubated
with the indicated concentrations of the GTP
S-bound form of ras p21 for 30 min at 4 °C. The mixtures were
immunoprecipitated with the anti-ras p21 antibody, and the
precipitates were probed with the anti-RalGDS antibody. The developed
bands were quantified by personal densitometer. The results shown are
representative of three independent
experiments.
Figure 4:
Effect of the phosphorylation of RalGDS on
its GDP/GTP exchange activity for ral p24. The
[H]GDP-bound form of ral p24 (5 pmol)
was incubated with the indicated concentrations of the unphosphorylated
(
) or phosphorylated (
) form of RalGDS for 10 min at 30
°C. The results shown are representative of three independent
experiments.
Figure 5:
Inhibitory action of Raf-1 for the
interaction of ras p21 and RalGDS. A, effect of the
phosphorylation of GST-(1-149)Raf on its binding to ras p21. The [-
P]GTP-bound form of ras p21 (2.5 pmol) was incubated with the indicated concentrations of
the unphosphorylated (
) or phosphorylated (
) form of
GST-(1-149)Raf for 30 min at 4 °C. The mixtures were
precipitated with glutathione-Sepharose 4B, and the radioactivities of
the precipitates were determined. B, effect of the
phosphorylation of GST-(1-149)Raf on its inhibitory action for
the interaction of ras p21 and RalGDS. The GTP
S-bound
form of ras p21 (10 pmol) was incubated with RalGDS (20 pmol
each) in the presence of the indicated concentrations of the
unphosphorylated (
) or phosphorylated (
) form of
GST-(1-149)Raf for 30 min at 4 °C. The mixtures were
immunoprecipitated with the anti-ras p21 antibody, and the
precipitates were probed with the anti-RalGDS antibody. The developed
bands were quantified by personal densitometer. The results shown are
representative of three independent
experiments.
Figure 6:
Effect of protein kinase A on the
interaction of ras p21 and RalGDS in COS cells. COS-7 cells
expressing both ras p21 and RalGDS were treated with the
indicated concentrations of forskolin for 15 min and then stimulated
with 100 ng/ml EGF for 10 min. The complex formation of ras p21 and RalGDS () was assayed as described in legend to Fig. 2. The amount of RalGDS bound to ras p21 was
expressed as percentage of that in the cells stimulated with EGF in the
absence of forskolin. Raf-1 activity (&cjs2113;) was measured using
GST-mitogen-activated protein kinase kinase and KNERK as substrates.
The results shown are representative of three independent
experiments.
Figure 7:
Stimulation of the interaction of ras p21 and RalGDS by protein kinase A in COS cells. COS-7 cells
expressing RalGDS alone were treated with () or without
(
) 50 µM forskolin for 15 min and then stimulated
with or without 100 ng/ml EGF for 10 min. The cells were lysed, and the
proteins of the lysates (2 ml, 1.6 mg of protein) were
immunoprecipitated with the anti-HA antibody. The precipitates were
probed with the anti-ras p21 antibody. The developed bands
were quantified by personal densitometer. The results shown are the
means ± S.D. of three independent experiments. *, p < 0.01.
We have demonstrated here that the interaction of ras p21 and RalGDS occurs in intact mammalian cells in response to an
extracellular signal. RalGDS makes a complex with v-ras p21
but not with ras p21 in COS cells. It is
believed that v-ras p21 is a GTP-bound form and that ras p21
is a GDP-bound form in intact
cells(1, 2, 39) . One ras p21
antibody, Y13-238, precipitates the ras p21-RalGDS complex,
but another antibody, Y13-259, does not. It is known that Y13-259 is a
neutralizing antibody and that this antibody does not recognize the ras p21-effector
complex(1, 2, 14, 29, 40) .
Furthermore, EGF induces the complex formation of RalGDS with c-ras p21, but not with ras p21
. ras p21
is an effector loop mutant of ras p21
and fails to interact with Raf-1 and
GAP(11, 12, 13, 14) . These
observations clearly show that RalGDS interacts with the GTP-bound form
of ras p21 through the effector loop of ras p21 by
stimulation with EGF in COS cells. Therefore, it is likely that RalGDS
is an effector protein of ras p21 in mammalian cells
Our results suggest that RalGDS provides a potential link between ras p21 and ral p24. The results showing that one small G protein act downstream of other small G proteins has been reported(46, 47) . Genetic analysis of yeast have demonstrated that cdc42sp, a member of small G protein of S. pombe, lies downstream of ras1 in S. pombe and that CDC42, a member of small G protein of S. cerevisiae, acts downstream of RSR1, another member of small G protein of S. cerevisiae(46) . It has been also shown that rac p21 is involved in the action of rho p21 to regulate the cytoskelton(47) . Although the function of ral p24 has not yet been understood, it is possible that ral p24 acts downstream of ras p21 and that ral p24 modulates some ras p21-dependent processes.
It has been reported that RalGDS is phosphorylated in COS cells and that phosphoserine, but not phosphotyrosine, is detected in the phosphorylated RalGDS(30) . Our results show that protein kinase A phosphorylates RalGDS. But, the phosphorylation of RalGDS affects neither its interaction with ras p21 nor its GDS activity for ral p24. The physiological significance of the phosphorylation of RalGDS by protein kinase A remains to be clarified. Among many small G proteins, rap1 is known to be phosphorylated by protein kinase A(45) . The GDP/GTP exchange reaction of rap1 is regulated by Smg GDP dissociation stimulator and the phosphorylation of rap1 enhanced the Smg GDP dissociation stimulator action(48) . Although we do not known whether ral p24 is phosphorylated by protein kinase A, ral p24 has consensus sequences of phosphorylation by protein kinase A. It is intriguing to speculate that the phosphorylation of ral p24 makes it sensitive to the action of RalGDS to stimulate the GDP/GTP exchange reaction.
Evidence has accumulated that there are several effector proteins of ras p21 (9, 11-16, 23, 26, 29, 31, 32). However, it has not yet been clarified how ras p21 distinguishes these effector proteins. Our results provide one possible model. It is known that Raf-1 is phosphorylated by protein kinase A and that phosphorylation of Raf-1 reduces its affinity for ras p21(35, 41, 42, 43) . The change of the characteristics of Raf-1 by phosphorylation could be one of the mechanisms by which protein kinase A inhibits ras p21-dependent Raf-1 activation. Our results show that the phosphorylation of RalGDS by protein kinase A does not affect its binding to ras p21 under the conditions that the phosphorylation of Raf-1 by protein kinase A inhibits its binding to ras p21 in vitro. Our results also show that when Raf-1 is phosphorylated by protein kinase A, the inhibitory action of Raf-1 for the interaction of RalGDS and ras p21 is attenuated in vitro. Furthermore, our results demonstrate that protein kinase A stimulates the interaction of ras p21 and RalGDS induced by EGF under the conditions that EGF-dependent Raf-1 activity is inhibited in COS cells. Taken together with the previous observations(35, 41, 42, 43) , these results indicate that protein kinase A inhibits the signal from ras p21 to Raf-1 but not to RalGDS. Therefore, it is likely that RalGDS and Raf-1 plays a role in cross-talk between the protein kinase A system and the tyrosine kinase-ras p21 system. Further studies are necessary to clarify the definitive function of RalGDS in signal transduction.