Effect of Phosphorylation on Activities of Rap1A to Interact with
Raf-1 and to Suppress Ras-dependent Raf-1 Activation*
Chang-Deng
Hu,
Ken-ichi
Kariya,
Tomoyo
Okada,
Xiaodong
Qi,
Chunhua
Song, and
Tohru
Kataoka
From the Department of Physiology II, Kobe University School of
Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
 |
ABSTRACT |
Rap1A is phosphorylated by
cAMP-dependent protein kinase (PKA), and this
phosphorylation has been shown to modulate its interaction with other
proteins. However, it is not known whether Rap1A phosphorylation is
involved in regulation of its cellular functions, including suppression
of Ras-dependent Raf-1 activation. We have previously shown
that this suppressive activity of Rap1A is attributable to its greatly
enhanced ability to bind to the cysteine-rich region (CRR, residues
152-184) of Raf-1 compared with that of Ras. Here, we show that
phosphorylation of Rap1A by PKA abolished its binding activity to CRR.
Furthermore, a mutant Rap1A(S180E), whose sole PKA phosphorylation
residue, Ser-180, was substituted by an acidic residue, Glu, to mimic
its phosphorylated form, failed to suppress Ras-dependent
Raf-1 activation in COS-7 cells. These results indicate that the CRR
binding activity and the Ras-suppressive function of Rap1A can be
modulated through phosphorylation and suggest that Rap1A may function
as a PKA-dependent regulator of Raf-1 activation, not
merely as a suppressor.
 |
INTRODUCTION |
Rap1A belongs to the Ras family of small GTP-binding proteins.
Accumulated evidences indicate that Rap1A is phosphorylated at its
C-terminal Ser-180 both in vitro and in vivo by
PKA1 (for reviews, see Refs.
1 and 2). Phosphorylation of Rap1A induced by cAMP-elevating agents is
observed in a number of cell types including Rat-1 cells (3), HL-60
cells (4), PC12 cells (5), and neutrophils (6, 7). The observation in
the neutrophils that the association of Rap1A with cytochrome
b is dramatically reduced by phosphorylation (7) suggests a
regulatory role of Rap1A phosphorylation in its interaction with other
proteins. However, there has been no report demonstrating the effect of phosphorylation on cellular functions of Rap1A.
Rap1A has a very high homology to Ras and associates with almost all
cellular effectors of Ras including Raf-1 (8), B-Raf (5), and RalGDS
(9-11) in mammalian cells as well as Caenorhabditis elegans
PLC210 (12). Raf-1 is a serine/threonine protein kinase regulating the
mitogen-activated protein kinase cascade (for a review, see Ref. 13).
Ras activates Raf-1 by physically associating with it at the plasma
membrane. This physical association is mediated mainly by the
interaction between RBD (residues 51-131) of Raf-1 and the effector
region of Ras (residues 32-40 of Ha-Ras) (13). In addition to RBD, we
and others have recently found that CRR (residues 152-184) of Raf-1
also interacts with Ras and shown that this novel interaction is
essential for activation of Raf-1 (Refs. 14-19; for a review, see Ref.
20).
In contrast to Ras, Rap1A cannot activate Raf-1. Instead, Rap1A
inhibits Ras-dependent Raf-1 activation. Because Rap1A has the identical effector region to that of Ras and associates well with
RBD (8), we speculated that it would have a defect in association with
CRR. However, to our surprise, we found that Rap1A has a greatly
enhanced ability to associate with CRR compared with Ras (21). Further,
the enhanced CRR binding property of Rap1A resulted in formation of a
ternary complex with Ras and Raf-1 in which Rap1A and Ras independently
associate with CRR and RBD of Raf-1, respectively. Raf-1 in this
complex cannot be activated by Ras, presumably because interaction of
CRR with Ras is hampered by Rap1A tightly bound to CRR.
Association with CRR seems to involve the C-terminal region of Rap1A,
because it was shown that the association was dependent on the
C-terminal posttranslational modification of Rap1A (21). We therefore
reasoned that phosphorylation of Rap1A at its C-terminal Ser-180 might
affect its ability to associate with CRR. Here we report that the
phosphorylation specifically abolished the activity of Rap1A to bind to
CRR. Further, a Rap1A mutant mimicking the phosphorylated form was
found to have lost the activity to suppress Ras-dependent
Raf-1 activation.
 |
EXPERIMENTAL PROCEDURES |
Preparation of Various Recombinant
Proteins--
MBP-Raf-1(51-131), MBP-Raf-1(132-206), and
MBP-Raf-1(48-206) are MBP fusion proteins containing the indicated
residues of human Raf-1 described before (14). MBP-PLC210 is an MBP
fusion protein containing the Ras-associating domain (residues
1570-1670) of PLC210 (12). MBP-RalGDS is an MBP fusion protein
containing the Ras-interacting domain (residues 724-852) of rat
RalGDSb (9-11). The MBP fusion proteins were expressed in
Escherichia coli harboring pMal vectors carrying the
corresponding cDNA fragments. The E. coli cells were
lysed and centrifuged at 100,000 × g for 30 min. The
MBP fusion proteins in the resulting supernatant fractions were
immobilized on amylose resin and used for the in vitro
binding assays as described below. cDNAs encoding
Rap1AV12(S180E) and Rap1AV12(S180A), carrying a
substitution of Glu or Ala, respectively, for Ser at residue 180 in
addition to an activating mutation of Gly to Val at residue 12, were
prepared by polymerase chain reaction using oligonucleotide primers
carrying the corresponding mutations and the Rap1AV12
cDNA as a template (21, 22). Cloning of the mutant Rap1A cDNAs
into a baculovirus transfer vector and preparation of the recombinant
baculoviruses expressing them were carried out as described before
(23). Procedures for purification of the posttranslationally modified
forms of Ras and Rap1A from Sf9 cells infected with
baculoviruses expressing the respective proteins were described before
(21). Expression of a GST fusion Ha-Ras (GST-Ha-Ras) in Sf9
cells using a baculovirus vector was described before (21).
In Vitro Binding and Complex Formation Assays--
The in
vitro binding reactions were carried out by incubating 20-30 µl
of amylose resin carrying various immobilized MBP fusion proteins with
GTP
S- or GDP-loaded Rap1A in a total volume of 100 µl of buffer A
(20 mM Tris/HCl, pH 7.4, 40 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 5 mM
MgCl2, and 0.1% Lubrol PX) as described (14, 21). After
incubation at 4 °C for 2 h, the resin was washed, and the bound
proteins were eluted with buffer A containing 10 mM maltose
and subjected to SDS-PAGE followed by Western immunoblot detection with
anti-Rap1A polyclonal antibody (Santa Cruz Biotechnology Inc., Santa
Cruz, California). The ECL system (Amersham Pharmacia Biotech) was used
for signal development. For examination of the ternary complex
formation, GST-Ha-Ras in Sf9 cell lysate was first immobilized
on glutathione-Sepharose resin and then loaded with GTP
S. The resin
was subsequently incubated with GTP
S-loaded Rap1A in the presence of
MBP-Raf-1(48-206). The experimental condition was the same as
described above, except that the bound proteins were eluted with 10 mM glutathione in buffer A. The eluate was probed with
anti-Ras monoclonal antibody Y13-259 (Oncogene Science Inc.,
Manhasset, New York), anti-MBP polyclonal antibody, or the anti-Rap1A
antibody. For examination of the effect of phosphorylation of Rap1A,
5-10 pmol of Rap1A were incubated with 5-10 units of recombinant PKA
catalytic subunit (Promega, Madison, Wisconsin) at 25 °C for 30 min.
After termination of the phosphorylation reaction by the addition of
0.1-0.2 µg of a synthetic peptide (TTYADFIASGRTGRRNAIHD)
corresponding to the active site of the rabbit PKA inhibitor (24)
(Sigma), the phosphorylated Rap1A was used for the assays described above.
Assay of Raf-1 Kinase Activity in COS-7 Cells--
COS-7 cells
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum and antibiotics. For the activation of
Raf-1 by Ras or Rap1A, cells in 60-mm dishes (50% confluency) were
cotransfected with a combination of a Raf-1 expression vector pH8-Raf-1
(25) and either one of pcDNA3.1-Ha-RasV12,
pSR
-Rap1AV12, pSR
-Rap1AV12(S180E), or
pSR
-Rap1AV12(S180A) by using SuperFect Transfection
Reagent (Qiagen GmbH, Germany). For examination of suppression of the
Ras-dependent Raf-1 activation by Rap1A or its mutants,
cells were cotransfected with a combination of
pcDNA3.1-Ha-RasV12, pH8-Raf-1, and either one of
pSR
-Rap1AV12, pSR
-Rap1AV12(S180E), or
pSR
-Rap1AV12(S180A). Twenty-four h after transfection,
cells were transferred to serum-free media and further cultured for
18 h. The membrane extract in 0.4 ml of the lysis buffer (20 mM Tris/HCl, pH 7.5, 137 mM NaCl, 0.5% Nonidet
P-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 20 mM
-glycerophosphate, and 1 mM sodium vanadate) was prepared from each dish as
described (25). One-half of the total membrane extract was subjected to
immunoprecipitation with the anti-Raf-1 antibody and protein A-agarose.
The Raf-1 kinase activity was determined by incubating the
immunoprecipitates in the presence of GST-mitogen-activated protein
kinase kinase (0.3 µg) and GST-KNERK2 (2 µg) in 30 µl of the
kinase reaction mixture {20 mM Tris/HCl, pH 7.5, 10 mM MnCl2, 10 mM MgCl2,
20 mM
-glycerophosphate, and 50 µM
[
-32P]ATP (4,000 cpm/pmol)} for 20 min at 25 °C
(25). After the incubation, proteins in the reaction mixture were
fractionated by SDS-PAGE. Phosphorylated proteins were either
visualized by autoradiograph or quantitated by using BAS2000 bioimaging
analyzer (Fujix, Tokyo, Japan). In parallel, a 20-µl aliquot of the
membrane extract was used for examination of the amounts of the
expressed proteins by Western immunoblotting with the anti-Ras,
anti-Rap1A, and anti-Raf-1 antibodies. Signals from the corresponding
endogenous proteins were negligible (data not shown).
 |
RESULTS |
Phosphorylation Specifically Abolishes the Activity of Rap1A to
Associate with CRR--
In the previous studies (14, 21), we found
that both Ha-Ras and Rap1A in posttranslationally modified form bound
in vitro to MBP-Raf-1(132-206) containing CRR in a
GTP-independent manner. This was in contrast to their
GTP-dependent binding to MBP-Raf-1(51-131) containing RBD.
In the present study, we tested the effect of phosphorylation of Rap1A
on its binding activities toward RBD and CRR in the same in
vitro binding assay. First, we incubated Rap1A with a recombinant
catalytic subunit of PKA and confirmed its quantitative phosphorylation
by observing its mobility shift on SDS-PAGE as reported (4) (Fig.
1A). After inactivation of PKA
by the protein kinase inhibitor peptide, Rap1A was loaded with GTP
S
and incubated with MBP-Raf-1(132-206). As shown in Fig. 1B,
the phosphorylated Rap1A exhibited no detectable activity to bind to
CRR in a sharp contrast to the unphosphorylated Rap1A. The same result
was obtained with the phosphorylated Rap1A loaded with GDP (data not
shown). In contrast, when tested with MBP-Raf-1(51-131), the
unphosphorylated and the phosphorylated Rap1A did not exhibit any
significant difference in their RBD binding activities (Fig. 1C). GTP dependence of the RBD binding was unaffected also.
These results indicate that the phosphorylation of Rap1A specifically impaired the Rap1A-CRR association.

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Fig. 1.
Effect of phosphorylation of Rap1A on its
association with Raf-1, RalGDS, and PLC210. A, 1 pmol
each of unphosphorylated Rap1A (Rap1A) or phosphorylated
Rap1A (Rap1A-P) was detected by Western immunoblotting with
the anti-Rap1A antibody. B, 5 pmol of GTP S-loaded
unphosphorylated Rap1A (Rap1A) or phosphorylated Rap1A
(Rap1A-P) were incubated with 20 pmol of immobilized
MBP-Raf-1(132-206). The amount of Rap1A protein bound to
MBP-Raf-1(132-206) was measured by Western immunoblotting with the
anti-Rap1A antibody. C, binding assay was carried out as in
B using 5 pmol of GTP S-loaded (T) or
GDP-loaded (D) unphosphorylated Rap1A (Rap1A) or
phosphorylated Rap1A (Rap1A-P) and 20 pmol of
MBP-Raf-1(50-131). D, similar binding assay was carried out
as in C using 20 pmol of MBP-RalGDS. E, a similar
binding assay was carried out as in C using 20 pmol of
MBP-PLC210. F, 10 pmol of immobilized GST-Ha-Ras were loaded
with GTP S and incubated with 10 pmol of GTP S-loaded
unphosphorylated (lane 1) or phosphorylated (lane
2) Rap1A in the presence of 10 pmol of MBP-Raf-1(48-206).
GST-Ha-Ras and its associated proteins were resolved by SDS-PAGE and
subjected to Western immunoblotting. Upper panel, immunoblot
detection of GST-Ha-Ras with the anti-Ras antibody; middle
panel, detection of associated MBP-Raf-1(48-206) with the
anti-MBP antibody; lower panel, detection of associated
Rap1A with the anti-Rap1A antibody. Experiments shown were repeated
three times yielding similar results.
|
|
In addition to Raf-1, we examined the effect of Rap1A phosphorylation
on interaction with other Ras effectors, RalGDS (9-11) and PLC210
(12). As shown in Fig. 1, D and E, we observed
GTP-dependent binding of Rap1A to both the Ras-interacting
domain of rat RalGDSb and the Ras-associating domain of C. elegans PLC210, which were produced as MBP fusions. However, we
did not observe any difference of binding between the phosphorylated
and the unphosphorylated forms of Rap1A (Fig. 1, D and
E). These results further indicate that the effect of Rap1A
phosphorylation is specific to Raf-1 CRR.
Phosphorylated Rap1A Lacks Its Ability to Form a Ternary Complex
with Ras and Raf-1--
We next examined the effect of Rap1A
phosphorylation on its ability to form a ternary complex with Ras and
Raf-1. In this complex, Ras and Rap1A were independently associated
with RBD and CRR, respectively. When Rap1A in the unphosphorylated form was incubated with GST-Ha-Ras immobilized on glutathione-Sepharose resin in the presence of MBP-Raf-1(48-206) containing both RBD and
CRR, all three proteins were trapped on the resin and thereby co-eluted
with 10 mM glutathione (Fig. 1F, lane
1). In contrast, when the phosphorylated Rap1A was used in the
same reaction in place of the unphosphorylated form, no Rap1A was
detectable in the eluate even though a similar amount of
MBP-Raf-1(48-206) was co-eluted with GST-Ha-Ras (Fig. 1F,
lane 2). These results indicate that the phosphorylation of
Rap1A abrogates its ability to form a ternary complex with Ha-Ras and
Raf-1.
Phosphorylation at Ser-180 of Rap1A Is Responsible for the Effect
on CRR Binding--
Accumulated evidences indicate that Ser-180 is a
sole phosphorylation site of Rap1A by PKA examined both in
vivo and in vitro (1, 2). To prove that phosphorylation
at Ser-180 was responsible for the observed effect of PKA treatment of
Rap1A, we examined the effect of substitution of Ala for Ser-180.
Rap1AV12(S180A) exhibited an activity to associate with
Raf-1 CRR comparable with wild-type Rap1AV12 (Fig.
2A). In contrast to the case
with wild-type Rap1AV12, no visible mobility shift was
observed after treatment of this Rap1A mutant by PKA, suggesting that
it was not phosphorylated by PKA (Fig. 2B). Furthermore, the
PKA treatment of Rap1AV12(S180A) did not affect its
activity to bind to CRR (Fig. 2C). This result indicates
that Ser-180 is indeed the sole phosphorylation site responsible for
the loss of the CRR binding activity of Rap1A.

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Fig. 2.
Binding activity of Rap1A mutants
Rap1AV12(S180E) and Rap1AV12(S180A) to Raf-1
CRR. A, binding assay was carried out as in Fig.
1B using 5 pmol of GTP S-loaded (T) or
GDP-loaded (D) Rap1AV12(S180E),
Rap1AV12(S180A) or wild-type Rap1AV12
(WT) and 20 pmol of MBP-Raf-1(132-206). B, 1 pmol each of untreated Rap1AV12(S180E) and
Rap1AV12(S180A) or PKA-treated Rap1AV12(S180E)
and Rap1AV12(S180A) was detected by Western immunoblotting
with the anti-Rap1A antibody. C, binding assay was carried
out as in A with 5 pmol of the above untreated or
PKA-treated GTP S-loaded Rap1A mutants and 20 pmol of
MBP-Raf-1(132-206).
|
|
Rap1A(S180E) Mutant Mimics the Phosphorylated Rap1A and Fails to
Suppress Raf-1 Activation by Ras--
Because we previously found that
the tight association of Rap1A with CRR is responsible for the
Ras-suppressive activity of Rap1A, the above findings prompted us to
test the effect of Rap1A phosphorylation on its cellular function, in
particular suppression of Ras-dependent Raf-1 activation
(1, 2). For this purpose, we could not employ the conventional approach
of stimulating cells with cAMP-elevating agents or overexpressing PKA
catalytic subunit, because it has been shown that Raf-1 is
phosphorylated by PKA and thereby rendered insensitive to activation by
Ras (26, 27). To circumvent this problem, we turned to test the
feasibility of using a mutant Rap1AV12(S180E) in which the
phosphorylation residue Ser-180 was replaced by an acidic residue Glu.
This mutant is similar to the reported mutant Rap1A(S180D), which was
shown to mimic the phosphorylated Rap1A (5).
Rap1AV12(S180E) was expressed in Sf9 cells,
purified, and examined for its activity to associate with CRR. As shown
in Fig. 2A, the mutant exhibited no binding activity toward
CRR, which is similar to the case with the phosphorylated Rap1A. This
convinced us that Rap1AV12(S180E) can mimic the
phosphorylated Rap1AV12. As expected, PKA treatment of this
mutant did not alter its electrophoretic mobility (Fig. 2B)
or its CRR binding property (Fig. 2C), further supporting
that Ser-180 is the sole phosphorylation site.
Then, we first examined the activities of the Rap1A mutants to
stimulate the kinase activity of Raf-1 (Fig.
3A).
Rap1AV12(S180E) as well as Rap1AV12(S180A) were
found to be incapable of activating Raf-1 like wild-type Rap1AV12. This result further supports our hypothesis that
association of Ras with CRR at an appropriate strength is required for
Raf-1 activation. Abnormally enhanced or attenuated binding to CRR is detrimental to Raf-1 activation (14, 21). Next, we examined the
activities of these Rap1A mutants to antagonize the
Ras-dependent Raf-1 activation. When
Rap1AV12(S180E) was co-expressed in COS-7 cells with Raf-1
and Ha-RasV12, no significant suppression of the
Ras-dependent Raf-1 activation was observed, in sharp
contrast to nearly 70% suppression observed with wild-type
Rap1AV12 (Fig. 3B) even though they were
expressed in a similar amount (Fig. 3C). In contrast,
Rap1AV12(S180A) was as active as wild-type
Rap1AV12 in suppressing the Ras-dependent Raf-1
activation. Considering that Rap1AV12(S180E) had its CRR
binding activity greatly attenuated and that Rap1AV12(S180A) retained the greatly enhanced CRR binding
activity, these results are consistent with our hypothesis that the
tight association of Rap1A with CRR, which results in blockade of Ras
access to CRR, accounts for the Ras-suppressive activity of Rap1A (21). Further, the data obtained with Rap1AV12(S180E), taken
together with the loss of CRR binding activity of the phosphorylated
Rap1A, strongly suggest that the phosphorylation of Rap1A abrogates its
activity to suppresses the Ras-dependent Raf-1
activation.

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Fig. 3.
Activation of Raf-1 and inhibition of
Ras-dependent Raf-1 activation by Rap1A and its mutants in
COS-7 cells. A, COS-7 cells were transfected with the
combination of plasmids pH8-Raf-1 (Raf-1) and either
pcDNA 3.1-Ha-RasV12, pSR -Rap1AV12
(WT), pSR -Rap1AV12(S180A), or
pSR -Rap-1AV12(S180E) as indicated. Raf-1 was
immunoprecipitated from the membrane lysate by the anti-Raf-1
antibody and examined for its activity to induce phosphorylation of
GST-KNERK2 in the presence of GST-mitogen-activated protein kinase
kinase, as described under "Experimental Procedures." Shown are the
autoradiograms of the phosphorylated GST-KNERK2. B, similar
transfection and Raf-1 kinase assays were done as in
A, with the combination of plasmids pH8-Raf-1
(Raf-1), pcDNA3.1-Ha-RasV12 and either
pSR -Rap1AV12 (WT),
pSR -Rap1AV12(S180A) or
pSR -Rap-1AV12(S180E) as indicated. The intensity of
phosphorylation of GST-KNERK2 shown in the inset was quantified and
expressed as fold increase with respect to cells transfected with
pH8-Raf-1 only. C, the amounts of Raf-1,
Ha-RasV12, and Rap1AV12 proteins in the
membrane lysate were measured by Western immunoblotting with the
anti-Raf-1, the anti-Ras, and the anti-Rap1A antibodies, respectively.
The arrows indicate positions of Raf-1,
Ha-RasV12 or Rap1AV12 proteins on the blot.
Lane numbers correspond to those in B.
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|
 |
DISCUSSION |
Rap1A has been reported to associate with almost all Ras effectors
and other proteins in cells. Although it is assumed that phosphorylation of Rap1A may be a physiological event, regulating its
interaction with other proteins based on the observation that phosphorylated Rap1A does not associate with cytochrome b
(7), there has been no report that phosphorylation also affects the association of Rap1A with Ras effectors. In the present study, we have
shown that the phosphorylated Rap1A lacks its ability to bind to CRR of
Raf-1. This represents the second direct evidence that phosphorylation
of Rap1A affects its interaction with other proteins. Consistent with
our previous finding that CRR binding requires the C-terminal lipid
modification of Rap1A (21), this result provides further support to the
idea that the C-terminal region of Rap1A is involved in CRR binding.
Significantly, we have shown that the S180E mutant, mimicking the
phosphorylated Rap1A, can no longer suppress Ras-dependent
Raf-1 activation. This result not only agrees well with our hypothesis
that the enhanced CRR binding is involved in suppression of
Ras-dependent Raf-1 activation by Rap1A but also, to our
knowledge, represents the first clear demonstration that the cellular
function of Rap1A is controlled by phosphorylation.
The first indication of Ras-antagonizing activity of Rap1A came from
its ability to induce reversion of Ras-transformed fibroblasts (28).
Subsequently, this antagonizing activity of Rap1A was also observed in
nontransformed cells (1, 2). Although these studies employed expression
of a large amount of Rap1A, it has recently been shown that expression
of Rap1AV12 at levels similar to the endogenous wild-type
protein also efficiently inhibits activation of mitogen-activated
protein kinase by growth factors (29). Because this mitogen-activated
protein kinase activation was found dependent on Ras, the observation
suggested that a physiological amount of endogenous Rap1A may be
sufficient for its antagonistic effect on Ras during normal cell growth
(29). Based on the present results that the antagonizing activity of Rap1A can be controlled by phosphorylation, we propose further that
Rap1A may function as a PKA-dependent regulator of Raf-1 activation, not merely as a suppressor.
In addition to Rap1A, PKA phosphorylates many cellular proteins
including Raf-1 (26, 27). Phosphorylation of Raf-1 by PKA renders it
incapable of associating with Ras, thereby precluding its activation by
Ras. Taken together with our present finding, one can hypothesize that
PKA may exert both positive and negative regulatory effects on
Ras-dependent Raf-1 activation. Although these ambivalent
effects appear puzzling, they may be required for fine-tuning the
signal output from Raf-1 in different cell-type or developmental
contexts. It should also be pointed out that there exists a possibility
that a protein kinase other than PKA may specifically phosphorylate
Rap1A, but not Raf-1, under control of a certain extracellular signal.
Identification of such a protein kinase might provide a further insight
into the regulatory mechanism of Raf-1 activity by Rap1A.
 |
ACKNOWLEDGEMENTS |
We thank A. Kikuchi at Hiroshima University
School of Medicine for providing GST-mitogen-activated protein kinase
kinase, GST-KNERK, and rat RalGDSb cDNA and X.-H. Deng for her
skillful technical assistance. We also thank A. Seki and A. Kawabe for help in preparation of this manuscript.
 |
FOOTNOTES |
*
This investigation was supported by Grants-in-aid for
Scientific Research on Priority Areas, for Scientific Research (B) and for Encouragement of Young Scientists from the Ministry of Education, Science, Sports, and Culture of Japan and by grants from the Yamanouchi Foundation for Research on Metabolic Disease and from the Mochida Memorial Foundation for Medical and Pharmaceutical Research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-78-341-7451 (ext. 3230); Fax: 81-78-341-3837; E-mail: kataoka{at}kobe-u.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
PKA, protein kinase
A (cAMP-dependent protein kinase);
RalGDS, Ral guanine
nucleotide dissociation stimulator;
RBD, Ras binding domain;
CRR, cysteine-rich region;
MBP, maltose-binding protein;
GST, glutathione
S-transferase;
KNERK, a kinase negative mutant of
extracellular signal-regulated kinase 2;
PAGE, polyacrylamide gel
electrophoresis;
GTP
S, guanosine
5'-O-(3-thiotriphosphate)..
 |
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