| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 274, Issue 53, 37815-37820, December 31, 1999
,From the Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
A yeast two-hybrid screening for Ras-binding
proteins in nematode Caenorhabditis elegans has identified
a guanine nucleotide exchange factor (GEF) containing a
Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and
its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ)
domain and a Ras exchanger motif (REM) domain in addition to the RA and
GEF domains. They also contained a region homologous to a cyclic
nucleotide monophosphate-binding domain, which turned out to be
incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to
Rap1A at its RA domain but also an activity to stimulate GDP/GTP
exchange of Rap1A both in vitro and in vivo at
the segment containing its REM and GEF domains. However, it did not
exhibit any binding or GEF activity toward Ras. On the other hand,
Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras
and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a
novel class of Rap1A GEF molecules, which are conserved through evolution.
Ras proteins are small guanine nucleotide-binding proteins that
serve as molecular switches in regulation of cellular proliferation and
differentiation by cycling between the active GTP-bound and the
inactive GDP-bound forms (for a review, see Ref. 1). In mammalian
cells, the GTP-bound Ras exerts its action by physically associating
with and activating effector proteins, such as the serine/threonine
kinase Raf-1, through its effector region (amino acid residues 32-40
in human Ha-Ras). In addition to Raf-1 and its isoforms B-Raf and
A-Raf, recent searches have identified a number of Ras effectors (or
effector candidates) that associate directly with Ras in a
GTP-dependent manner (for a review, see Ref. 2). Two of
them, RalGDS1 and
AF-6/Afadin, have been shown to possess homologous motifs of about 100 amino acids in their Ras-associating regions, termed RA domains (3). It
has been shown that the RA domain of RalGDS and the RBD of Raf-1 share
a similar tertiary structure, the ubiquitin superfold (4-6).
Rap1A, another member of Ras family small GTP-binding proteins,
possesses an identical effector region with that of Ras (for a review,
see Ref. 7). Like Ras, Rap1A associates with Raf-1 when it is in a
GTP-bound form. However, it fails to activate Raf-1 and, when
overexpressed, even suppresses the Ras-dependent activation
of Raf-1. Although conflicting reports exist, certain cellular
responses, such as the interleukin-2 gene transcription in T cells and
the insulin-induced mitogen-activated protein kinase activation in CHO
cells, are presumed to be regulated by both the positive and negative
actions on Raf-1 exerted by Ras and Rap1A, respectively (7). On the
other hand, Rap1A activates B-Raf and may cooperate with Ras in
regulation of B-Raf-mediated responses in some cell types (8, 9). In
addition to Raf-1 and B-Raf, a majority of other Ras effector molecules
are capable of associating with Rap1A as well, suggesting that both Ras
and Rap1A are involved in a complex regulation of signaling networks downstream of them (7).
The upstream regulatory mechanisms of Ras and Rap1A appear also complex
and await further clarification. The activities of Ras and Rap1A are
regulated positively and negatively by specific GEFs and GAPs,
respectively (1). The transition of Ras from its GDP- to GTP-bound form
is stimulated by different types of GEFs such as Sos (10), RasGRFs (11,
12), and CalDAGGEFII/RasGRP (13, 14). Similarly, multiple GEFs acting
on Rap1A have been identified, including C3G (15), Epac/cAMP-GEF (16,
17), and CalDAGGEFI (13). On the other hand, both Ras and Rap1A have very low intrinsic GTPase activities, and the activities are stimulated by GAPs. Neurofibromin and p120GAP were identified as
specific GAPs for Ras (18), while Rap1A has specific GAPs, Rap1GAP (19)
and the recently discovered Rap1GAPII (20).
In this report, we describe the isolation and characterization of a
novel type of Rap1A GEF conserved between nematode Caenorhabditis elegans and humans. It is clearly distinct from known Rap1A GEFs in that it contains a functional RA domain and is hence designated RA-GEF. The observed good structural conservation between the C. elegans form (Ce-RA-GEF) and the human form (Hs-RA-GEF) suggests their essential roles in biological processes of multicellular organisms.
Cloning of Ce-RA-GEF--
The yeast two-hybrid screening for
Ras-associating proteins in C. elegans has been performed as
described in the previous report (21) by using a cDNA library
provided by Dr. Robert Barstead (Oklahoma Medical Research Foundation,
Oklahoma City, OK). The "spliced leader sequence PCR" for obtaining
the 5'-end of Ce-RA-GEF cDNA was carried out exactly as described
previously, except that the 3'-primer corresponded to the sequence
within the cDNA insert of a positive clone pACT5-7 (21, 22) (Fig.
1A). The nucleotide sequence of the PCR-amplified cDNA
was confirmed by subcloning and sequencing multiple clones. A cDNA
clone containing the full-length protein-coding sequence of Hs-RA-GEF
was provided by Dr. Takahiro Nagase (Kazusa DNA Research Institute,
Chiba, Japan).
In Vitro Ras/Rap1A Association Assays--
The
post-translationally modified forms of human Ha-Ras and Rap1A were
purified from Spodoptera frugiperda Sf9 cells
infected with baculoviruses expressing respective proteins as described previously (23, 24). A fragment of Hs-RA-GEF cDNA encoding amino
acid residues 540-710, encompassing the RA domain, was amplified by
PCR and cloned into pMal-c (New England Biolabs, Inc.) for expression
as an MBP fusion protein, MBP-Hs-RA-GEF-RA, in Escherichia coli. The in vitro association assay was carried out by
incubating 20 µl of amylose resin carrying MBP-Hs-RA-GEF-RA with
GTP In Vitro Association with cAMP and cGMP--
A fragment of
Hs-RA-GEF cDNA encoding amino acid residues 123-243, encompassing
the cNMP-binding domain, was amplified by PCR and cloned into pGEX-2T
(Amersham Pharmacia Biotech) for expression as a GST fusion protein,
GST-Hs-RA-GEF-cNMP. A full protein-coding sequence of human PKA
regulatory subunit I In Vitro GEF Assays--
Fragments of Ce-RA-GEF cDNA,
corresponding to amino acid residues 470-1300, and of Hs-RA-GEF
cDNA, corresponding to residues 258-1147, were amplified by PCR,
fused to a DNA fragment encoding the FLAG peptide, and cloned into
pBluebacIII (Pharmingen, San Diego, CA). The recombinant baculoviruses
expressing the FLAG fusion proteins, FLAG-Ce-RA-GEF and FLAG-Hs-RA-GEF,
in Sf9 cells were constructed as described previously (23). The
FLAG fusion proteins were affinity-purified from Sf9 cell
extracts with resin conjugated with anti-FLAG monoclonal antibody M2
(Sigma). GEF assays were performed as described (15). Briefly, 2 pmol
of Ha-Ras or Rap1A loaded with [3H]GDP (3,000 cpm/pmol)
were incubated with 1 µg of the FLAG fusion protein in 50 µl of a
reaction buffer containing 20 mM Tris/HCl, pH 7.4, 3 mM MgCl2, 50 mM NaCl, 10 mM 2-mercaptoethanol, 5% glycerol, 5 mg/ml bovine serum
albumin, and 3 µM unlabeled GTP. The reaction was
terminated by the addition of 2 ml of ice-cold stop buffer containing
20 mM Tris/HCl, pH 8.0, 100 mM NaCl, and 5 mM MgCl2, and the sample was subjected to
filtration through a nitrocellulose membrane (0.22-µm pore size).
After washing with the same stop buffer, the membrane-trapped
radioactivity was measured by liquid scintillation counting. In another
set of experiments, Ha-Ras or Rap1A loaded with unlabeled GDP was
incubated with the FLAG fusion proteins in the same reaction buffer
containing 3 µM [ In Vivo GEF Assays--
The full-length Hs-RA-GEF cDNA was
cloned into the mammalian expression vector pcDNA3.1HisC
(Invitrogen, San Diego, CA), yielding pcDNA3.1HisC-Hs-RA-GEF, for
expression as a fusion with the N-terminal Xpress epitope tag. The
Rap1A and Ha-Ras cDNAs were cloned into the pEF-BOS-HA vector for
expression with the N-terminal HA epitope tag. A 291-base pair cDNA
fragment of human RalGDS encoding its RID was amplified by PCR from a
human brain cDNA library (CLONTECH) and cloned
into pGEX-2T for expression as a GST fusion protein, GST-RalGDS-RID.
MBP-Raf-1-RBD, an MBP fusion protein of human Raf-1 RBD, has been
described previously (24, 25). The in vivo GEF activity of
Hs-RA-GEF on Rap1A was examined by using the RalGDS-RID pull-down assay
as described before (28). Briefly, COS-7 cells (50% confluent) in
100-mm plates were cotransfected with pEF-BOS-HA-Rap1A and either
pcDNA3.1HisC-Hs-RA- GEF or pcDNA3.1HisC vector. After
incubation for 24 h in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum and antibiotics, the cells were
washed and starved for another 24 h in the same medium containing
0.1% serum. The cells were then harvested and lysed in a buffer
containing 50 mM Tris/HCl, pH 7.4, 200 mM NaCl,
2.5 mM MgCl2, 10% glycerol, 1% Nonidet P-40,
1 mM phenylmethylsulfonyl fluoride, and 1 µM
leupeptin (28), and 800 µl of the clarified lysate were incubated
with 5 µg of GST-RalGDS-RID immobilized on glutathione-agarose resin.
After incubation for 60 min at 4 °C, the resin was washed four times
with the same buffer, and the bound proteins were eluted with 10 mM glutathione and subjected to SDS-polyacrylamide gel
electrophoresis (12% gel) followed by Western immunoblot detection
with anti-HA monoclonal antibody 12CA5 (Roche Molecular Biochemicals).
Essentially the same condition was employed for the assay of GEF
activity on Ha-Ras except that pEF-BOS-HA-Ha-Ras was transfected in
place of pEF-BOS-HA-Rap1A, the cell lysates were incubated with
MBP-Raf-1-RBD to pull down Ha-Ras, and the bound proteins were eluted
with 10 mM maltose.
We also analyzed the effect of Hs-RA-GEF expression on Rap1A- and
Ha-Ras-bound GDP/GTP ratios. Cotransfections of COS-7 cells were
performed as described above except that pEF-BOS-FLAG-Rap1A and
pEF-BOS-FLAG-Ha-Ras were used instead of pEF-BOS-HA-Rap1A and
pEF-BOS-HA-Ha-Ras, respectively. After serum starvation, the cells were
washed twice with phosphate-free Dulbecco's modified Eagle's medium
and incubated in 4 ml of the same medium supplemented with 0.5 mCi/ml
of [32P]orthophosphate (Amersham Pharmacia Biotech) for
4 h. The cells were lysed in 0.75 ml of lysis buffer containing 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.5% Triton
X-100, 0.1% Nonidet P-40, 10 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 µM
leupeptin, and 10 µg/ml aprotinin, and centrifuged at 100,000 × g for 30 min. FLAG-Rap1A or FLAG-Ha-Ras was
immunoprecipitated from the supernatant with 20 µl of anti-FLAG M2
resin for 90 min at 4 °C and, after washing four times with the
lysis buffer, eluted with FLAG peptide (Sigma). Nucleotides bound to
FLAG-Rap1A and FLAG-Ha-Ras were released by treating the eluate with 20 mM Tris/HCl, pH 7.4, 10 mM EDTA, and 2% SDS
containing 0.5 mM GDP and GTP for 20 min at 68 °C, and
subjected to thin layer chromatography on a polyethyleneimine-cellulose plate with 1 M LiCl as solvent (29, 30). The
radioactivities associated with the GDP and GTP spots on the plate were
detected and quantified by using a BAS2000 bioimaging analyzer (Fujix, Tokyo, Japan).
Cloning and Structures of Ce-RA-GEF and Hs-RA-GEF--
In a
previous report (21), we carried out a yeast two-hybrid screening for
C. elegans proteins associating with Ras (encoded by the
let-60 gene in this organism) and isolated a novel
phosphoinositide-specific phospholipase C, PLC210. PLC210 contained two
tandemly arranged RA domains, one of which associated in
vitro with Ras directly in a GTP-dependent manner. In
addition to PLC210, the same screening has identified another novel
protein encoded by 10 overlapping partial cDNA clones. The longest
clone, pACT5-7, encoded a protein truncated at both its N and C
termini (Fig. 1A). A cDNA
coding for the upstream sequence was isolated by the spliced leader
sequence PCR. A putative initiator ATG was identified in this cDNA
as that matching the Kozak consensus sequence (31) and preceded by
in-frame stop codons. Also, a BLAST search (32) of
GenBankTM entries identified a C. elegans
expressed sequence tag clone yk17d8.3 coding for the 3'-portion of this
protein. A composite cDNA encoding the full-length protein
consisting of 1,470 amino acid residues was reconstructed by joining
the three cDNAs. The deduced Ce-RA-GEF protein contained, from the
N terminus to the C terminus, a cNMP-binding domain, a REM domain, a
PDZ domain, a RA domain and a GEF domain, all of which were predicted
based on their sequence homology to the corresponding functional
domains already characterized (Fig. 1, A and B).
A BLAST search of the GenBankTM data base identified a
cDNA encoding an uncharacterized 1,499-amino acid human protein
(gene name KIAA0313; accession number AB002311), containing
all of these domains in the same order as in Ce-RA-GEF, and the protein
was termed Hs-RA-GEF (Fig. 1, A and B).
Direct Association of Hs-RA-GEF with Rap1A but Not with cAMP and
cGMP--
Ce-RA-GEF associated with human Ha-Ras and Rap1A in
addition to LET-60 but not with human R-Ras, RalA, RhoA, Cdc42, and
Rac1 as judged by the two-hybrid assay using the clone pACT5-7 (data not shown). However, direct and GTP-dependent association
of its RA domain with Ras and Rap1A could not be tested in
vitro, since an MBP fusion protein of this RA domain was found to
be insoluble when expressed in E. coli. On the other hand, a
similar fusion protein derived from Hs-RA-GEF, MBP-Hs-RA-GEF-RA, was
found to be soluble and could be used for the in vitro
association assay with Ha-Ras and Rap1A. As shown in Fig.
2B, the association of the
immobilized MBP-Hs-RA-GEF-RA with Ha-Ras was barely detectable and was
independent of the guanine nucleotide configuration. In contrast, it
associated efficiently with Rap1A, and the association exhibited a
clear GTP dependence (Fig. 2A). In agreement with this,
quantitative analyses with the radiolabeled Ha-Ras and Rap1A indicated
that MBP-Hs-RA-GEF-RA was capable of specific association with the
GTP-bound form of Rap1A but not with the GTP-bound form of Ha-Ras or
the GDP-bound form of Rap1A (Fig. 2C). The amount of the
bound Rap1A increased almost linearly to the concentration of 500 nM and reached to the level where about 35% of the input RA domain established the association. Unavailability of a higher concentration preparation of Rap1A precluded the assessment of the
dissociation constant for Rap1A.
Next, we tested the ability of Hs-RA-GEF to bind cAMP or cGMP in
vitro. GST-Hs-RA-GEF-cNMP, encompassing the putative cNMP-binding domain, was immobilized on glutathione-agarose and examined for association with increasing concentrations of radiolabeled cAMP and
cGMP as described under "Experimental Procedures" (Fig.
3). In contrast to the nearly
stoichiometric binding of cAMP to GST-PKA-RI GEF Activities of Ce-RA-GEF and Hs-RA-GEF in Vitro and in
Vivo--
We first examined the catalytic activities of Ce-RA-GEF and
Hs-RA-GEF in vitro. First, fragments of these proteins
encompassing from the REM domains to the GEF domains were purified as
those fused to the FLAG epitope, and the resulting FLAG-Ce-RA-GEF and FLAG-Hs-RA-GEF were incubated with Ras and Rap1A, both of which had
been loaded with radiolabeled GDP, in the presence of unlabeled GTP. As
shown in Fig. 4A, both
FLAG-Ce-RA-GEF and FLAG-Hs-RA-GEF exhibited an activity to stimulate
the release of GDP from Rap1A. FLAG-Ce-RA-GEF also stimulated release
of GDP from Ha-Ras (Fig. 4B), and the extent of this
stimulation was comparable with that observed for Rap1A. In contrast,
FLAG-Hs-RA-GEF exhibited very little activity on Ha-Ras (Fig.
4B) compared with that on Rap1A. In the second set of
experiments, in which Ha-Ras and Rap1A were loaded with unlabeled GDP
and incubated with the FLAG-tagged proteins in the presence of
35S-labeled GTP
We next examined the in vivo GEF activities of Hs-RA-GEF
toward Rap1A and Ha-Ras by the following two experiments. First, the
amounts of the GTP-bound forms of Rap1A and Ha-Ras in mammalian cells
were measured by using the pull-down assays. For this purpose, the
full-length Hs-RA-GEF tagged with the Xpress peptide was expressed in
COS-7 cells together with the HA-tagged Rap1A. The cell lysate was
incubated with the immobilized GST-RalGDS-RID, which contained the RA
domain of RalGDS and associated specifically with the GTP-bound form of
Rap1A but not with its GDP-bound form (28). As shown in Fig.
5A, the lysates of cells
expressing Hs-RA-GEF contained an increased amount of the GTP-bound
HA-Rap1A compared with the lysate of cells expressing HA-Rap1A alone.
In contrast, when the lysate of cells expressing Hs-RA-GEF and
HA-Ha-Ras was incubated with MBP-Raf-1-RBD, no increase was observed in
the GTP-bound Ha-Ras compared with the lysate of cells expressing
Ha-Ras alone (Fig. 5B). We also analyzed the effect of
Hs-RA-GEF expression on Rap1A- and Ha-Ras-bound GDP/GTP ratios. For
this purpose, the full-length Hs-RA-GEF tagged with the Xpress peptide
was expressed in COS-7 cells together with FLAG-tagged Rap1A or Ha-Ras,
and the cells were metabolically labeled with
[32P]orthophosphate. The FLAG-tagged Rap1A and Ha-Ras
were immunoprecipitated from the cell lysates with the anti-FLAG
antibody, the bound guanine nucleotides were separated, and the
radioactivities associated with GDP and GTP were quantified (Fig.
5C). More than 2-fold increase was observed in the
Rap1A-bound GTP concomitant with the coexpression of Hs-RA-GEF (Fig.
5C, lanes 2 and 3), whereas
no increase was observed in the Ha-Ras-bound GTP (Fig. 5C,
lanes 4 and 5). These results
unambiguously demonstrated that Hs-RA-GEF has a GEF activity toward
Rap1A but not toward Ha-Ras.
The recent discovery of multiple forms of Rap1A GEF has revealed
an unexpected complexity of Rap1A regulation. Each of the Rap1A GEF
molecules has been shown to possess distinct regulatory elements: C3G
contains proline-rich regions that associate with SH3-containing
adaptor proteins (15); Epac/cAMP-GEF contains a binding site for cAMP
(16, 17); and CalDAGGEFI contains domains that bind Ca2+
and diacylglycerol (13). The presence of the RA domains clearly distinguishes Ce-RA-GEF and Hs-RA-GEF from these known Rap1A GEF molecules. In addition, the PDZ domains, which are found in a number of
proteins localized at cellular junctions (33), represent another unique
structural feature of RA-GEFs, although the function of these PDZ
domains remains to be clarified.
On the other hand, Ce-RA-GEF and Hs-RA-GEF share some elements with the
other Rap1A GEF proteins. REM domains are found in all the GEF proteins
acting on Ras family small GTP-binding proteins (34). In agreement with
previous reports on the other GEFs (34, 35), fragments of both
Ce-RA-GEF and Hs-RA-GEF carrying the GEF domains but lacking the REM
domains were inactive in the in vitro GEF assays, suggesting
that the REM domains are required for the catalytic
activities.2 In addition,
both Ce-RA-GEF and Hs-RA-GEF possess the putative cNMP-binding domains,
whose amino acid sequences exhibit a considerable homology to the
cAMP-binding domain of Epac/cAMP-GEF (Fig. 1B). Epac/cAMP-GEF was reported to be activated directly by cAMP (16, 17).
However, the cNMP-binding domain of Hs-RA-GEF failed to exhibit any
binding to both cAMP and cGMP. This may be explained by specific amino
acid substitutions found in the cNMP-binding domains of both Ce-RA-GEF
and Hs-RA-GEF. A PRAA motif is present in both the slow and fast
cAMP-binding pockets of the two PKA regulatory subunits and is also
conserved in Epac/cAMP-GEF (residues 278-281) (16, 17). The first Ala
of this motif is considered to confer specificity for cAMP as opposed
to Thr, which is found in the cNMP-binding domains of proteins that
bind cGMP instead of cAMP (36, 37). The PRAA motif is totally missing
in the corresponding regions of both Ce-RA-GEF and Hs-RA-GEF (Fig.
1B).
The RA domain of Hs-RA-GEF was found to associate with Rap1A but not
with Ras. This kind of binding specificity of the RA domain is not
unprecedented; the RalGDS RA domain was reported to associate with
Rap1A much more strongly than with Ras (38). These binding
specificities are presumably determined by the nature of certain amino
acids within or flanking the RA domains. More importantly, the GEF
activity of Hs-RA-GEF also exhibits clear specificity for Rap1A,
whereas Ce-RA-GEF has GEF activity toward both Ras and Rap1A. In this
line, it may be interesting to note that CalDAGGEFII/RasGRP, a
Ras-specific GEF, and CalDAGGEFI, a Rap1A-specific GEF, share an
identical domain organization with each other, suggesting that they
might have diverged from a common ancestral protein. Hs-RA-GEF might
have acquired the specificity toward Rap1A in the course of evolution
from an ancestral GEF protein that may have exhibited no selectivity
toward Ras and Rap1A. Ce-RA-GEF may be a direct descendant of this
ancestral protein, still retaining the original substrate specificity.
This raises an interesting possibility that there may exist another isoform of Hs-RA-GEF that is specific for Ras in humans.
The unique and remarkable feature of Hs-RA-GEF is that it possesses two
domains that are capable of interacting with different forms of Rap1A;
the RA domain associates with the GTP-bound form, whereas the GEF
domain uses the GDP-bound form as its substrate. The role of the RA
domain in a physiological function of Hs-RA-GEF remains to be
clarified. One possibility is that Hs-RA-GEF is translocated to a
Rap1A-containing membrane compartment through association with the
GTP-bound Rap1A and catalyzes activation of other GDP-bound Rap1A
molecules present in the compartment, thereby causing an amplification
or a sustained activation of the Rap1A-mediated cellular responses.
Experiments with Hs-RA-GEF molecules carrying RA domain mutations that
alter its Rap1A-binding property may provide further insights into this possibility.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S- or GDP
S-loaded Ha-Ras or 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). 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-polyacrylamide gel electrophoresis (12% gel)
followed by Western immunoblot detection with anti-Ha-Ras monoclonal
antibody F235 (Oncogene Science Inc, Manhasset, NY) or anti-Rap1A
polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) as
described previously (24, 25). For the quantitative in vitro
association assays, Ha-Ras and Rap1A were loaded with
[-35S]GTPS (3,500 cpm/pmol) or
[3H]GDP (1,100 cpm/pmol) and incubated with
MBP-Hs-RA-GEF-RA as described above except that unlabeled GTPS or
GDP (0.1 mM), respectively, was included in the binding
reaction. The eluates from amylose resin were counted for
35S or 3H label, respectively.
was amplified by PCR from a human brain
cDNA library (CLONTECH, Palo Alto, CA) and cloned into pGEX-2T for expression as a GST fusion, GST-PKA-RI. The
two proteins were expressed in an adenylyl cyclase-deficient E. coli strain CA8306 (26). The cAMP binding assay was carried out
essentially as described (27). GST-Hs-RA-GEF-cNMP or GST-PKA-RI (0.5 µg each) immobilized on glutathione-agarose resin was incubated in a
100-µl reaction mixture containing 10 mM potassium
phosphate, pH 6.8, 2 M NaCl, 1 mM EDTA, 100 µg/ml bovine serum albumin, 25 mM 2-mercaptoethanol, and
various concentrations of [2,8-3H]cAMP (15,000 cpm/pmol)
(Moravek Biochemicals Inc., Brea, CA) at room temperature for 90 min
with gentle shaking. After incubation, the resin was washed, and the
bound proteins were eluted with 10 mM glutathione and
counted for 3H label. The cGMP-binding assay was carried
out similarly as described above, except that [8-3H]cGMP
(5,700 cpm/pmol) (Moravek Biochemicals Inc.) replaced cAMP.
-35S]GTPS (30,000 cpm/pmol) instead of unlabeled GTP.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (54K):
[in a new window]
Fig. 1.
Structural features of Ce-RA-GEF and
Hs-RA-GEF. A, schematic representations of various
domains of Ce-RA-GEF and Hs-RA-GEF. cNMP, cNMP-binding
domain; REM, REM domain; PDZ, PDZ domain;
RA, RA domain; GEF, GEF domain. The region
encoded by pACT5-7 and those expressed as GST, MBP, and FLAG fusions
are also indicated. B, the amino acid sequences of the
various domains of Ce-RA-GEF (Ce) and Hs-RA-GEF
(Hs) are aligned with those of other human proteins carrying
the corresponding domains. EP, Epac/cAMP-GEF; C3,
C3G; ZO, ZO-1; RG, RalGDS. Numbers on
the left represent amino acid positions within the
respective proteins. All of the alignments were generated with
GENETYX-MAC 6.2.0 program (Software Development Co., Japan) and
adjusted manually. Asterisks and plus
signs above the aligned residues represent
identities and similarities, respectively, between Ce-RA-GEF and
Hs-RA-GEF, while those below the aligned residues represent
identities and similarities, respectively, among all the three
proteins. The three regions (SCR1 to SCR3), which
are structurally conserved among the catalytic domains of all the known
GEFs for Ras family small GTP-binding proteins, are
underlined.
View larger version (18K):
[in a new window]
Fig. 2.
Direct association of the RA domain of
Hs-RA-GEF with Rap1A. A, 6 pmol of GTP S-loaded Rap1A
(T) or GDPS-loaded Rap1A (D) were incubated
with the indicated amounts of MBP-Hs-RA-GEF-RA (MBP-RA) or
MBP alone (MBP) immobilized on amylose resin, and the bound
proteins were eluted with 10 mM maltose as described under
"Experimental Procedures." Rap1A in the eluate (Bound
Rap1A, upper panel) and 
aliquot of Rap1A
used in the assay (Input of Rap1A, lower panel)
were detected by Western immunoblotting with the anti-Rap1A antibody.
B, the in vitro association assays were carried
out as in A except that 10 pmol of Ha-Ras were incubated
with MBP-Hs-RA-GEF-RA and that the anti-Ha-Ras antibody was used for
Western immunoblotting. The assays in A and B
were performed three times, giving equivalent results. C, 25 pmol of MBP-Hs-RA-GEF-RA immobilized on amylose resin were incubated
with increasing amounts of Rap1A loaded with
[-35S]GTPS (
) or [3H]GDP (
), or
of Ha-Ras loaded with [-35S]GTPS (×) as described
under "Experimental Procedures." The bound Rap1A and Ha-Ras
proteins were quantitated by counting for 35S or
3H label. Mean values obtained from two independent
experiments performed in duplicate are shown with S.E. values.
used as a positive
control, GST-Hs-RA-GEF-cNMP failed to exhibit any detectable binding to
both cAMP and cGMP.
View larger version (18K):
[in a new window]
Fig. 3.
Absence of the cAMP/cGMP binding activity in
the putative cNMP-binding domain of Hs-RA-GEF. GST-Hs-RA-GEF-cNMP
( ) or GST-PKA-RI () (0.5 µg each) immobilized on
glutathione-agarose resin was examined for in vitro
association with increasing concentrations of cAMP as described under
"Experimental Procedures." The association of GST-Hs-RA-GEF-cNMP
with cGMP was also measured similarly (×).
S, FLAG-Ce-RA-GEF stimulated the
replacement of the bound GDP with GTPS for both Ha-Ras and Rap1A
(Fig. 4, C and D). Again, FLAG-Hs-RA-GEF
exhibited a clear specificity for Rap1A (Fig. 4, C and
D).
View larger version (26K):
[in a new window]
Fig. 4.
Measurements of the in vitro
GEF activities of Ce-RA-GEF and Hs-RA-GEF. A, Rap1A
loaded with [3H]GDP was incubated with FLAG-Ce-RA-GEF
( ), FLAG-Hs-RA-GEF () or buffer (×) in the presence of unlabeled
GTP for the indicated times. [3H]GDP that remained bound
to Rap1A was measured and expressed as the percentage of the value at
the zero time point. Mean values obtained from three independent
experiments are shown with S.E. values. B, the assays were
carried out as in A except that Ha-Ras was employed in place
of Rap1A. C, Rap1A loaded with unlabeled GDP was incubated
with FLAG-Ce-RA-GEF (), FLAG-Hs-RA-GEF (), or buffer (×) in the
presence of [-35S]GTPS, and
[-35S]GTPS bound to Rap1A was measured at the
indicated times. Mean values obtained from three independent
experiments are shown with S.E. values. D, the assays were
carried out as in C except that Ha-Ras was employed in place
of Rap1A.
View larger version (21K):
[in a new window]
Fig. 5.
Measurements of the in vivo
GEF activities of Hs-RA-GEF. A, COS-7 cells were
co-transfected with pcDNA3.1HisC-Hs-RA-GEF and pEF-BOS-HA-Rap1A.
The cell lysates were incubated with GST-RalGDS-RID, and Rap1A
associated with GST-RalGDS-RID (Bound Rap1A, upper
panel) and that in aliquot of the cell lysate
(Input of Rap1A, lower panel) were detected by
Western immunoblotting with the anti-HA antibody. Numbers
indicate the amounts (in µg) of the transfected expression plasmids.
B, the experiments were carried out as in A
except that pEF-BOS-HA-Ha-Ras was used in place of pEF-BOS-HA-Rap1A and
that MBP-Raf-1-RBD was used in place of GST-RalGDS-RID. C,
COS-7 cells were co-transfected with the following combinations of the
plasmids: pEF-BOS-FLAG-Rap1A and pcDNA3.1HisC (lane 2),
pEF-BOS-FLAG-Rap1A and pcDNA3.1HisC-Hs-RA-GEF (lane 3),
pEF-BOS-FLAG-Ha-Ras and pcDNA3.1HisC (lane 4), and
pEF-BOS-FLAG-Ha-Ras and pcDNA3.1HisC-Hs-RA-GEF (lane 5).
Then cells were metabolically labeled with
[32P]orthophosphate. Cells without transfection were also
treated similarly (lane 1). The FLAG-tagged Rap1A or Ha-Ras
was immunoprecipitated from the cell lysates with the anti-FLAG
antibody, the bound guanine nucleotides were separated by thin layer
chromatography, and the radioactivities associated with GDP and GTP
were quantified as described under "Experimental Procedures." The
number above each lane represents the
molar ratio of GTP in percentage, which was calculated by the following
formula: (radioactivity of GTP/(the radioactivity of GTP + 1.5 × the radioactivity of GDP)) × 100%. The experiments in
A, B, and C were performed three
times, giving equivalent results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
ACKNOWLEDGEMENTS |
|---|
We thank R. Barstead for providing the pACT-RB2 cDNA library, Y. Kohara for the C. elegans expressed sequence tag clone, and T. Nagase for the KIAA0313 clone. We also thank T. Inagaki for excellent technical assistance and A. Seki and A. Kawabe for help in preparation of this manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by grants-in-aid for scientific research in priority areas and for scientific research B and C from the Ministry of Education, Science, Sports, and Culture of Japan and by grants from the Yamanouchi Foundation for Research on Metabolic Disease, from the Sankyo Foundation of Life Science, 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF170796.
Present address: Dept. of Biochemistry II, School of Medicine,
University of the Ryukyus, 207 Uehara, Nishihara-cho, Okinawa 903-0215, Japan.
§ To whom correspondence should be addressed. Tel.: 81-78-382-5380; Fax: 81-78-382-5399; E-mail: kataoka@kobe-u.ac.jp.
2 Y. Liao and T. Kataoka, unpublished observations.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
RalGDS, Ral guanine
nucleotide dissociation stimulator;
RA, Ras-associating or RalGDS/AF-6;
RBD, Ras-binding domain;
GEF, guanine nucleotide exchange factor;
GAP, GTPase-activating protein;
PCR, polymerase chain reaction;
MBP, maltose-binding protein;
GTPS, guanosine
5'-O-(3-thiotriphosphate);
GDPS, guanosine
5'-O-(2-thiodiphosphate);
cNMP, cyclic nucleotide
monophosphate;
PKA, protein kinase A;
GST, glutathione
S-transferase;
RID, Rap1A-interacting domain;
PDZ, PSD-95,
DlgA, and ZO-1;
REM, Ras exchanger motif.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Lowy, D. R., and Willumsen, B. M. (1993) Annu. Rev. Biochem. 62, 851-891[CrossRef][Medline] [Order article via Infotrieve] |
| 2. | Katz, M. E., and McCormick, F. (1997) Curr. Opin. Genet. Dev. 7, 75-79[CrossRef][Medline] [Order article via Infotrieve] |
| 3. | Ponting, C. P., and Benjamin, D. R. (1996) Trends Biochem. Sci. 21, 422-425[CrossRef][Medline] [Order article via Infotrieve] |
| 4. | Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1995) Nature 375, 554-560[CrossRef][Medline] [Order article via Infotrieve] |
| 5. | Huang, L., Weng, X., Hofer, F., Martin, G. S., and Kim, S. H. (1997) Nat. Struct. Biol. 4, 609-614[CrossRef][Medline] [Order article via Infotrieve] |
| 6. | Huang, L., Hofer, F., Martin, G. S., and Kim, S. H. (1998) Nat. Struct. Biol. 5, 422-426[CrossRef][Medline] [Order article via Infotrieve] |
| 7. | Bos, J. L. (1998) EMBO J. 17, 6776-6782[CrossRef][Medline] [Order article via Infotrieve] |
| 8. | Vossler, M. R., Yao, H., York, R. D., Pan, M. G., Rim, C. S., and Stork, P. J. S. (1997) Cell 89, 73-82[CrossRef][Medline] [Order article via Infotrieve] |
| 9. | York, R. D., Yao, H., Dillon, T., Ellig, C. L., Eckert, S. P., McCleskey, E. W., and Stork, P. J. S. (1998) Nature 392, 622-626[CrossRef][Medline] [Order article via Infotrieve] |
| 10. |
Chardin, P.,
Camonis, J. H.,
Gale, N. W.,
Van Aelst, L.,
Schlessinger, J.,
Wigler, M. H.,
and Bar-Sagi, D.
(1993)
Science
260,
1338-1343 |
| 11. | Farnsworth, C. L., Freshney, N. W., Rosen, L. B., Ghosh, A., Greenberg, M. E., and Feig, L. A. (1995) Nature 376, 524-527[CrossRef][Medline] [Order article via Infotrieve] |
| 12. | Fam, N. P., Fan, W.-T., Wang, Z., Zhang, L.-Z., Chen, H., and Moran, M. F. (1997) Mol. Cell. Biol. 17, 1396-1406[Abstract] |
| 13. |
Kawasaki, H.,
Springett, G. M.,
Toki, S.,
Canales, J. J.,
Harlan, P.,
Blumenstiel, J. P.,
Chen, E. J.,
Bany, I. A.,
Mochizuki, N.,
Ashbacher, A.,
Matsuda, M.,
Housman, D. E.,
and Graybiel, A. M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
13278-13283 |
| 14. |
Ebinu, J. O.,
Bottorff, D. A.,
Chan, E. Y. W.,
Stang, S. L.,
Dunn, R. J.,
and Stone, J. C.
(1998)
Science
280,
1082-1086 |
| 15. | Gotoh, T., Hattori, S., Nakamura, S., Kitayama, H., Noda, M., Takai, Y., Kaibuchi, K., Matsui, H., Hatase, O., Takahashi, H., Kurata, T., and Matsuda, M. (1995) Mol. Cell. Biol. 15, 6746-6753[Abstract] |
| 16. | de Rooij, J., Zwartkruis, F. J. T., Verheijen, M. H. G., Cool, R. H., Nijman, S. M. B., Wittinghofer, A., and Bos, J. L. (1998) Nature 396, 474-477[CrossRef][Medline] [Order article via Infotrieve] |
| 17. |
Kawasaki, H.,
Springett, G. M.,
Mochizuki, N.,
Toki, S.,
Nakaya, M.,
Matsuda, M.,
Housman, D. E.,
and Graybiel, A. M.
(1998)
Science
282,
2275-2279 |
| 18. | Boguski, M. S., and McCormick, F. (1993) Nature 366, 643-653[CrossRef][Medline] [Order article via Infotrieve] |
| 19. | Rubinfeld, B., Munemitsu, S., Clark, R., Conroy, L., Watt, K., Crosier, W. J., McCormick, F., and Polakis, P. (1991) Cell 65, 1033-1042[CrossRef][Medline] [Order article via Infotrieve] |
| 20. | Mochizuki, N., Ohba, Y., Kiyokawa, E., Kurata, T., Murakami, T., Ozaki, T., Kitabatake, A., Nagashima, K., and Matsuda, M. (1999) Nature 400, 891-894[CrossRef][Medline] [Order article via Infotrieve] |
| 21. |
Shibatohge, M.,
Kariya, K.,
Liao, Y.,
Hu, C.-D.,
Watari, Y.,
Goshima, M.,
Shima, F.,
and Kataoka, T.
(1998)
J. Biol. Chem.
273,
6218-6222 |
| 22. | Krause, M. (1995) in Caenorhabditis elegans: Modern Biological Analysis of an Organism (Epstein, H. F. , and Shakes, D. C., eds) , pp. 513-529, Academic Press, Inc., San Diego |
| 23. | Kuroda, Y., Suzuki, N., and Kataoka, T. (1993) Science 259, 683-686[Abstract] |
| 24. |
Hu, C.-D.,
Kariya, K.,
Tamada, M.,
Akasaka, K.,
Shirouzu, M.,
Yokoyama, S.,
and Kataoka, T.
(1995)
J. Biol. Chem.
270,
30274-30277 |
| 25. |
Hu, C.-D.,
Kariya, K.,
Kotani, G.,
Shirouzu, M.,
Yokoyama, S.,
and Kataoka, T.
(1997)
J. Biol. Chem.
272,
11702-11705 |
| 26. |
Brickman, E.,
Soll, L.,
and Beckwith, J.
(1973)
J. Bacteriol.
116,
582-587 |
| 27. | Rannels, S. R., and Corbin, J. D. (1983) Methods Enzymol. 99, 168-175[Medline] [Order article via Infotrieve] |
| 28. |
Wolthuis, R. M. F.,
Franke, B.,
Van Triest, M.,
Bauer, B.,
Cool, R. H.,
Camonis, J. H.,
Akkerman, J.-W. N.,
and Bos, J. L.
(1998)
Mol. Cell. Biol.
18,
2486-2491 |
| 29. | Gibbs, J. B. (1995) Methods Enzymol. 255, 118-125[Medline] [Order article via Infotrieve] |
| 30. |
Noguchi, T.,
Matozaki, T.,
Horita, K.,
Fujioka, Y.,
and Kasuga, M.
(1994)
Mol. Cell. Biol.
14,
6674-6682 |
| 31. | Blumenthal, T., and Steward, K. (1997) in C. elegans II (Riddle, D. L. , Blumenthal, T. , Meyer, B. J. , and Priess, J. R., eds) , pp. 117-145, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
| 32. | Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410[CrossRef][Medline] [Order article via Infotrieve] |
| 33. | Fanning, A. S., and Anderson, J. M. (1999) J. Clin. Invest. 103, 767-772[Medline] [Order article via Infotrieve] |
| 34. |
Lai, C.-C.,
Boguski, M.,
Broek, D.,
and Powers, S.
(1993)
Mol. Cell. Biol.
13,
1345-1352 |
| 35. | Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D., and Kuriyan, J. (1998) Nature 394, 337-343[CrossRef][Medline] [Order article via Infotrieve] |
| 36. |
Taylor, S. S.
(1989)
J. Biol. Chem.
264,
8443-8446 |
| 37. |
Shabb, J. B.,
and Corbin, J. D.
(1992)
J. Biol. Chem.
267,
5723-5726 |
| 38. |
Herrmann, C.,
Horn, G.,
Spaargaren, M.,
and Wittinghofer, A.
(1996)
J. Biol. Chem.
271,
6794-6800 |
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Shibasaki, H. Takahashi, T. Miki, Y. Sunaga, K. Matsumura, M. Yamanaka, C. Zhang, A. Tamamoto, T. Satoh, J.-i. Miyazaki, et al. Essential role of Epac2/Rap1 signaling in regulation of insulin granule dynamics by cAMP PNAS, December 4, 2007; 104(49): 19333 - 19338. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Hisata, T. Sakisaka, T. Baba, T. Yamada, K. Aoki, M. Matsuda, and Y. Takai Rap1-PDZ-GEF1 interacts with a neurotrophin receptor at late endosomes, leading to sustained activation of Rap1 and ERK and neurite outgrowth J. Cell Biol., August 27, 2007; 178(5): 843 - 860. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Yoshikawa, T. Satoh, T. Tamura, P. Wei, S. E. Bilasy, H. Edamatsu, A. Aiba, K. Katagiri, T. Kinashi, K. Nakao, et al. The M-Ras-RA-GEF-2-Rap1 Pathway Mediates Tumor Necrosis Factor-{alpha} dependent Regulation of Integrin Activation in Splenocytes Mol. Biol. Cell, August 1, 2007; 18(8): 2949 - 2959. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. M. Amsen, N. Pham, Y. Pak, and D. Rotin The Guanine Nucleotide Exchange Factor CNrasGEF Regulates Melanogenesis and Cell Survival in Melanoma Cells J. Biol. Chem., January 6, 2006; 281(1): 121 - 128. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Seino and T. Shibasaki PKA-Dependent and PKA-Independent Pathways for cAMP-Regulated Exocytosis Physiol Rev, October 1, 2005; 85(4): 1303 - 1342. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. J. Mandell, B. A. Babbin, A. Nusrat, and C. A. Parkos Junctional Adhesion Molecule 1 Regulates Epithelial Cell Morphology through Effects on {beta}1 Integrins and Rap1 Activity J. Biol. Chem., March 25, 2005; 280(12): 11665 - 11674. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. P.-v. Berkel, M.H.G. Verheijen, E. Cuppen, M. Asahina, J. de Rooij, G. Jansen, R.H.A. Plasterk, J. L. Bos, and F.J.T. Zwartkruis Requirement of the Caenorhabditis elegans RapGEF pxf-1 and rap-1 for Epithelial Integrity Mol. Biol. Cell, January 1, 2005; 16(1): 106 - 116. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Taira, M. Umikawa, K. Takei, B.-E. Myagmar, M. Shinzato, N. Machida, H. Uezato, S. Nonaka, and K.-i. Kariya The Traf2- and Nck-interacting Kinase as a Putative Effector of Rap2 to Regulate Actin Cytoskeleton J. Biol. Chem., November 19, 2004; 279(47): 49488 - 49496. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Pak, N. Pham, and D. Rotin Direct Binding of the {beta}1 Adrenergic Receptor to the Cyclic AMP-Dependent Guanine Nucleotide Exchange Factor CNrasGEF Leads to Ras Activation Mol. Cell. Biol., November 15, 2002; 22(22): 7942 - 7952. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. H. Lee, K. S. Cho, J. Lee, D. Kim, S.-B. Lee, J. Yoo, G.-H. Cha, and J. Chung Drosophila PDZ-GEF, a Guanine Nucleotide Exchange Factor for Rap1 GTPase, Reveals a Novel Upstream Regulatory Mechanism in the Mitogen-Activated Protein Kinase Signaling Pathway Mol. Cell. Biol., November 1, 2002; 22(21): 7658 - 7666. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kido, F. Shima, T. Satoh, T. Asato, K.-i. Kariya, and T. Kataoka Critical Function of the Ras-associating Domain as a Primary Ras-binding Site for Regulation of Saccharomyces cerevisiae Adenylyl Cyclase J. Biol. Chem., January 25, 2002; 277(5): 3117 - 3123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Saito, J. Murai, H. Kajiho, K. Kontani, H. Kurosu, and T. Katada A Novel Binding Protein Composed of Homophilic Tetramer Exhibits Unique Properties for the Small GTPase Rab5 J. Biol. Chem., January 25, 2002; 277(5): 3412 - 3418. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Pham and D. Rotin Nedd4 Regulates Ubiquitination and Stability of the Guanine-Nucleotide Exchange Factor CNrasGEF J. Biol. Chem., December 7, 2001; 276(50): 46995 - 47003. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Gao, T. Satoh, Y. Liao, C. Song, C.-D. Hu, K.-i. Kariya, and T. Kataoka Identification and Characterization of RA-GEF-2, a Rap Guanine Nucleotide Exchange Factor That Serves a |
