From the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ras is a master GTPase switch controlling multiple signal transduction cascades in the regulation of cell proliferation and differentiation. Rab5 is a local GTPase switch that is localized on early endosomes and controls early endosome fusion. This study demonstrates that the catalytic domain of p120 GTPase-activating protein (GAP), a well known Ras GAP, is able to interact physically with Rab5 and stimulate its GTPase activity. This GAP activity toward Rab5, however, cannot be extended to other Rab GTPases such as Rab3, Rab4, and Rab6, indicating that it is not a generic GAP for the Rab family of GTPases that regulate intracellular membrane fusion during endocytosis and exocytosis. The findings indicate a level of structural similarity between Ras and Rab5 unexpected from their primary sequences. They also suggest a possible signal transduction regulation of the Rab5-dependent endosome fusion via the Ras GAP.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ras and Rab5 are members of the monomeric GTPase superfamily that function as molecular switches in regulating diverse cell functions such as cell proliferation, cytoskeleton organization, and intracellular trafficking (1, 2). The ability to alternate between GTP-bound and GDP-bound conformations provides a molecular basis for these GTPase switches (3, 4). In a GTPase cycle, it is usually the GTP-bound conformation that promotes a biological event. GTP hydrolysis converts it into an inactive GDP-bound conformation. When the GDP molecule is replaced by a GTP molecule, the GTPase is ready again to turn on the cellular function. The conversion between GTP-bound and GDP-bound conformations are facilitated by GTPase-activating proteins (GAP)1 and guanine nucleotide exchange factors in the biological environment of cells.
Ras and Rab5 belong to the Ras and Rab subfamilies, respectively. In the hierarchy of cell functions, Ras is a master switch that coordinates multiple signal transduction cascades (5, 6) in preparation for cell proliferation. Each Rab is a local switch that controls a specific membrane fusion event during endocytosis and exocytosis (7-10). Rab5 in particular is localized on the cytoplasmic side of early endosomes (11) and regulates endosome fusion (12-15), an important step of endocytosis. Endosome fusion is a dynamic process that ceases during mitosis (16-18), suggesting a link between endosome fusion and cell proliferation. In this study, we demonstrate that the catalytic domain of p120 GAP, a well known Ras GAP, is able to bind with high affinity to Rab5 and stimulates its GTPase activity. The data reveal an unexpected level of structural similarity between Rab5 and Ras, since Rab5 shares no more sequence similarity with Ras than the other Rab proteins such as Rab3, Rab4, and Rab6, which are not responsive to the Ras GAP. The data also suggest a possible coordination between the Ras GTPase cycle, which generates cell proliferation signals, and the Rab5 GTPase cycle, which controls endosome fusion and endocytosis.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression and Purification of Recombinant Proteins in
Bacteria--
The cDNA of human Ha-Ras in the bacterial expression
vector pGEX-3X was kindly provided by Dr. Jonathan Cooper of Fred
Hutchinson Cancer Research Center. The recombinant human Ras and Rab
proteins were expressed in the Escherichia coli strain
MC1061 as glutathione S-transferase (GST) fusion proteins
and purified by using the glutathione-Sepharose 4B resin (Amersham
Pharmacia Biotech) (14, 15). Free Rab5 was generated by the Xa protease
cleavage following the manufacture's instructions (Amersham Pharmacia
Biotech). GAPette (the catalytic domain of human p120 GAP) was tagged
with a KT3 epitope (TPPPEPET) at the C terminus, expressed in the
E. coli strain MC1061 and purified by using the KT3 affinity
resin (19). The purified proteins were dialyzed overnight against the
storage buffer (20 mM Tris-HCl, pH 8, 100 mM
NaCl, 1 mM DTT, 1 mM EDTA, and 50% glycerol)
and stored at 20 °C before use.
GAP Assays--
Two assays were employed to measure the GAP
activity on Rab5 and other related GTPases. In the first assay, Rab5
was cleaved from GST-Rab5 by the Xa protease (Amersham Pharmacia
Biotech) and purified according to the manufacture's instructions. The Rab5 protein (200-500 nM) was loaded with
[-32P]GTP (85-200 nM) (Amersham Pharmacia
Biotech) for 30 min at 25 °C in 20 µl of the loading buffer (20 mM Tris-HCl, pH 8, 2 mM EDTA, and 1 mM DTT). The GTP hydrolysis reaction was initiated by
adding MgCl2 (to 10 mM) and various amounts of
GAPette as indicated. The GTP hydrolysis reaction was conducted at
25 °C for the indicated times. Samples were immediately solubilized
in the elution buffer (0.2% SDS, 5 mM EDTA, 5 mM GDP, 5 mM GTP) by heating at 65 °C for 2 min. The eluted GTP and GDP were separated by thin layer chromatography
on polyethyleneimine-cellulose sheets (J. T. Baker) with 0.75 M KH2PO4, pH 3.5, as the developing
solvent, followed by autoradiography and PhosphorImager analysis
(Molecular Dynamics) to quantify the radioactive counts of the GTP and
GDP spots and calculate the amounts of GDP produced. The phosphorus
content of GDP was corrected as two-thirds that of GTP.
In Vitro Binding Assay-- 15 µl of the KT3 affinity resin with bound GAPette were mixed with various amounts of GST-Rab5, and other GST fusion proteins as indicated, in 50 µl of the binding buffer (20 mM phosphate buffer, pH7.4, containing 1 mM DTT and 1% Triton X-100). The binding reaction was conducted at room temperature (25 °C) for 30 min. The resins were then washed three times with the binding buffer to remove unbound proteins. The bound proteins were analyzed by SDS-PAGE and visualized by Coomassie Blue staining. The KT3 resin without bound GAPette served as a negative control in these experiments.
Kinetic Competition Assay--
GST-Ras (10 nM) was
bound to the glutathione-Sepharose 4B resin and loaded with
[-32P]GTP in 50 µl of the loading buffer (20 mM Tris-HCl, pH 8, 2 mM EDTA, and 1 mM DTT). The resin was washed twice with the loading buffer
to remove unbound radioactivity and then resuspended in 20 µl of the
same buffer. The GTP hydrolysis reaction was initiated by adding
MgCl2 (to 10 mM) in the absence and presence of
GAPette (10 nM) and various amounts of the Rab5-GTP
S
complex (1.6-16 µM). The reaction was conducted at
25 °C for 10 min, and the products were separated by thin layer
chromatography, followed by autoradiography and quantification of the
radioactive GTP and GDP spots using a PhosphorImager (Molecular
Dynamics). IC50 was determined as the Rab5 concentration
that reduced the GAPette stimulation of Ras GTPase activity by
50%.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The p120 GAP was the very first GAP discovered (20, 21). Its catalytic domain is close to the C terminus of the protein and is termed GAPette (amino acid residues 714-1047) (19, 22). Bacterially expressed recombinant GAPette was purified as described previously (19) and analyzed by SDS-PAGE (Fig. 1). H-Ras, Rab4, Rab5, and Rab5 mutants (Rab5:Q79L and Rab5:S34N) were expressed in bacteria as GST fusion proteins, purified through GST affinity resin (14, 15), and analyzed by SDS-PAGE (Fig. 1). The Ras and Rab proteins could be separated from GST by digestion with the Xa protease. The production of Rab5 by this treatment is shown in Fig. 1.
|
We first determined if GAPette interacts with Rab5 in the biochemical binding assay with the purified proteins. In the assay, GAPette was bound to the KT3 affinity resin, followed by incubation with the purified Rab5 and Rab4 GST fusion proteins. After extensive washing to remove unbound proteins, the bound proteins were analyzed by SDS-PAGE and visualized by Coomassie Blue staining.
Rab5 was able to bind to GAPette in a concentration-dependent manner (Fig. 2A). At the saturation concentration, Rab5 associated with GAPette in about 1:1 stoichiometry, suggesting high affinity of binding. There was little background binding of Rab5 to the KT3 affinity resin alone under the same binding condition (Fig. 2B). Using this binding assay, we further determined if GAPette interacts with the GTP- and GDP-bound Rab5 mutants and other Rab proteins such as Rab4. The Rab5:Q79L mutant is locked in the GTP-bound conformation due to the Gln79 to Leu mutation that decreases the GTPase activity (Fig. 6D) (23, 24). Like Rab5, this Rab5:Q79L mutant was also able to interact with GAPette (Fig. 2B). In contrast, the Rab5:S34N mutant, which contains the Ser34 to Asn mutation and is kept in the GDP-bound state (23, 24), interacted only weakly with GAPette and the binding was greatly reduced by 70% (Fig. 2B). Rab4 did not bind to GAPette under the same condition (Fig. 2B).
|
The interaction between GAPette and Rab5 was further quantified by
using the kinetic competition assay (25). In these experiments, GST-Ras
(10 nM) was loaded with [-32P]GTP, and the
GTP hydrolysis reaction was initiated by the addition of GAPette
(10 nM) in the absence and presence of various amounts of
Rab5 complexed with GTP
S (1.6-16 µM). The
Rab5-GTP
S complex was prepared using the procedure of Schaber and
Gibbs (25). The reactions were conducted at room temperature (25 °C)
for 10 min, and the products were analyzed by thin layer
chromatography.
Competition was observed and Rab5 was a potent inhibitor of the GAPette-Ras interaction (Fig. 3). At 16 µM, Rab5 reduced the GAPette-stimulated GTP hydrolysis of Ras by more than 90% (Fig. 3) and the IC50 value was determined at 6 µM. This high affinity binding to GAPette was comparable with that of the GTP-bound Ras mutant (Ras:Q61L). Although Ras:Q61L interacted with the full-length GAP at an IC50 value of 2 µM (25), the affinity for GAPette was 3-4-fold lower (26). In comparison, the wild-type Ras showed a much lower affinity for the GAP (IC50 = 100 µM). The only other GTPase that showed detectable physical interaction with the Ras GAP was the Rap1 protein. Like Rab5, the wild-type Rap1 was able to bind to the GAP with high affinity (29). However, this physical interaction did not translate into a stimulation of the GTP hydrolysis by Rap1.
|
To determine if GAPette exhibits GAP activity toward Rab5, purified
Rab5 (200 nM) was loaded with [-32P]GTP,
followed by measuring the GTP hydrolysis rate in the presence of
various concentrations of purified GAPette. The reaction products were
analyzed by thin layer chromatography and Fig.
4A shows the conversion of GTP
to GDP after 10 min at room temperature (25 °C). GAPette stimulated
the GTP hydrolysis by Rab5 in a concentration-dependent manner, and this stimulatory effect was evident at a GAPette
concentration as low as 50 nM. Fig. 4B shows the
quantification of the GAPette mediated stimulation of Rab5 GTPase
activity as the amount of GDP produced per min.
|
To ascertain the catalytic nature of the GAPette action, the GAPette
concentration was lowered to 20 nM, while the substrate (Rab5) concentration was increased to 500 nM and loaded
with 200 nM [-32P]GTP. The GTP hydrolysis
reaction was conducted at 25 °C for different times as indicated,
followed by thin layer chromatography to separate the reaction products
(Fig. 5A). The results
indicated again a stimulation of the Rab5 GTPase activity by GAPette
during the time course. This stimulation was quantified as the amount of GDP produced during the time course (Fig. 5B).
|
To characterize further the GAPette activity on Rab5, a second GAP
assay was conducted to compare the GTP hydrolysis by Ras, Rab5, and
other Rab proteins. In these experiments, purified GST-Ras, GST-Rab5,
GST-Rab5:Q79L (a GTP hydrolysis-defective Rab5 mutant), and other GST
Rab proteins were used. These GST fusion proteins (3.6 µM) were bound to GST affinity resin and loaded with
[-32P]GTP. After washing to remove unbound
[
-32P]GTP, GTP hydrolysis was determined in the
absence and presence of GAPette (0.8 µM). In these GAP
assays, Ras was used as a positive control and indeed the GTP
hydrolysis by Ras was strongly stimulated by GAPette (Fig.
6A). Rab5 showed higher
intrinsic GTPase activity (Fig. 6B), consistent with
previous results (23, 24). Importantly the Rab5 GTPase activity was
further stimulated by GAPette (Fig. 6B). The Gln to Leu
mutation at residue 79 (Q79L) inhibited the intrinsic GTPase activity
of Rab5 (Fig. 6C). However, this Q79L mutation did not
completely block the stimulation by GAPette (Fig. 6C),
indicating that the Gln79 residue is not absolutely
necessary for the GAPette-stimulated GTP hydrolysis by Rab5. A similar
result was found for the equivalent Ras mutant (Ras:Q61L) (27).
|
Since this was the first time that a Ras GTPase cycle regulator was shown to act on a Rab protein, it raised a question as to whether GAPette could also act on other Rab GTPases, in addition to Rab5. We determined the GTPase activity of three other Rab proteins (Rab3A, Rab4, and Rab6) in the absence and presence of GAPette, and the results indicated that none of these Rab proteins was sensitive to GAPette (Fig. 6D). Another Rab protein, the yeast Ypt1p (homologous to mammalian Rab1), was also insensitive to the GAP (21). Taken together, the results indicate that GAPette is not a generic GAP for the Rab GTPases.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have identified an unexpected interaction between the catalytic domain (GAPette) of the p120 Ras-GAP and the Rab5 GTPase. The data indicate that GAPette prefers GTP-bound Rab5 to GDP-bound Rab5 for the interaction and this interaction results in a stimulation of Rab5 GTPase activity. In addition to Rab5, it is known that the GAP interacts with Ras and Rap1. In the case of Ras, the GTP-bound Ras:Q61L mutant shows a 50-fold higher affinity for the GAP than the wild-type Ras (28). In the case of Rap1, the GAP is able to bind to the wild-type Rap1 with high affinity, similar to the Rab5-GAP interaction, but does not stimulate its GTPase activity (29). Unlike Rab5, however, Rap1 shares high sequence identity with Ras (50%) and their effector domains are identical (30).
Rab5 shares little sequence homology with Ras outside the GTP-binding domain (31). The finding that Rab5 interacts with a Ras GAP suggests a level of structural similarity that is not predicted from their primary sequences (31). This structural similarity between Rab5 and Ras is not shared by other Rab proteins (Fig. 6). Ras interacts with the GAP through the L1 loop (amino acid residues Gly12 and Gly13) (21), the effector loop (residues 32-40), and the L4 loop (residues 60-65) in Ras (32-35). In these regions, Rab5 is not more similar to Ras than Rab3A, Rab4, Rab6, and Ypt1p (31) that do not interact with and respond to the GAP. We are generating chimeric proteins of Rab5 and other Rab GTPases in order to identify the domains responsible for the GAPette-Rab5 interaction. Further mechanistic understanding may require solving the atomic structures of Rab5 and other Rab GTPases and making comparisons to the Ras structure (35).
At the cellular level, the data imply a signal transduction mechanism controlling the endosome dynamics. Like Rab5, p120 GAP is widely distributed in many cell types. It is also an important intermediate during receptor tyrosine kinase-mediated signal transduction processes (36-39) many of which ultimately promote cell proliferation. The intracellular localization and activity of p120 GAP is highly regulated during the signal transduction processes (36-39). In the case of EGF signal transduction, the ligand-bound EGF receptor recruits the cytosolic p120 GAP to the plasma membrane where it may interact with Ras. The EGF receptor is then rapidly endocytosed to intracellular endosomes. The p120 GAP remains associated with the endocytosed EGF receptor and is localized on the cytoplasmic side of endosomes (40), where Rab5 is localized (11). This physical co-localization suggests that p120 GAP may provide a temporal control of the Rab5 GTPase cycle and endosome fusion at this stage of the signal transduction process. Endosome fusion is a dynamic process that ceases during mitosis (16-18). It is an attractive possibility that p120 GAP may contribute to the regulation of Rab5 and endosome fusion in preparation for cell proliferation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Zhimin Liang for technical assistance, Gideon Bollag for reagents and comments on the manuscript, and Richard Cummings and Robert Steinberg for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by an Oklahoma Center for the Advancement of Science and Technology grant (to G. L.).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.: 405-271-2227 (ext. 1232); Fax: 405-271-3092; E-mail: guangpu-li{at}ouhsc.edu.
1
The abbreviations used are: GAP,
GTPase-activating protein; GAPette, the catalytic domain of the p120
GAP; GST, glutathione S-transferase; DTT, dithiothreitol;
PAGE, polyacrylamide gel electrophoresis; GTPS, guanosine
5'-O-(3-thiotriphosphate).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|