From the Department of Biology, Faculty of Science,
and Graduate School of Science and Technology, Chiba University,
Yayoicho, Inageku, Chiba 263-8522, the § Division of
Biochemistry and ¶ Division of Cancer Genomics, Institute of
Medical Science, University of Tokyo, Shirokanedai, Minatoku, Tokyo
108-8639, and ** CREST and
PRESTO, Japan
Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan
Received for publication, August 25, 2002, and in revised form, November 1, 2002
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ABSTRACT |
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Macropinocytosis is an efficient process for the
uptake of nutrients and solute macromolecules into cells from the
external environment. Macropinosomes, which are surrounded by actin,
are formed from the cell surface membrane ruffles and migrate toward the cell center. We have cloned the entire coding sequence of a member
of the Rab family small GTPases, Rah/Rab34. It lacked a consensus
sequence for GTP-binding/GTPase domain. Although wild-type Rah
exhibited extremely low GTPase activity in vitro, it
exerted appreciable GTPase activity in vivo. In
fibroblasts, Rah was colocalized with actin to the membrane ruffles and
membranes of relatively large vesicles adjacent to the ruffles. These
vesicles were identified as macropinosomes on the basis of several
criteria. Rah and Rab5 coexisted in some, but not all, macropinosomes.
Rah was predominantly associated with nascent macropinosomes, whereas
Rab5 was present in endosomes at later stages. The number of
macropinosomes in the cells overexpressing Rah increased about 2-fold.
The formation of macropinosomes by the treatment of platelet-derived
growth factor or phorbol ester was also facilitated by Rah but
suppressed by a dominant-negative Rah. Rah-promoted macropinosome
formation was retarded by dominant-negative mutants of Rac1 and WAVE2,
which are essential for membrane ruffling. These results imply that Rah
is required for efficient macropinosome formation from the membrane ruffles.
Endocytosis in eukaryotic cells serves to maintain cellular and
organismal homeostasis by taking up fluids and macromolecules, signaling molecules, and their receptors from the external environment (1). There are at least five independent endocytic processes: clathrin-dependent endocytosis mediated by clathrin-coated
vesicles (100-150 nm in diameter), caveolin-dependent
endocytosis mediated by caveolae (50-80 nm), clathrin- and
caveolin-independent endocytosis, macropinocytosis, and phagocytosis
(2, 3). Among these processes, macropinocytosis is carried out with
relatively large vesicles (0.2-5 µm in diameter) formed from cell
surface membrane ruffles folding back on the plasma membrane (4, 5).
Macropinosomes are not coated with clathrin or caveolin but surrounded
by actin at early stages. Macropinocytosis provides an efficient
process for non-selective uptake of nutrients and solute
macromolecules. It also accounts for internalization of extracellular
antigens by professional antigen-presenting cells like dendritic cells. Furthermore, macropinocytosis is postulated to play important roles in
chemotaxis by regulating plasma membrane-actin cytoskeleton interaction
and membrane trafficking. Some pathogenic bacteria such as
Salmonella typhimurium and Shigella flexneri also
exploit macropinocytosis to invade the cells (6).
Treatment of various types of cultured cells with growth
factors, cytokines, phorbol esters such as phorbol 12-myristate
13-acetate (PMA),1 or
diacylglycerol elicits rapid and dramatic membrane ruffling and
macropinocytic responses (7-13). Introduction of small GTPases, Ras or
Rac1, or Tiam1, a guanine nucleotide exchange factor (GEF) for Rac1,
also induces membrane ruffling and macropinocytosis in fibroblasts
(13-15). The dominant-negative mutant Rac1(T17N) interferes with
membrane ruffling and macropinosome formation induced by growth
factors, PMA, Ras, or Tiam1 (13, 15). This implies that the ruffling
and macropinosome formation by these agents are mediated by Rac1.
Rac1 causes membrane ruffling through its target protein IRSp53, which
activates WAVE2/Scar2 (16-18). WAVE2 is involved in the formation of
branched actin filament meshwork in membrane ruffles by activating
Arp2/3 complex (18-20).
Rab family small GTPases play essential roles in the endocytic and
exocytic pathways. It is well established that Rab proteins function in
the targeting and docking of the vesicles to their acceptor membranes.
However, it has become evident that at least some of them exert their
functions in multiple steps of vesicular trafficking, including vesicle
formation, vesicle motility, membrane remodeling, and vesicle fusion as
well as vesicle targeting and docking (21-24). In mammalian cells,
>50 members of Rab family proteins have been identified.
These proteins are associated with particular vesicle
membrane compartments and function in specific stages of the diverse
vesicle trafficking events. Among them, Rab5 is located to the
membranes of clathrin-coated vesicles and early endosomes. It is
involved in receptor-mediated endocytosis and fluid-phase pinocytosis
(25-27). Introduction of a constitutively active Rab5 mutant in cells
stimulates the rate of endocytosis and homotypic fusion of early
endosomes, whereas its dominant-negative mutant prevents the vesicle
fusion (26). Rab4 is also present in early endosome membrane and
implicated in recycling pathway from endosomes to the plasma membrane
(28). Rab7 is present in late endosome membrane and participates in
transport from early to late endosomes and lysosomes (29, 30). Although
a mutant Rab5 protein defective in GTP-binding ability retards
fluid-phase pinocytosis in addition to receptor-mediated endocytosis
(25), there has been no report indicating that any Rab family proteins are specifically located to the membrane ruffles or macropinosomes and
play direct roles in macropinosome formation.
Rah is a small GTPase postulated to be a member of Rab family due to
the similarity of its effector domain and C-terminal sequences to those
of several Rab proteins (31). Because only a truncated cDNA lacking
the sequence encoding N-terminal portion was available (31), we have
cloned in the present study the cDNA containing the entire coding
region of mouse Rah. Rah was colocalized with actin to the membrane
ruffles and macropinosome membrane. During macropinosome biogenesis,
Rah associated with nascent macropinosomes seemed to be replaced by
Rab5 on the early endosomes. Overexpression of Rah elevated the number
of macropinosomes, whereas the expression of a dominant-negative form
of Rah prevented the formation of macropinosomes induced by
platelet-derived growth factor (PDGF) or PMA. On the other hand,
Rah-promoted macropinosome formation was retarded by dominant-negative
mutants of Rac1 and WAVE2. Thus, Rah is likely to play crucial roles in
the formation of macropinosomes from the membrane ruffles.
Cloning of Rah and Rab5--
Mouse heart total RNA was prepared
according to Chomczynski and Sacchi (32), and poly(A)+ RNA
was isolated from the total RNA with Oligotex-dT30 Super (Roche
Diagnostics). A single-stranded cDNA pool was synthesized with
SuperScript II RNase H( Expression and Purification of Recombinant Proteins--
Point
mutations to generate Rah(Q111L) and Rah(T66N) analogous to a
constitutively active form of Ras(Q61L) and a dominant-negative form of
Ras(S17N), respectively, were introduced in Rah cDNA with a
Transformer site-directed mutagenesis kit
(Clontech). Similarly, point mutations to generate
constitutively active Rab5(Q79L) and dominant-negative Rab5(S34N) were
introduced in Rab5 cDNA. Coding sequences of the wild-type (wt) or
the mutated Rah and Rab5 were fused in-frame to glutathione
S-transferase (GST) in pGEX-2T (Amersham Biosciences). These
recombinant fusion proteins were expressed in Escherichia
coli strain XL1-Blue and affinity-purified with glutathione-Sepharose (Amersham Biosciences) as described previously (34).
GTP Binding/GTPase Assay--
An in vitro
GTP binding/GTPase assay of the recombinant GST fusion proteins by
thin-layer chromatography was carried out as described previously (33).
Briefly, each GST fusion protein bound to glutathione-Sepharose was
incubated with 370 kBq of [
An in vivo GTP binding/GTPase assay was conducted
essentially according to Muroya et al. (35). The entire
coding region of the wt or the constitutively active Rah and Rab5 was
fused in-frame to the Myc tag in pEF-BOS vector. COS-1 cells (36) cultured in Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum (growth medium) were transfected with these plasmids by
using FuGENE 6 transfection reagent (Roche Molecular Biochemicals).
Twelve hours after the transfection, the medium was replaced with
phosphate-free Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum dialyzed against 0.15 M NaCl and 10 mM HEPES-NaOH (pH 7.9). Phosphorus 32 (32Pi) (ICN Biomedicals, Inc.) was added to the
medium at 9.25 MBq/ml and incubated for a further 12 h. The cells
were lysed with the lysis buffer (150 mM NaCl, 20 mM MgCl2, 50 mM Tris-HCl, pH 7.5, 0.5% Nonidet P-40, 1 mM Na3VO4,
and 2 µg/ml aprotinin). The anti-Myc monoclonal antibody Myc1-9E10
(37) (American Type Culture Collection) was added to the cell lysates
and incubated at 4 °C for 60 min, and then Protein G-Sepharose 4 Fast Flow (Amersham Biosciences) was mixed and incubated for a further
60 min. After thorough washing with the lysis buffer, bound nucleotides
were dissociated with the dissociation buffer (20 mM
Tris-HCl, pH 7.5, 20 mM EDTA, 2% SDS, 0.5 mM
GDP, and 0.5 mM GTP) at 65 °C for 5 min. Four
microliters each of the dissociated samples was analyzed by thin-layer
chromatography followed by autoradiography.
Epitope Tagging, EGFP Tagging, and Fluorescence
Microscopy--
Entire coding region of the wt or the mutated Rah and
Rab5 were fused in-frame to the Myc tag in pCMVmyc vector (33), to the
hemagglutinin (HA) tag in pEF-BOS vector, and to the enhanced green
fluorescent protein (EGFP) sequence in pEGFP-C1 vector
(Clontech). Human WAVE2 and its dominant-negative
mutant WAVE2( Time-lapse Microscopy--
Living 10T1/2 cells transfected with
the pEGFP-C1 constructs were observed with a Zeiss Axiovert 135TV
microscope equipped with phase-contrast and epifluorescence optics. The
EGFP fluorescence was recorded through a CoolSNAP digital
charge-coupled device camera (Roper Scientific) and analyzed by
IPLab/Mac imaging software (Scanalytics, Inc.).
Sequence and Functional Domains of Rah--
Because Rah cDNA
available at that time had lacked the sequence corresponding to the
N-terminal region (31), we tried to obtain cDNA containing the
entire coding region and cloned a 1.6-kb cDNA (accession number
AB082927) from the mouse C2 skeletal muscle myoblast cDNA library.
Because there was an in-frame termination codon (T416AA) upstream of
A503TG in this clone, this ATG is most likely to serve the initiation
codon. The coding region of the former truncated clone contains 208 amino acids (31), whereas that of this clone consisted of 259 amino
acids and its calculated molecular weight was 29,128. Because Rah was
most similar to Rab family proteins among small GTPase families and its
amino acid sequence was identical to that of mouse Rab34 (accession
number BC038638, the data of which were submitted while we were
submitting this report), we compared its amino acid sequence
with those of several other Rab family proteins (Fig.
1A). Rah/Rab34 had the longest
N-terminal sequence among Rab family proteins except for Rab36 (see
Fig. 1B). Small GTPases generally contain conserved four
sequence motifs for GTP/GDP-binding and GTPase activities (42, 43).
Although Rah contained the first three motif sequences, it lacked the
consensus sequence for the fourth motif (EXSA)
(X, any amino acid) (Fig. 1A). The sequence of
the putative effector domain of Rah was distinct from those of the
other Rab family proteins, suggesting that Rah exerts unique cellular
functions. The C termini of unprocessed Rab proteins usually end with
-CC, -CCX1-3, or -CXC, and
the cysteine residues in these motifs are postulated to be
geranylgeranylated (44). Because the C-terminal end of Rah was
-CCP, it coincides with the motifs.
Rab36 is a recently identified Rab family protein encoded by the gene
locating at 22q11.2, but its function has totally been unknown (45). It
is the largest Rab family small GTPase to date consisting of 333 amino
acids with extremely long N-terminal sequence (Fig. 1B). Rah
was most closely related to Rab36, and the identity between the two
proteins was 57% over the entire length of Rah (Fig. 1B).
Even the long N-terminal portion of Rah showed a similarity to the
corresponding region of Rab36. Notably, the sequences of their putative
effector domains were identical, and, in addition, sequences around the
effector domains were also relatively well conserved between these two
proteins. The fourth motif for GTP/GDP binding was abrogated also in
Rab36. The consensus sequence for phosphorylation by tyrosine kinases
((R/K)X2-3(D/E)X2-3Y) was present in Rah as well as in Rab36. This putatively
phosphorylatable tyrosine corresponds to the first amino acid of
the effector domain. This might suggest that binding of target proteins
to Rah and Rab36 is regulated by the tyrosine phosphorylation.
Rah Exerts Low GTPase Activity in Vitro but Substantial Activity in
Vivo--
Small GTPases basically exhibit GTP/GDP-binding and
intrinsic GTPase activities. We analyzed these activities of Rah and
Rab5 in vitro by thin-layer chromatography. Point mutations
were introduced in the cDNA to generate Rah(Q111L) and Rah(T66N),
which are equivalent to a constitutively active form (a mutant
deficient in GTP hydrolysis) of Ras(Q61L) or Rab5(Q79L) (26) and a
dominant-negative form (a mutant stabilized in GDP-bound
form) of Ras(S17N) or Rab5(S34N) (26), respectively. The GST-tagged
proteins of the wt and these mutants expressed in E. coli
were used for the analyses of the activities. Although Rab5(wt) easily
hydrolyzed bound GTP into GDP, Rah(wt) hydrolyzed bound GTP much less
efficiently (Fig. 2A).
Rah(Q111L) and Rab5(Q79L) were deficient in GTP-hydrolyzing activity.
Rah(T66N) was lacking in GTP-binding ability as was Rab5(S34N). The low
intrinsic GTPase activity of Rah(wt) might be ascribable to the lack of
the fourth motif for GTP/GDP-binding and GTPase activities.
We further analyzed these activities of Rah and Rab5 in
vivo. Myc-tagged wt and constitutively active mutants of Rah and
Rab5 were transiently transfected to COS-1 cells. Each of these
proteins labeled with 32Pi was
immunoprecipitated with the anti-Myc mAb, and the bound guanine
nucleotides were analyzed by thin-layer chromatography. Rab5(wt) bound
a high amount of GDP and much less GTP, whereas Rab5(Q79L)
bound almost equivalent amounts of GTP and GDP (Fig. 2B).
Remarkably, Rah(wt) bound a low amount of GTP and a high amount of GDP
as did Rab5(wt). Furthermore, Rah(Q111L) bound less GTP than did
Rab5(Q79L). These results imply that, in contrast with the low GTPase
activity in vitro, Rah(wt) exhibits a considerable level of
GTPase activity in vivo comparable to the activity of Rab5(wt). They also indicate that even the constitutively active mutants exert appreciable GTPase activity in vivo. This is
presumably due to the presence of GTPase-activating protein (GAP) in
cells. Indeed, GAP activity stimulates GTPase activity of the
constitutively active Rab3A in vivo (46). Thus, specific
GAPs are likely to exert their functions not only on Rab5(wt)
but also on Rah(wt) and on both these constitutively active mutants
in vivo.
Rah Is Colocalized with Actin to Membrane Ruffles and Nascent
Endosome Membranes--
To infer cellular functions of Rah, we first
determined the localization of Rah. Myc-epitope-tagged or EGFP-tagged
Rah and its point mutants were transiently expressed in mouse 10T1/2
fibroblasts by transfection. Myc-tagged wt Rah was predominantly
located to the cell surface membrane ruffles and membranes of
relatively large (usually several µm in diameter) vesicles adjacent
to the ruffles (Fig. 3, a and
b). Staining with rhodamine-phalloidin showed that actin
coexisted with Rah in these membrane structures (Fig. 3, c
and d). Myc-Rah(Q111L) was also located to these membrane structures, and the phenotype of Myc-Rah(Q111L)-expressing cells was
indistinguishable from that of the Myc-Rah(wt)-expressing cells (Fig.
3, e-h). On the other hand, most of the
Myc-tagged dominant-negative Rah(T66N) was diffusely distributed
throughout the cytoplasm and also associated with membrane ruffles but
not with the vesicles, even if they existed (Fig. 3,
i-l). EGFP-tagged Rah(wt) and the point mutants
distributed in the cells similarly to each of the Myc-tagged proteins
(data not shown).
Rab5 is located to the membranes of early endosomes, and expression of
the constitutively active Rab5(Q79L) causes the formation of unusually
large endosomes by the homotypic fusion of early endosomes (25, 26). To
identify the Rah-associated vesicles, Myc- or EGFP-tagged Rab5(wt),
Rab5(Q79L), and the dominant-negative Rab5(S34N) were expressed in
10T1/2 cells. Both Rab5(wt) and Rab5(Q79L) were localized to the early
endosome membranes, and actin was associated with the membranes of only
some but not all of the endosomes (Fig.
4A,
a-f). The endosomes associated with actin seem
to be macropinosomes and those without actin association are likely to
be either receptor-mediated endosomes or macropinosomes at later
stages. Expression of Rab5(Q79L) resulted in the formation of large
endosomes as much as 10-20 µm in diameter (Fig. 4A,
d-f). In contrast, Rab5(S34N) was apparently
diffusely distributed throughout the cytoplasm (Fig. 4A,
g-i), consistent with the former results (26).
None of these Rab5 proteins were integrated into the membrane ruffles.
Coexpression of Myc-Rah and EGFP-Rab5(Q79L) showed that these two Rab
family proteins coexisted in a subset of vesicular membranes (Fig.
4B). Furthermore, there were vesicles containing either Rah
or Rab5(Q79L). The Rah-containing vesicles were present peripherally in
the cells, whereas the Rab5-containing endosomes occupied more central
areas of the cells. In these coexpressing cells, Rah but not Rab5 was
associated with membrane ruffles. These results suggest that the
Rah-associated vesicles are endosomes and that they are nascent
endocytic vesicles preceding early endosomes or are a part of early endosomes.
To further analyze the temporal and spatial relationship between Rah-
and Rab5-associated endosomes, we examined whether Rah and Rab5 were
colocalized to the endosome membranes with Lamp1, a marker membrane
protein for late endosomes and lysosomes (41). Cells transfected with
EGFP-Rah or EGFP-Rab5(Q79L) were stained with the anti-Lamp1 mAb H4A3.
The antibody detected small vesicles in the central areas of the cells
but did not the Rah-associated peripheral endosomes (Fig.
5A,
a-c). In Rab5(Q79L)-expressing cells, some of
the Rab5(Q79L)-associated enlarged vesicles were labeled by the
anti-Lamp1 (Fig. 5A, d-f). In
addition, enlarged vesicles that did not contain Rab5(Q79L) were also
labeled by the antibody. These results corroborate the above
postulation that Rah-associated vesicles are nascent endocytic vesicles
or a part of early endosomes.
Rah Is Associated with Macropinosome Membrane--
The vesicles
where Rah is located are predicted to be macropinosomes among various
types of endosomes because of their size, the location close to the
membrane ruffles, and coexistence with actin (4). Because fluorescent
dextrans are used to detect fluid-phase pinocytosis (47), we added
RITC-conjugated dextran to the culture medium of Rah- or
Rab5(Q79L)-transfected 10T1/2 cells and incubated for 1 h.
RITC-dextran was taken up in many of the Rah-associated vesicles (Fig.
5B, a-c). It was incorporated into
some of the Rab5(Q79L)-associated enlarged vesicles as well (Fig.
5B, d-f).
Macropinosomes are formed from the membrane ruffles (4). To determine
whether Rah-associated endosomes were formed from the membrane ruffles,
we exploited time-lapse microscopy on the living cells expressing
EGFP-Rah. In the EGFP-Rah-expressing cells, EGFP fluorescence was
detected at the membrane ruffles (Fig.
6). Relatively large vesicles were
generated at the membrane ruffles and they migrated away from the cell
margin toward the cell center (Fig. 6). Taken together, these results
imply that the Rah-associated vesicles are macropinosomes.
Rah Facilitates the Formation of Macropinosomes--
Next we
examined which step of macropinocytosis Rah is involved in,
i.e. in the formation of macropinosomes, in the movement of
macropinosomes, or in any other steps. For convenience, relatively large vesicles coated by actin, which was recognized by the staining with rhodamine-phalloidin, were regarded as macropinosomes. When EGFP-Rah(wt) or EGFP-Rah(Q111L) was expressed in 10T1/2 cells, the
number of actin-coated macropinosomes per cell increased more than
2-fold over a mock-transfected cell (Fig.
7A). On the other hand,
expression of EGFP-Rah(T66N) resulted in a slight decrease in the
number of macropinosomes per cell. Consequently, exogenously expressed
Rah facilitates the macropinosome formation over the control level, and
intrinsic Rah may contribute to a limited degree to the control level
formation. In contrast with the number of macropinosomes, the degree of
membrane ruffling was not affected by the expression of any of these
Rah proteins (see Figs. 3-6).
Treatment of diverse types of cultured cells with growth factors or PMA
induces prominent membrane ruffling and subsequent macropinocytosis
(7-9, 11-13). When 10T1/2 fibroblasts were treated with PDGF or PMA,
membrane ruffling and macropinosome formation were
facilitated (Fig. 7B). The number of macropinosomes per cell elevated about 2-fold over a control level. To elucidate the
relationship between Rah-promoted macropinocytosis and PDGF- or
PMA-induced macropinocytosis, EGFP-Rah(wt)-transfected cells were
treated with PDGF or PMA. The number of macropinosomes per cell
elevated more than 3-fold over the control level in either case (Fig.
7B). Next, EGFP-Rah(T66N)-transfected cells were treated
with each of these reagents. Expression of the dominant-negative Rah
highly reduced the number of actin-associated macropinosomes per cell both in the PDGF- and the PMA-treated cells, but the levels remained higher than the control level (Fig. 7B). These results
indicate that Rah participates in the macropinosome formation
synergistically to these reagents.
Introduction of Ras or Rac1 induces membrane ruffling and
macropinocytosis (13, 14). WAVE2 activated by IRSp53, a target protein
of Rac1, is responsible for the Arp2/3 complex-mediated formation of
the branched actin filament meshwork in membrane ruffles (16-18, 20).
In addition, Dictyostelium discoideum Scar, an ortholog of
mammalian WAVE2, is required for macropinocytosis (48). Thus, we
examined the relationship among Ras, Rac1, WAVE2, and Rah in
macropinocytosis. The number of actin-associated macropinosomes induced
by Rah was reduced to ~72% by the coexpression of the dominant-negative H-Ras(S17N), but the level was over the control level
(Fig. 7C). In contrast, coexpression of the
dominant-negative Rac1(T17N) resulted in remarkable reduction in the
number of macropinosomes to ~8%. EGFP-tagged WAVE2 coexisted with
Rah in the membrane ruffles and macropinosomes (Fig. 7D).
When FLAG-tagged WAVE2( We have cloned the entire coding sequence of Rah/Rab34. It
consists of 259 amino acids, and its large size is ascribed to its long
N-terminal sequence. Usually the N- and C-terminal sequences are
diverged even among the same family of small GTPases. Rah was most
closely related to Rab36, the largest Rab family protein, over its
entire length and in its N terminus. Although roles of the N-terminal
sequences of small GTPases have not been well understood, some small
GTPases bind particular proteins at their N termini. For instance, RalA
interacts at its N terminus with phospholipase D1 in collaboration with
Arf1 (49, 50). Rnd1 and RhoE interfere with both Rho- and Rac-mediated
reorganization of the actin cytoskeleton. Deletion of the N-terminal
six amino acids in Rnd1, however, results in deprivation of the
antagonistic effects on the cytoskeleton (51). In addition, Rnd2, which
lacks the N-terminal extending sequence present in RhoE and Rnd1, has
no observable effects on the cytoskeleton (51). Thus, the long
N-terminal sequence of Rah seems to be involved in the interaction with
some proteins to exert its cellular functions. The similarity between
the N terminus of Rah and the corresponding sequence of Rab36 raises the possibility that these proteins share the proteins interacting with
these sequences. Furthermore, Rah and Rab36 exhibit the identity between their core effector domains and close similarity between their
extended effector domains. This may imply that these proteins also
share several target proteins.
Although small GTPases have four conserved motifs for GTP/GDP-binding
and GTPase activities, Rah lacked the fourth motif. In addition, the
fourth motif was abrogated in Rab36 as well. The low intrinsic GTPase
activity of Rah(wt) in vitro might be ascribable to the lack
of this motif. However, Rah(wt) exhibited substantial GTPase activity
in vivo comparable to the activity of Rab5(wt). In addition,
even the constitutively active Rah(Q111L) and Rab5(Q79L) exerted
appreciable GTPase activity in vivo. This is presumably due
to the presence of specific GAP activities in cells as has been shown
with Rab3A (46). Particularly, the GAP activity acting on Rah seems to
be strong, because Rah(Q111L) showed higher GTPase activity than did
Rab5(Q79L) and almost comparable to that of Rah(wt) in vivo.
Furthermore, certain functions, including endocytic ability, are
indistinguishable between Rab5(wt) and Rab5(Q79L) (52, 53). This may
explain why the phenotype of the cells transfected with Rah(wt) and
that with Rah(Q111L) were indistinguishable.
Rah was colocalized with actin to the membrane ruffles and
membranes of relatively large vesicles adjacent to the ruffles. In
addition, time-lapse microscopy showed that the vesicles were actually
formed from the membrane ruffles. These vesicles are identified as
macropinosomes because of their large size, formation from the membrane
ruffles, incorporation of dextran from the medium, and colocalization
with actin. Although >50 members of Rab family proteins have been
identified in mammalian cells (22-24), Rah is the first Rab family
protein, to our knowledge, associated specifically with both the
membrane ruffles and macropinosome membranes. Rab5 is located to the
membranes of clathrin-coated vesicles and early endosomes. It
participates in receptor-mediated endocytosis and fluid-phase
pinocytosis (25-27). Coexpression of Rah and Rab5(Q79L) showed that
Rah but not Rab5 was associated with membrane ruffles. Although Rah and
Rab5 were colocated in some vesicles, the Rah-containing vesicles were
generally present more peripherally in the cells, whereas the
Rab5-containing vesicles occupied more central areas of the cells.
These results suggest that, during macropinosome biogenesis, Rah acts
at early stages and Rab5 functions at later stages and that Rah is
replaced by Rab5 on macropinosomes.
The formation of membrane ruffles and macropinosomes are
induced by the treatment of cells with growth factors or PMA (7-9, 11-13). Introduction of constitutively activated Ras or Rac1 or Tiam1,
a GEF for Rac1, also results in prominent membrane ruffling and the
formation of macropinosomes (13-15). This action of Ras is mediated by
Rac1, because the dominant-negative Rac1(T17N) prevents this effect of
Ras (13). Tiam1 is directly associated with GTP-bound Ras and causes
activation of Rac1 in a phosphatidylinositol 3-kinase
(PI3K)-independent manner (54). Alternatively, PI3K, a target protein
of Ras, may link Ras with Rac1 through its lipid product
phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). This lipid binds to and activates Vav and Sos, which serve as the GEFs
for Rac1 (55, 56). Rac1 activated by these pathways, in turn, induces
membrane ruffling by activating WAVE2 through the target protein IRSp53
(16-18). Induction of membrane ruffling and macropinocytosis by a
short-term treatment of cells with PMA is mediated by Rac (13) or
presumably by protein kinase C
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) reverse transcriptase (Invitrogen) from the
poly(A)+ RNA primed with an oligo(dT) primer. A Rah
cDNA fragment was cloned from the cDNA pool by reverse
transcription-polymerase chain reaction (RT-PCR) with Taq
DNA polymerase (Qiagen) by using a sense primer
5'-AAGGTCATCGTTGTGGGAGA-3' (nucleotides 7-26 of the
DDBJ/EMBL/GenBankTM accession number S72304) and an
antisense primer 5'-GGGACAACATGTGGCCTTTT-3' (nucleotides 605-624). The
cDNA library of the mouse skeletal muscle cell line C2 myoblasts
constructed in
ZAPII (33) was screened with the PCR product. Rab5b
cDNA containing the entire coding region was cloned from the heart
cDNA pool by RT-PCR using a sense primer 5'-GGAGGACATATGACTAGCAG-3'
(nucleotides 42-61 of the accession number X84239) and an antisense
primer 5'-CACCCCTCAGTTGCTACAAC-3' (nucleotides 685-704). Mouse H-Ras
and Rac1 cDNAs were cloned as described previously (33). The
nucleotide sequence of the cloned cDNAs was determined with a
LI-COR LI-COR 4000 automated DNA sequencing system using a SequiTherm
Long-Read Cycle Sequencing Kit-LC (Epicentre Technologies). The
nucleotide and amino acid sequences were analyzed with GENETYX-Mac
software (version 10.1, Software Development Co.).
-32P]GTP (>111 TBq/mmol,
ICN Biomedicals, Inc.) in the incubation buffer (50 mM
NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.1 mM EGTA, 0.1 mM dithiothreitol (DTT), and 10 µM ATP) at 37 °C for 10 min. After washing with the
wash buffer (20 mM MgCl2, 50 mM Tris-HCl, pH 7.5, 1 mM DTT, and 1 mg/ml bovine serum
albumin), a GTPase reaction was carried out in the assay buffer (50 mM NaCl, 5 mM MgCl2, 50 mM Tris-HCl, pH 7.5, 5 mM DTT, and 1 mM GTP) at 37 °C for 30 min and stopped by the addition
of the ice-cold wash buffer. After washing with the wash buffer, bound
nucleotides were eluted by incubating the Sepharose beads in 1% SDS
and 20 mM EDTA at 65 °C for 5 min. Dissociated samples
were spotted onto polyethyleneimine cellulose plates (Macherey-Nagel).
The released nucleotides were resolved by thin-layer chromatography in
0.75 M KH2PO4 and visualized by autoradiography.
V) were fused to the FLAG tag in pEF-BOS vector and to
EGFP in pEGFP-C1 vector (38). The mouse C3H/10T1/2 (10T1/2) fibroblasts
(39), cultured on glass coverslips in the growth medium, were
transfected with these plasmids by the calcium phosphate-mediated
method as described previously (40). To detect macropinosomes,
rhodamine B isothiocyanate (RITC)-dextran (Mr ~ 70,000, Sigma) was added to the medium at the concentration of 1 mg/ml. To induce macropinocytosis, the cells were treated with 5 units/ml PDGF (Calbiochem) or 0.1 µM PMA (Sigma) for 30 min. The transiently transfected cells were processed for fluorescence
microscopy 24 h after the transfection (40). The fixed and
permeabilized cells transfected with Myc-, HA-, and FLAG-tagging
constructs were incubated with the mAb Myc1-9E10, anti-HA rabbit
polyclonal antibody (MBL), and anti-FLAG tag mAb (Sigma), respectively.
Then they were incubated with fluorescein isothiocyanate- or
rhodamine-conjugated goat anti-mouse or anti-rabbit IgG
(affinity-purified, Cappel). Actin filaments were detected by the
staining with rhodamine-phalloidin (Molecular Probes, Inc.). Lamp1 was
detected by the staining with mAb H4A3 (41) (Developmental Studies
Hybridoma Bank) followed by the incubation with rhodamine-goat anti-mouse IgG. The specimens were observed with a Zeiss Axioskop microscope equipped with phase-contrast and epifluorescence optics. The
number of macropinosomes was estimated by counting relatively large
vesicles (0.2-5 µm in diameter) surrounded by actin in >100 transfected cells in each transfection experiment.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequence homology among Rah and
other Rab family proteins. A, the amino acid sequence
of mouse Rah and comparison of its sequence with those of several Rab
family proteins. The N-terminal sequence of Rah upstream from Ser-53,
which is indicated by an arrow, is not included in the
previously reported truncated clone (31). The first five nucleotides
(AATTC) in the truncated cDNA clone in Morimoto et al.
(31) seem to be derived from an EcoRI linker. The compared
Rab family proteins are human Rab1 (accession number, M28209), Rab3A
(M28210), Rab4 (M28211), Rab5 (M28215), Rab7 (X93499), and Rab9
(U44103). The amino acid sequences are aligned for search
similarity using the method of Lipman and Pearson (64) and by eye.
Amino acids at positions of >50% identity are shown in
white on black. G1-G4,
conserved core motifs for GTP/GDP-binding and GTPase activities;
E, effector domain; asterisks, conserved
C-terminal Cys residues. B, comparison of Rah and Rab36
sequences.
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Fig. 2.
GTP-binding and GTPase activities of Rah and
Rab5. A, in vitro GTP-binding/GTPase assay.
GST-tagged wt, constitutively active mutants (CA), and
dominant-negative mutants (DN) of Rah and Rab5 were loaded
with [ -32P]GTP and incubated for 30 min at 37 °C.
Bound GTP/GDP were analyzed by thin-layer chromatography. The positions
of GTP and GDP are indicated. B, in vivo
GTP-binding/GTPase assay. Myc-tagged wt and constitutively active
mutants (CA) of Rah and Rab5 as well as the empty vector
(control) were transiently transfected to COS-1 cells. The cells were
incubated with 32Pi for 12 h to label the
proteins. Each of these proteins was immunoprecipitated with the
anti-Myc mAb and the bound GTP/GDP were analyzed by thin-layer
chromatography.
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Fig. 3.
Colocalization of Rah with actin to membrane
ruffles and membranes of large vesicles adjacent to the ruffles.
Myc-tagged Rah(wt) (a-d), Rah(Q111L)
(e-h), or Rah(T66N) (i-l)
was expressed in 10T1/2 cells. The cells were doubly stained with
anti-Myc mAb Myc1-9E10 and rhodamine-phalloidin. Shown are
phase-contrast images (a, e, and i),
Myc1-9E10 staining locating Rah (b, f, and
j), rhodamine-phalloidin staining locating actin filaments
(c, g, and k), and their merged
fluorescent images (d, h, and l). In
the merged images, colocalization of Rah and actin filaments is seen in
yellow. Arrowheads, chevrons, and
arrows point to the membrane ruffles, vesicles neighboring
the ruffles, and vesicles away from the ruffles, respectively.
Scale bar, 20 µm.
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Fig. 4.
Partial coexistence of Rah and Rab5 in
endosome membranes. A, localization of Rab5 in endosome
membranes. Myc-tagged Rab5(wt) (a-c), Rab5(Q79L)
(d-f), or Rab5(S34N)
(g-i) was expressed in 10T1/2 cells. The cells
were doubly stained with Myc1-9E10 to locate Rab5 (a,
d, and g) and rhodamine-phalloidin to locate
actin filaments (b, e, and h). In the
merged images (c, f, and i),
colocalization of Rab5 and actin filaments is seen in
yellow. Only some but not all of the Rab5-linked endosomes
are associated with actin. Arrows point to the Rab5-linked
endosomes partially colocalized with actin. B, distribution
of Rah- and Rab5-containing vesicles. EGFP-tagged Rab5(Q79L) and
Myc-Rah were coexpressed in 10T1/2 cells. Shown are EGFP fluorescence
locating Rab5(Q79L) (a and d), Myc1-9E10
staining locating Rah (b and e), and their merged
images (c and f). Arrowheads,
chevrons, white arrows, and black
arrows point to the Rah-containing membrane ruffles, the vesicles
exclusively containing Rah, those containing both Rah and Rab5, and
those exclusively containing Rab5, respectively. Scale bar,
20 µm.
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Fig. 5.
Association of Rah with nascent
macropinosomes but not with late endosomes or lysosomes.
A, distribution of Rah and Rab5 compared with that of Lamp1
in late endosomes and lysosomes. EGFP-tagged Rah
(a-c) or Rab5(Q79L) (d-f)
was expressed in 10T1/2 cells. Shown are EGFP fluorescence
(a and d), anti-Lamp1 staining (b and
e), and their merged images (c and f).
Arrows indicate late endosomes, where Rab5(Q79L) and Lamp1
coexist. A chevron denotes putative lysosome, where
Lamp1 but not Rab5(Q79L) exists. B, incorporation of
rhodamine-dextran in Rah- or Rab5-associated endosomes. EGFP-tagged Rah
(a-c) or Rab5(Q79L) (d-f)
was expressed in 10T1/2 cells. RITC-dextran was added to the medium and
incubated for 1 h. Shown are EGFP fluorescence (a and
d), RITC-dextran incorporation (b and
e), and their merged images (c and f).
Arrows point to RITC-dextran-incorporating endosomes
(macropinosomes). Scale bar, 20 µm.
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Fig. 6.
Association of Rah with macropinosome
membranes formed from the membrane ruffles. EGFP-tagged Rah(wt)
was expressed in 10T1/2 cells, and EGFP fluorescence was recorded at
intervals of several minutes each at 37 °C. The numbers
indicate minutes after starting the observation. Shown are selected
figures of reverse color images. An arrow indicates a
Rah-associated macropinosome formed from the membrane ruffle and
migrating toward the cell center.
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Fig. 7.
Promotion of macropinosome
formation by Rah. A, increase in the number of
macropinosomes in Rah-expressing cells. Myc-tagged Rah(wt), Rah(Q111L),
or Rah(T66N) was transfected to 10T1/2 cells. The number of
actin-coated macropinosomes per cell was counted. The values are the
means ± S.D. of triplicate experiments. B, elevation
and reduction in the number of macropinosomes by the expression Rah(wt)
and Rah(T66N), respectively, in PDGF- or PMA-treated cells.
Myc-Rah(wt)- or Myc-Rah(T66N)-transfected 10T1/2 cells were treated
with 5 units/ml PDGF or 0.1 µM PMA for 30 min. The number
of actin-coated macropinosomes per cell was counted. The values are the
means ± S.D. of triplicate experiments. C, influence
of dominant-negative mutants of H-Ras, Rac1, and WAVE2 on Rah-promoted
macropinosome formation. HA-tagged Rah(wt) was cotransfected with
Myc-H-Ras(S17N), Myc-Rac1(T17N), or FLAG-WAVE2( V) in 10T1/2 cells.
The number of actin-coated macropinosomes per cell was counted. The
values are the means ± S.D. of triplicate experiments.
D, colocalization of Rah and WAVE2 to membrane ruffles and
macropinosomes. Myc-Rah(wt) and EGFP-WAVE2 were cotransfected to 10T1/2
cells. Shown are EGFP fluorescence locating WAVE2 (a),
Myc1-9E10 staining locating Rah (b), and their merged image
(c). An arrowhead and arrows point to
the membrane ruffles and macropinosomes, respectively, containing both
WAVE2 and Rah.
V), a dominant-negative WAVE2 mutant lacking
verprolin-homology domain (35), was coexpressed with Rah, the number of
macropinosomes was declined close to the control level (Fig.
7C). Taken together, these results imply that Rah
facilitates the macropinosome formation triggered by
Rac1-IRSp53-WAVE2-induced membrane ruffling and that
Rac1-IRSp53-WAVE2-mediated actin polymerization and subsequent membrane
ruffling are required for the Rah-promoted macropinosome formation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, which is activated by the PI3K
product PI(3,4,5)P3 (57). A presumable pathway for growth
factor-induced membrane ruffling and macropinocytosis is summarized in
Fig. 8.
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Fig. 8.
A scheme of synergistic effect of Rah on the
growth factor-induced macropinocytosis. Growth factor-induced
membrane ruffling, which is mediated by Rac1-IRSp53-WAVE2, leads to a
basal level macropinosome formation. Rah seems to be synergistically
concerned with efficient macropinosome formation possibly by closing
the membrane ruffles.
We showed here that the expression of Rah(wt) and Rah(Q111L) facilitated the formation of macropinosomes over the control level. In contrast, the dominant-negative Rah(T66N) slightly retarded the macropinosome formation under the control level. When the cells were treated with PDGF or PMA, membrane ruffling and macropinosome formation were promoted. Expression of Rah(wt) in the PDGF- or PMA-treated cells further promoted the macropinosome formation. Expression of the dominant-negative Rah reduced the macropinosome formation, but the levels remained higher than the control level. On the other hand, the degree of membrane ruffling was not affected by any of these Rah proteins. These results indicate that Rah is concerned with macropinosome formation synergistically with growth factors (including serum growth factors in the growth medium) or PMA. Because Rah is not involved in membrane ruffling, however, Rah does not seem to constitute the signaling pathway activated by growth factors (Fig. 8).
We further showed that Rah-induced macropinosome formation was retarded
to some degree by the dominant-negative H-Ras(S17N). On the other hand,
Rac1(T17N) almost completely inhibited macropinosome formation, whereas
the dominant-negative WAVE2(V) suppressed it close to the control
level. The only moderate retardation by H-Ras(S17N) might be ascribed
to the presence in 10T1/2 cells of multiple endogenous Ras
proteins (H-Ras, K-Ras, and N-Ras), which are structurally closely
related but functionally distinctive (58, 59). Indeed, K-Ras generates
membrane ruffles and macropinosomes more prominently than does H-Ras,
probably because K-Ras activates Rac1 more efficiently than H-Ras (60).
Thus, if GEFs specifically act on each member of Ras in vivo
and H-Ras(S17N) sequester a particular GEF, H-Ras(S17N) may not
efficiently interfere with macropinosome formation. Although WAVE2 is
activated by Rac1-IRSp53 to induce membrane ruffling and subsequent
macropinocytosis, there are two other WAVE isoforms, WAVE1 and 3 (18,
20, 38). In addition, other Rac1 target proteins such as PAK1 may be
implicated in the macropinocytosis pathway by inducing membrane
ruffling (61, 62). These facts seem to be responsible for the
incomplete inhibition of macropinosome formation by WAVE2(
V).
Because macropinosomes are formed from membrane ruffles, macropinosome
formation primarily requires membrane ruffling (4). Membrane ruffling
induced by growth factors or PMA may result in spontaneous basal level
macropinosome formation, which does not require the aid of Rah. Rah was
colocalized with actin to the membrane ruffles and macropinosome
membranes. Thus, the primary role of Rah seems to be the formation of
macropinosomes by closing membrane ruffles through regulating actin
reorganization. In this manner, Rah might be concerned with efficient
macropinosome formation synergistically to the action of growth factors
or PMA. This postulation is supported by the findings that Rah(T66N) is
associated with membrane ruffles but not with macropinosomes and that
both Rah and the actin coat disappear from macropinosomes
as the vesicles migrate to the cell center. In this context, it should
be noted that PI3K is not necessary for membrane ruffling but rather
functions in the closure of membrane ruffles to form macropinosomes and phagosomes in macrophages (63). If this is also the case for fibroblasts, PI3K and PI(3,4,5)P3 may participate in the
activation of Rah (Fig. 8). The closure of membrane ruffles is
accompanied by a membrane fusion process. Some Rab family proteins,
including Rab5, are involved in vesicle membrane fusion (23, 24). Thus, it is of interest to examine whether Rah is required for the plasma membrane fusion at the ruffles. Identification of target proteins of Rah and their binding proteins may help to answer these questions.
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ACKNOWLEDGEMENTS |
---|
We appreciate Drs. Chihiro Sasakawa and Toshihiko Suzuki for valuable discussions.
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FOOTNOTES |
---|
* This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by Research Grants (11B-1 and 14B-4) for Nervous and Mental Disorders from the Ministry of Health, Labor, and Welfare of Japan.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/EBI Data Bank with accession number(s) AB082927
To whom correspondence should be addressed: Dept. of Biology,
Faculty of Science, Chiba University, 1-33 Yayoicho, Inageku, Chiba
263-8522, Japan. Tel.: 81-43-290-3911; Fax: 81-43-290-3911; E-mail:
t.endo@faculty.chiba-u.jp.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M208699200
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ABBREVIATIONS |
---|
The abbreviations used are: PMA, phorbol 12-myristate 13-acetate; GEF, guanine nucleotide exchange factor; PDGF, platelet-derived growth factor; RT, reverse transcription; wt, wild-type; GST, glutathione S-transferase; DTT, dithiothreitol; mAb, monoclonal antibody; HA, hemagglutinin; EGFP, enhanced green fluorescent protein; 10T1/2, C3H/10T1/2; RITC, rhodamine B isothiocyanate; GAP, GTPase-activating protein; PI3K, phosphatidylinositol 3-kinase; PI(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; CMV, cytomegalovirus.
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REFERENCES |
---|
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---|
1. | Mellman, I. (1996) Annu. Rev. Cell Dev. Biol. 12, 575-625[CrossRef][Medline] [Order article via Infotrieve] |
2. | Robinson, M. S., Watts, C., and Zerial, M. (1996) Cell 84, 13-21[Medline] [Order article via Infotrieve] |
3. | Riezman, H., Woodman, P. G., van Meer, G., and Marsh, M. (1997) Cell 91, 731-738[Medline] [Order article via Infotrieve] |
4. | Swanson, J. A., and Watts, C. (1995) Trends Cell Biol. 5, 424-428[CrossRef] |
5. | Cardelli, J. (2001) Traffic 2, 311-320[CrossRef][Medline] [Order article via Infotrieve] |
6. | Isberg, R. R., and Tran Van Nhieu, G. (1994) Annu. Rev. Genet. 28, 395-422[CrossRef][Medline] [Order article via Infotrieve] |
7. | Brunk, U., Schellens, J., and Westermark, B. (1976) Exp. Cell Res. 103, 295-302[Medline] [Order article via Infotrieve] |
8. |
Davies, P. F.,
and Ross, R.
(1978)
J. Cell Biol.
79,
663-671 |
9. | Haigler, H. T., McKanna, J. A., and Cohen, S. (1979) J. Cell Biol. 83, 82-90[Abstract] |
10. | Racoosin, E. L., and Swanson, J. A. (1989) J. Exp. Med. 170, 1635-1648[Abstract] |
11. | Keller, H. U. (1990) J. Cell. Physiol. 145, 465-471[Medline] [Order article via Infotrieve] |
12. |
Sandvig, K.,
and van Deurs, B.
(1990)
J. Biol. Chem.
265,
6382-6388 |
13. | Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A. (1992) Cell 70, 401-410[Medline] [Order article via Infotrieve] |
14. | Bar-Sagi, D., and Feramisco, J. R. (1986) Science 233, 1061-1068[Medline] [Order article via Infotrieve] |
15. | Michiels, F., Habets, G. G. M., Stam, J. C., van der Kammen, R. A., and Collard, J. G. (1995) Nature 375, 338-340[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Miki, H.,
Suetsugu, S.,
and Takenawa, T.
(1998)
EMBO J.
17,
6932-6941 |
17. | Miki, H., Yamaguchi, H., Suetsugu, S., and Takenawa, T. (2000) Nature 408, 732-735[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Takenawa, T.,
and Miki, H.
(2001)
J. Cell Sci.
114,
1801-1809 |
19. | Higgs, H. N., and Pollard, T. D. (2001) Annu. Rev. Biochem. 70, 649-676[CrossRef][Medline] [Order article via Infotrieve] |
20. | Suetsugu, S., Miki, H., and Takenawa, T. (2002) Cell Motil. Cytoskel. 51, 113-122[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Rodman, J. S.,
and Wandinger-Ness, A.
(2000)
J. Cell Sci.
113,
183-192 |
22. |
Takai, Y.,
Sasaki, T.,
and Matozaki, T.
(2001)
Physiol. Rev.
81,
153-208 |
23. | Zerial, M., and McBride, H. (2001) Nat. Rev. Mol. Cell. Biol. 2, 107-117[CrossRef][Medline] [Order article via Infotrieve] |
24. | Segev, N. (2001) Curr. Opin. Cell Biol. 13, 500-511[CrossRef][Medline] [Order article via Infotrieve] |
25. | Bucchi, C., Parton, R. G., Mather, I. H., Stunnenberg, H., Simons, K., Hoflack, B., and Zerial, M. (1992) Cell 70, 715-728[Medline] [Order article via Infotrieve] |
26. | Stenmark, H., Parton, R. G., Steele-Mortimer, O., Lütcke, A., Gruenberg, J., and Zerial, M. (1994) EMBO J. 13, 1287-1296[Abstract] |
27. | Bucchi, C., Lütcke, A., Steele-Mortimer, O., Olkkonen, V. M., Dupree, P., Chiariello, M., Bruni, C. B., Simons, K., and Zerial, M. (1995) FEBS Lett. 366, 65-71[CrossRef][Medline] [Order article via Infotrieve] |
28. | van der Sluijs, P., Hull, M., Webster, P., Mâle, P., Goud, B., and Mellman, I. (1992) Cell 70, 729-740[Medline] [Order article via Infotrieve] |
29. | Feng, Y., Press, B., and Wandinger-Ness, A. (1995) J. Cell Biol. 131, 1435-1452[Abstract] |
30. |
Méresse, S.,
Gorvel, J.-P.,
and Chavrier, P.
(1995)
J. Cell Sci.
108,
3349-3358 |
31. | Morimoto, B. H., Chuang, C.-C., and Koshland, D. E., Jr. (1991) Genes Dev. 5, 2386-2391[Abstract] |
32. | Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve] |
33. | Matsumoto, K., Asano, T., and Endo, T. (1997) Oncogene 15, 2409-2417[CrossRef][Medline] [Order article via Infotrieve] |
34. | Tsubakimoto, K., Matsumoto, K., Abe, H., Ishii, J., Amano, M., Kaibuchi, K., and Endo, T. (1999) Oncogene 18, 2431-2440[CrossRef][Medline] [Order article via Infotrieve] |
35. | Muroya, K., Hattori, S., and Nakamura, S. (1992) Oncogene 7, 277-281[Medline] [Order article via Infotrieve] |
36. | Gluzman, Y. (1981) Cell 23, 175-182[Medline] [Order article via Infotrieve] |
37. | Evan, G. I., Lewis, G. K., Ramsay, G., and Bishop, J. M. (1985) Mol. Cell. Biol. 5, 3610-3616[Medline] [Order article via Infotrieve] |
38. | Suetsugu, S., Miki, H., and Takenawa, T. (1999) Biochem. Biophys. Res. Commun. 260, 296-302[CrossRef][Medline] [Order article via Infotrieve] |
39. | Reznikoff, C. A., Brankow, D. W., and Heidelberger, C. (1973) Cancer Res. 33, 3231-3238[Medline] [Order article via Infotrieve] |
40. |
Endo, T.,
Matsumoto, K.,
Hama, T.,
Ohtsuka, Y.,
Katsura, G.,
and Obinata, T.
(1996)
J. Biol. Chem.
271,
27855-27862 |
41. | Chen, J. W., Murphy, T. L., Willingham, M. C., Pastan, I., and August, J. T. (1985) J. Cell Biol. 101, 85-95[Abstract] |
42. | Bourne, H. R., Sanders, D. A., and McCormick, F. (1991) Nature 349, 117-127[CrossRef][Medline] [Order article via Infotrieve] |
43. | Valencia, A., Chardin, P., Wittinghofer, A., and Sander, C. (1991) Biochemistry 30, 4637-4648[Medline] [Order article via Infotrieve] |
44. | Glomset, J. A., and Farnsworth, C. C. (1994) Annu. Rev. Cell Biol. 10, 181-205[CrossRef] |
45. | Mori, T., Fukuda, Y., Kuroda, H., Matsumura, T., Ota, S., Sugimoto, T., Nakamura, Y., and Inazawa, J. (1999) Biochem. Biophys. Res. Commun. 254, 594-600[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Brondyk, W. H.,
McKiernan, C. J.,
Burstein, E. S.,
and Macara, I. G.
(1993)
J. Biol. Chem.
268,
9410-9415 |
47. | Oliver, J. M., Berlin, R. D., and Davis, B. H. (1984) Methods Enzymol. 108, 336-347[Medline] [Order article via Infotrieve] |
48. | Seastone, D. J., Harris, E., Temesvari, L. A., Bear, J. E., Saxe, C. L., and Cardelli, J. (2001) J. Cell Sci. 114, 2673-2683[Medline] [Order article via Infotrieve] |
49. | Jiang, H., Luo, J.-Q., Urano, T., Frankel, P., Lu, Z., Foster, D. A., and Feig, L. A. (1995) Nature 378, 409-412[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Luo, J.-Q.,
Liu, X.,
Frankel, P.,
Rotunda, T.,
Ramos, M.,
Flom, J.,
Jiang, H.,
Feig, L. A.,
Morris, A. J.,
Kahn, R. A.,
and Foster, D. A.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
3632-3637 |
51. |
Nobes, C. D.,
Lauritzen, I.,
Mattei, M.-G.,
Paris, S.,
Hall, A.,
and Chardin, P.
(1998)
J. Cell Biol.
141,
187-197 |
52. |
Li, G.,
and Stahl, P. D.
(1993)
J. Biol. Chem.
268,
24475-24480 |
53. |
Mukhopadhyay, A.,
Barbieri, A. M.,
Funato, K.,
Roberts, R.,
and Stahl, P. D.
(1997)
J. Cell Biol.
136,
1227-1237 |
54. | Lambert, J. M., Lambert, Q. T., Reuther, G. W., Malliri, A., Siderovski, D. P., Sondek, J., Collard, J. G., and Der, C. J. (2002) Nat. Cell Biol. 4, 621-625[Medline] [Order article via Infotrieve] |
55. |
Han, J.,
Luby-Phelps, K.,
Das, B.,
Shu, X.,
Xia, Y.,
Mosteller, R. D.,
Krishna, U. M.,
Falck, J. R.,
White, M. A.,
and Broek, D.
(1998)
Science
279,
558-560 |
56. |
Nimnual, A. S.,
Yatsula, B. A.,
and Bar-Sagi, D.
(1998)
Science
279,
560-563 |
57. |
Derman, M. P.,
Toker, A.,
Hartwig, J. H.,
Spokes, K.,
Falck, J. R.,
Chen, C.-S.,
Cantley, L. C.,
and Cantley, L. G.
(1997)
J. Biol. Chem.
272,
6465-6470 |
58. |
Yan, J.,
Roy, S.,
Apolloni, A.,
Lane, A.,
and Hancock, J. F.
(1998)
J. Biol. Chem.
273,
24052-24056 |
59. |
Voice, J. K.,
Klemke, R. L., Le, A.,
and Jackson, J. H.
(1999)
J. Biol. Chem.
274,
17164-17170 |
60. |
Walsh, A. B.,
and Bar-Sagi, D.
(2001)
J. Biol. Chem.
276,
15609-15615 |
61. | Sells, M. A., Knaus, U. G., Bagrodia, S., Ambrose, D. M., Bokoch, G. M., and Chernoff, J. (1997) Curr. Biol. 7, 202-210[Medline] [Order article via Infotrieve] |
62. |
Frost, J. A.,
Khokhlatchev, A.,
Stippec, S.,
White, M. A.,
and Cobb, M. H.
(1998)
J. Biol. Chem.
273,
28191-28198 |
63. | Araki, N., Johnson, M. T., and Swanson, J. A. (1996) J. Cell Biol. 135, 1249-1260[Abstract] |
64. | Lipman, D. J., and Pearson, W. R. (1985) Science 227, 1435-1441[Medline] [Order article via Infotrieve] |