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From the Departments of
Received for publication, June 26, 2001
Stimulation of HIRcB fibroblasts with insulin
leads to accumulation of active components of the
mitogen-activated protein kinase cascade in endocytic compartments.
However, the factors that regulate the mobilization of these components
through the endocytic pathway and the relevance of this event to
cellular signaling remain unclear. Here we report that Ras proteins are associated with lipid rafts in resting HIRcB fibroblasts. Ras is
rapidly internalized into the endocytic compartment following stimulation with insulin. The redistribution of Ras is independent of
its activation. Attachment of the C-terminal 20 amino acids of
Ha-Ras to green fluorescent protein was sufficient to target this construct to the same loci as the endogenous Ras protein, indicating that Ras distribution is a consequence of the association of
its lipid modified C terminus with membranes. Depletion of plasma
membrane cholesterol delocalized Ras and blocked
insulin-dependent Ras traffic. Cholesterol depletion also
blocked insulin-dependent phosphorylation of MEK and
mitogen-activated protein kinase (MAPK) but had no effects on the
translocation and activation of Raf-1. A second inhibitor of
endocytosis, cytochalasin D, also blocked insulin-dependent
MAPK phosphorylation. Taken together, these results suggest that
mobilization of active Raf-1 through the endocytic compartment is
required for completion of the MAPK cascade.
Stimulation of growth factor receptors leads to the activation of
the mitogen-activated protein kinase
(MAPK)1 cascade. Central to
this process is the activation of the small GTPase Ras (1), which, upon
binding GTP, associates with the serine/threonine kinase Raf-1.
Ras-Raf-1 association results in the activation of the latter. Raf-1
then phosphorylates and activates MEK, which in turn phosphorylates and
activates the extracellular signal-regulated kinase family of
MAPKs. Genetic models have provided exceptional evidence that supports
this view (2). However, a number of events must occur to get efficient
transduction of the signal. In a resting cell, the components of the
MAPK cascade exist in different subcellular compartments. Raf-1 must
translocate to the plasma membrane (3, 4) to interact with
membrane-bound Ras proteins. This translocation is regulated by its
association with phosphatidic acid. Disruption of the interaction
between Raf-1 and phosphatidic acid, either by mutation of Raf (5) or
by blocking the activation of phospholipase (6), prevents agonist-dependent activation of the MAPK cascade. In
general, most current models of signal transduction propose the
formation of multimeric complexes at specified loci as critical steps
in the regulation of signaling pathways.
Acidic lipid second messengers, such as polyphosphoinositides (7)
and phosphatidic acid (6), are important regulators of protein
compartmentalization. Many proteins, including EEA-1 (8), protein
kinase B (9), and Raf-1 (5, 6, 10), require association with acidic
phospholipids for proper targeting. In addition to their role in
protein recruitment, acidic phospholipids also possess distinct
biophysical properties that are relevant to cellular signaling. In the
presence of divalent cations, such as calcium, many acidic
phospholipids cluster together and phase separate in model membrane
systems. Not surprisingly, at least some of these lipids have been
found in cellular lipid domains that contain similar biophysical
characteristics (11). These domains, which include lipid rafts, are
characterized by phase separation, resistance to solubilization in
non-ionic detergents, and cholesterol enrichment.
The biological functions of lipid rafts have not been well defined.
However, the metabolism of acidic phospholipids and the maintenance of
the structure of lipid rafts have been found to be crucial to vesicular
traffic from the plasma membrane. Depletion of plasma membrane
cholesterol disrupts clathrin-mediated endocytosis (12, 13), and
enzymes that generate acidic phospholipids play a critical role in
vesicle fission (14). Furthermore, the generation of
phosphatidylinositol phosphates and phosphatidic acid promotes internalization of vesicles from the plasma membrane (15). Both lipid
raft structures and acidic phospholipids are clearly important elements
in vesicle transport from the plasma membrane.
There is a substantial body of evidence linking endocytosis to the
regulation of signaling events. In fact, it has been proposed that some
of these, such as the Ras-MAPK cascade, might occur on endocytic
vesicles. For instance, Pol et al. (16) have reported that
the components of the MAPK are associated with endosomes in epidermal
growth factor-treated rat livers. Previous work from our laboratory has
shown similar results in insulin-stimulated HIRcB cells (5, 6).
Therefore, the components of the MAPK cascade appear to accumulate on
the surface of endosomes. However, the mechanisms through which these
proteins associate to endocytic vesicles and the functional relevance
of the redistribution of these signaling molecules are not well understood.
Therefore, we examined the redistribution of Ras from the plasma
membrane to the endocytic compartment. Endogenous Ras in HIRcB
fibroblasts colocalized with cholesterol and glycosylphosphatidyl inositol (GPI)-anchored proteins, two markers for lipid rafts. After
stimulation with insulin, Ras was found associated with immunopurified
vesicles that were resistant to extraction with Triton X-100,
suggesting that agonist-dependent Ras traffic occurs through a subset of cholesterol-rich endosomes, probably derived from
lipid rafts. Furthermore, green fluorescent protein tagged Ha-Ras
(GFP-Ras) was also localized in lipid rafts and was mobilized through
the endocytic compartment in response to insulin.
Agonist-dependent GFP-Ras traffic was independent of its
association with GTP, and a 20-amino acid peptide containing the
farnesylation and acylation motifs of Ha-Ras was sufficient to target
GFP to the correct subcellular localization. Depletion of cellular
cholesterol by cyclodextrin treatment disrupted traffic of GFP-Ras.
Furthermore, cyclodextrin treatment also inhibited
insulin-dependent MEK and MAPK phosphorylation without
affecting Raf-1 activation. Cytochalasin D also inhibited Ras
internalization and MAPK phosphorylation. These results indicate that
translocation of the components of the MAPK to the surface of endocytic
vesicles is required for activation of MEK by Raf-1.
Materials and Constructs--
Ha-Ras and the Ha-Ras
CAAX motif (C-terminal 20 amino acids) were cloned into
pEGFP-C1 (CLONTECH). Anti-Ras antibodies (clone Ras10) were obtained from Upstate Biotechnologies. Mouse anti-c-Raf-1 antibodies were obtained from Transduction Laboratories.
Phospho-specific MEK and MAPK antibodies were obtained from New England
Biolabs, and anti-GFP antibodies were from
CLONTECH. All other materials were purchased from
Sigma unless otherwise noted. Anti-GPI antibodies were prepared as
described previously (17).
Cell Culture--
Rat-1 fibroblasts that overexpress insulin
receptors (HIRcB cells) were cultured in Dulbecco's modified Eagle's
medium/Ham's F-12 medium (Life Technologies, Inc.) supplemented with
10% fetal bovine serum (Life Technologies, Inc.) as described
previously (6). Superfect transfection reagent (Qiagen) was used to
introduce plasmid DNA into HIRcB cells for imaging studies, whereas
LipofectAMINE (Life Technologies, Inc.) was used for biochemical
studies. Transfection efficiencies greater than 70% were achieved
using LipofectAMINE as determined by fluorescence and
differential interference contrast microscopy of the transfected cells.
Fluorescence Microscopy--
Cells grown on
poly-L-lysine coated coverslips (6) were fixed in 3%
paraformaldehyde/phosphate-buffered saline (30 min, 4 °C) prior to
permeabilization with 0.1% Triton X-100 (2 min) or filipin (150 µg/ml in 3% bovine serum albumin). Permeabilized cells were stained
with anti-Ras (1:500 in 3% bovine serum albumin, phosphate-buffered
saline) followed by Cy5-conjugated secondary (Jackson Immunoresearch)
or with anti-Ras and rabbit anti-GPI antibodies with fluorescein
isothiocyanate- and Cy5-conjugated secondaries. Observation of fixed
specimens was with a Leica-TCS confocal microscope using filters and
laser lines appropriate for fluorescein isothiocyanate and Cy5 or with
an Olympus Provis equipped with an Optronics "Magnafire" camera
with filters appropriate for filipin, fluorescein isothiocyanate, and Cy5.
Live cell epifluorescence images were acquired on a Nikon Diaphot
inverted microscope equipped with a cooled CCD camera (Princeton Instruments) and appropriate excitation and emission filters for GFP.
Images were captured and processed using Inovision ISEE software. Experiments on this system were performed at 37 °C as described (6).
Evanescent wave microscopy was performed as described by Han et
al. (18). Total cellular fluorescence was quantitated using
Inovision ISEE software. Background fluorescence was subtracted from
each image. Each image was then normalized to the total cellular fluorescence prior to cellular stimulation (100%). The images were
processed and prepared using Adobe Photoshop 5.0 software.
Immunoisolation of Vesicles--
Endocytic vesicles were
purified from HIRcB cells as previously (6). Prior to immunoisolation,
purified vesicles from six dishes were split into three equal
fractions. Immunoisolation of endocytic vesicles was performed as
described previously (6) in the absence of detergent, with 1% Triton
X-100, or with 1% cholate. Immunoisolates were then analyzed by
Western blot as described previously (6).
Cholesterol Depletion--
Cells were treated with 2%
We have previously reported that insulin stimulates the
redistribution of Ras to early endosomes in Rat-1 fibroblasts that overexpress the human insulin receptor (HIRcB cells) (5). However, the
dynamics of this process and the mechanisms involved in targeting Ras
to endosomes have not been described. Cholesterol-rich microdomains known as lipid rafts have been shown to contain significant levels of
Ras (20) and to play a role in the regulation of endocytic processes
(12, 13, 21). Therefore, the specific association of Ras with
cholesterol-rich structures may be relevant to the mechanisms that
regulate intracellular Ras traffic. We therefore examined the
distribution of Ras on the plasma membrane of HIRcB fibroblasts and
compared it with the distribution of two known components of lipid
rafts, cholesterol, and GPI-anchored proteins (Fig.
1A). GPI-anchored proteins
were clearly localized on discrete regions of the plasma membrane, as
described by others (22). These regions were stained with filipin and
anti-Ras antibodies, indicating that these structures also contained
cholesterol and Ras. However, cholesterol and Ras staining was not
limited to regions containing GPI-anchored proteins (Fig.
1A, arrows 2). These results suggest that only a
pool of Ras is associated with lipid rafts.
We have observed Ras redistribution to endosomes shortly after the
stimulation of cells with insulin. To further examine these endosomes,
endocytic vesicles were purified by sucrose gradient followed by
immunoisolation using antibodies specific to the insulin receptor.
Vesicles containing Ras, Raf-1 and phosphorylated MAPK were resistant
to extraction with Triton X-100 (Fig. 1B), suggesting that
these vesicles were highly enriched in cholesterol. Furthermore, these
vesicles were soluble in cholate, a bile salt detergent that
efficiently solubilizes cholesterol-rich membranes.
These data suggested the hypothesis that a subpopulation of Ras
primarily associated to cholesterol-rich microdomains traffics while
attached to the cytoplasmic surface of endosomes in response to
insulin. To further characterize this phenomenon, we examined the
dynamics of a green fluorescent protein-Ras fusion protein (GFP-Ras) in
resting and insulin-treated live cells. Like the endogenous protein,
GFP-Ras fluorescence and filipin staining were colocalized in punctate
regions of the plasma membrane prior to agonist stimulation (Fig.
2A). After insulin
stimulation, a significant fraction of GFP-Ras was also found
associated to endocytic vesicles that were positively stained with
filipin (Fig. 2A).
Agonist-dependent Traffic of Raft-associated Ras and
Raf-1 Is Required for Activation of the Mitogen-activated Protein
Kinase Cascade*,
§,,, and
Pharmacology and
¶ Cell Biology and Physiology, University of
Pittsburgh, Pittsburgh, Pennsylvania 15261
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-methyl cyclodextrin (CD)/Dulbecco's modified Eagle's
medium/Ham's F-12 medium for 30 min and washed three times with
phosphate-buffered saline to deplete plasma membrane cholesterol. CD
treatment reduced total cholesterol content from 62.69 ± 8.64 to
11.97 ± 2.72 ng/µg protein as determined by the Amplex Red
Cholesterol Oxidase Assay (Molecular Probes). Cholesterol-CD inclusion
complexes were prepared as described by Klein et al.
(19).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (31K):
[in a new window]
Fig. 1.
Ras is present in lipid rafts.
A, the distribution of cholesterol, Ras, and GPI-anchored
proteins was observed in serum-starved HIRcB cells using fluorescent
immunostaining techniques as described under "Experimental
Procedures." Arrows 1 indicate colocalization of all three
markers, and arrows 2 indicate colocalization of Ras and
cholesterol in the absence of staining for GPI-anchored proteins. The
results are representative of at least three separate experiments.
B, to determine whether localization of Ras to rafts was
relevant to traffic of Ras through the endocytic pathway, endocytic
vesicles were purified from insulin-treated cells (200 nM,
5 min) by sucrose gradient and immunoisolated using antibodies specific
for the insulin receptor. Vesicles from a single preparation were split
into three equal fractions and immunopurified in the absence of
detergent, with 1% Triton X-100, or 1% cholate. Immunopurified
pellets were analyzed by Western blot using antibodies specific for
Ras, Raf-1, and phosphorylated MAPK.
View larger version (51K):
[in a new window]
Fig. 2.
Agonist-dependent internalization
of GFP-Ras. A, the distribution of cholesterol and
GFP-Ras in fixed cells was examined by epifluorescence microscopy.
Insulin stimulation was for 5 min prior to fixation. B,
cells expressing GFP-Ras, GFP-Ras(S17N), or GFP-CAAX were
imaged before and after insulin stimulation (200 nM, 5 min). Frames after insulin stimulation are sequential (10 s). Movies
are available in the supplemental information. C, the
distribution of Ras was examined in insulin-treated (5 min)
GFP-CAAX expressing cells by immunostaining and confocal
microscopy.
Several interesting observations arise from the examination of the dynamics of agonist-dependent GFP-Ras traffic in live cells. Prior to stimulation, GFP-Ras was associated to the plasma membrane and to a perinuclear region that has been previously identified as the Golgi apparatus (23). Following insulin stimulation, plasma membrane-bound GFP-Ras was rapidly internalized. Interestingly, GFP-Ras internalization was most conspicuous on the leading edge of the fibroblast. This is similar to the findings of Chen et al. (24), who found that the Ras activator Sos was preferentially recruited to the leading edge of fibroblasts, although the causes of this apparent polarization are unclear. A GFP-Ras mutant that is locked in the inactive conformation was also found to traffic like the wild-type protein, indicating that regulation of Ras localization is independent of its state of activation. These observations suggested that the specific association of Ras to cholesterol-rich microdomains might be determined by the interactions of the lipid-modified C terminus of Ras with the lipid membrane. To test this hypothesis, a fragment containing the C-terminal 20 amino acids of Ha-Ras, which include the CAAX farnesylation motif and the palmitoylation sites (25), was fused to GFP (GFP-CAAX). The localization of GFP-CAAX was very similar to the localization of GFP-Ras. Furthermore, the traffic of GFP-CAAX in response to insulin was identical to that of the full-length protein. In fact, internalized GFP-CAAX was found in endocytic vesicles and colocalized with endogenous Ras (Fig. 2C), indicating that the post-translational modifications of the C terminus are sufficient to target GFP to the correct subcellular localization.
Evancescent wave microscopy was used to further examine the dynamics of
GFP-Ras on the plasma membrane. In this technique, an internally
reflected laser is used to illuminate a very thin section adjacent to
the coverslip, essentially restricting the plane of observation to a
thin slice that includes the plasma membrane (26). Two pools of GFP-Ras
were detected using this technique. A fraction of plasma membrane-bound
GFP-Ras was found localized in punctate microdomains, whereas the bulk
of GFP-Ras was found dispersed on the membrane (Fig.
3A). Stimulation with insulin
resulted in the disappearance of some of the Ras-containing microdomains (Fig. 3A, arrows). Furthermore,
insulin treatment caused a decrease in the total amount of fluorescence
associated to the plasma membrane section (Fig. 3B). Control
experiments showed that: 1) the total area of the section did not
change significantly during the course of experimentation and 2) the
excitation protocol did not produce detectable photobleaching.
Therefore, we conclude that the decrease in the fluorescence of the
plasma membrane was a result of the internalization of Ras and its
subsequent movement away from the plane of illumination. This motion is
consistent with the formation of endocytic vesicles. To confirm that
the disappearance of GFP-Ras from the membrane was a consequence of endocytic traffic, the cells were treated with cytochalasin D, which
inhibits endocytosis by destabilizing actin filaments (11, 27). As
shown in Fig. 3B, treatment with cytochalasin D inhibited insulin-dependent decrease in plasma membrane associated
GFP-Ras, therefore confirming that the decrease in fluorescence
intensity of the plasma membrane section is due to the traffic of
GFP-Ras during endocytosis.
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Because cholesterol is an essential structural component of lipid rafts
and of other specific lipid microdomains, drugs that acutely deplete
plasma membrane cholesterol and disrupt the structure of these domains
have been used to investigate their functional role (28). Therefore,
the cholesterol depleting drug, CD, was used to deplete membrane
cholesterol in HIRcB cells. The distribution of GFP-Ras on the plasma
membrane of CD-treated cells was diffuse, and no punctate structures
containing GFP-Ras were observed (Fig. 4A). Furthermore, CD treatment
blocked insulin-dependent GFP-Ras internalization.
Epifluorescence images showed that CD treatment resulted in a smooth
distribution of GFP-Ras on the plasma membrane. Insulin treatment of
cholesterol-depleted cells did not promote the traffic of GFP-Ras (Fig.
4B). However, repletion of cellular cholesterol by addition
of cholesterol-CD inclusion complexes (19) restored GFP-Ras the
internalization, indicating that Ras traffic requires the presence of
cholesterol. Taken together, these data support a model in which
insulin stimulates endocytosis of lipid microdomains with structural
and biological properties akin to those of lipid rafts. Proteins that
reside in these domains, such as Ras or the GFP-CAAX
construct, are subsequently internalized along with the raft.
|
The role of Ras traffic in the regulation of the Ras-MAPK pathway was
then examined. Because Ras binds and activates the serine/threonine kinase Raf-1, we examined the effect of cholesterol depletion on Raf-1
activation. It has been established that Raf-1 must associate with
membranes for activation. Cholesterol depletion with CD resulted in
accumulation of Raf-1 on the plasma membrane (Fig.
5A) rather than on endosomal
membranes (Fig. 5A and Ref. 5).
Insulin-dependent Raf-1 activation was not significantly
affected by CD treatment (Fig. 5B) as determined by an
in vitro kinase assay. Because Raf-1 activation in this
system requires interaction with Ras proteins, this indicates that the
capacity of Ras to become active and interact with effectors does not
require lipid raft-like structures. However, CD treatment inhibited
insulin-dependent MEK and MAPK phosphorylation. This
result is consistent with a general requirement for endocytosis in
agonist-dependent MAPK activation that has been reported in many systems (29-32). Likewise, cytochalasin D also inhibited
insulin-dependent MAPK phosphorylation in this system (Fig.
5B), supporting the conclusion that CD treatment inhibits
insulin-dependent MAPK phosphorylation through its effects
on endocytosis of the Ras-Raf complex.
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DISCUSSION
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Lipid rafts and other cholesterol-rich microdomains are important structural elements of the membrane that play a role in the regulation of both endocytic and signal transduction processes. According to the classical signal transduction models, following receptor activation, specific signals are passed off to other components that remain attached to the plasma membrane, such as Ras, phosphoinositide 3-kinase and the heterotrimeric G-proteins. Activation of these elements then leads to the stimulation of specific enzymatic activities on the plasma membrane that result in the generation of soluble or membrane-bound second messengers or in the phosphorylation of specific target proteins. Endocytosis, in contrast, was primarily thought to provide signal termination by removing the active receptor from the cell surface and promoting deactivation of the receptor-ligand complex (33). Recently, it has become apparent that endocytosis and signal transduction share many components and may actually be aspects of a single phenomenon. For example, Akt/protein kinase B has been shown to participate in the activation of the endocytic machinery (34), as well as regulating Raf-1 activity (35). Likewise, MAPK has recently been shown to phosphorylate members of the Rab5 subfamily of GTPases (36), which are important regulators of endocytosis.
Here we have provided direct evidence that, during insulin signaling, endocytosis and the activation of the MAPK cascade are coupled. Prior to insulin stimulation, Ha-Ras is distributed between the bulk membrane and cholesterol-rich microdomains that share many of the properties of lipid rafts and appear as distinct punctate structures. The dynamic studies we report in this paper show that, after stimulation with insulin, Ha-Ras moves to these punctate structures and soon afterward to the endocytic compartment. Isolated endosomes are insoluble in Triton X-100, therefore suggesting high cholesterol contents. This suggests that the endosomes are derived directly from the cholesterol-rich structures observed in the resting cells.
Ha-Ras appears to be recruited to lipid rafts by virtue of its lipid modified tail. Ha-Ras is modified by both palmitoylation and farnesylation, which likely strengthen interaction with lipid raft structures. It was originally suggested, mostly on the basis of in vitro studies, that isoprenyl modifications may result in exclusion from cholesterol-rich structures (37) and liquid ordered domains (38). However, further work has supported the view that prenylated proteins, especially Ha-Ras, are sensitive to alterations in the cholesterol content of plasma membranes and associate with lipid rafts (20, 39). However, only a fraction of Ha-Ras appears to be recruited to lipid rafts and the fraction of Ha-Ras that localizes in rafts appears to decrease upon GTP binding (40). Our observations partially agree with this view. We show that Ha-Ras exists in discrete, cholesterol-rich, and detergent-resistant membrane domains that have all the properties of lipid rafts. However, we show that, rather than redistributing to the bulk lipid of the plasma membrane upon activation, as suggested by Prior et al. (40), Ha-Ras is translocated to an internal pool while attached to the surface of endocytic vesicles that remain insoluble in Triton X-100. These vesicles contain insulin receptors, and Ras, Raf-1, MEK and MAPK appear to be associated to their surface (see also Ref. 5). The most relevant feature of these vesicles is that they appear to be essential functional units. This conclusion is derived from the fact that, although functional Ras-Raf-1 complexes may exist on the plasma membrane, phosphorylated MEK is found exclusively on these endosomes. It follows from these observations that the traffic of Ras and Raf-1 is required not for the activation of Raf-1 kinase, but for the interaction of Raf-1 with its main endogenous substrate. The structural and biochemical reasons for this still remain unclear.
The linkage between endocytosis and the MAPK cascade was initially proposed on the basis of the effects of dynamin negative mutants on the activation of the MAPK cascade by receptor tyrosine kinases (41, 42) and G-protein-linked receptors (29, 30). More recent work has suggested that the relationship between receptor endocytosis and the activation of the MAPK cascade is casual rather than causal and that it is the inhibition of the traffic of other cellular components that causes the effects of dynamin (43). The data described here support this hypothesis. Effective receptor-dependent activation of Ras-Raf-1 complexes occurs at the plasma membrane level. However, other components are necessary for the coupling of Raf-1 activation to the phosphorylation of MEK. A plausible explanation for this observation is that the effective coupling of the MAPK can only occur on the surface of endosomes. Specific recruitment of cellular proteins to the surface of endosomes is not a new phenomenon (for instance, proteins containing FYVE domains are exclusively recruited to endosomes) (44). We therefore propose that the effective formation of scaffolding complexes on the surface of endocytic vesicles is a requirement for the coupling of Ras-Raf-1 activation to the phosphorylation of MAPK.
Many of the spatial-temporal features of the recruitment of cytosolic
proteins to the surface of cell membranes are regulated by the
appearance and disappearance of lipid second messengers. We have shown,
for instance, that Raf-1 binding to membranes is primarily driven by
its interactions with phosphatidic acid (5, 6). Coincidentally, a
putative scaffolding protein that binds MEK and MAPK, Ksr (the
so-called kinase suppressor of Ras)
(45, 46), has a sequence that is almost identical to the phosphatidic acid binding domain of Raf-1. Therefore, the model that emerges is one
in which phosphatidic acid brings Ksr and Raf-1 into close contact on
the surface of endosomes that are enriched in phosphatidic acid,
cholesterol, and possibly phosphatidylinositol derivatives. Further
work is needed to validate the correctness of this model.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R01-DK51183, R01-DK54782, and T32-GM54813.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 on-line version of this article (available at
http://www.jbc.org) contains movies as referred to in the
figure legends.
§ Supported by a predoctoral fellowship from the Susan B. Komen Foundation.
Recipient of Independent Investigator Award K01-DK02465 from
the National Institutes of Health. To whom correspondence should be addressed: Dept. of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261. Tel.: 412-648-9408; Fax: 412-648-1945;
E-mail: ggr@pitt.edu.
Published, JBC Papers in Press, July 20, 2001, DOI 10.1074/jbc.M105918200
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ABBREVIATIONS |
|---|
The abbreviations used are:
MAPK, mitogen-activated protein kinase;
GFP, green fluorescent protein;
CD, -methyl cyclodextrin;
GPI, glycosylphosphatidyl inositol;
MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase.
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REFERENCES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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