Download The C-terminal end of R-Ras contains a focal adhesion targeting

Research Article
3729
The C-terminal end of R-Ras contains a focal adhesion
targeting signal
Johanna Furuhjelm and Johan Peränen*
Institute of Biotechnology, Program in Cellular Biotechnology, PO Box 56 (Viikinkaari 9), FIN-00014 University of Helsinki, Finland
*Author for correspondence (e-mail: [email protected])
Accepted 28 May 2003
Journal of Cell Science 116, 3729-3738 © 2003 The Company of Biologists Ltd
doi:10.1242/jcs.00689
Summary
R-Ras promotes cell adhesion and activation of integrins
through a process that is yet unknown. We show here that
active R-Ras (38V) promotes the formation of focal
adhesions and a spread cell shape. By contrast, the
dominant-negative mutant of R-Ras (43N) reduces the
number of focal adhesions, leading to the formation of
refractile cells. In adherent cells wild-type R-Ras, activated
(38V) R-Ras and endogeous R-Ras were preferentially
targeted to focal adhesions, whereas the dominant-negative
mutant (43N) of R-Ras was excluded from these structures.
Activated mutants of H-Ras and K-Ras were not found in
focal adhesions. We dissected R-Ras to find out the
determinants that are important for the targeting process.
The outermost region in the N-terminus of R-Ras, as well
as the intact proline-rich sequence in the C-terminus of RRas that mediates binding to Nck, were not essential.
Mutating the potential palmitoylation site (C213A) of RRas results in depalmitoylation and accumulation of R-Ras
in the Golgi. Using H-Ras/R-Ras, R-Ras/H-Ras and RRas/K-Ras hybrid molecules we showed that the C-termini
(175-218 amino acids) of R-Ras contains the signal for focal
adhesions targeting. Exchanging the hypervariable region
of H-Ras to R-Ras inhibited the targeting of R-Ras to focal
adhesions, whereas H-Ras obtained the ability to localize to
focal adhesions after receiving the hypervariable region of
R-Ras. This indicates that R-Ras targeting is mediated both
by the nucleotide binding status as well as through a
specific region in the C-terminus of R-Ras. These results
indicate that targeting and activation of R-Ras are linked
processes in the formation of focal adhesions.
Introduction
Ras proteins are important regulators of cell differentiation and
growth that localize to the inner surface of the plasma
membrane (Crespo and Leon, 2000). Ras acts as a molecular
switch, converting signals from the cell surface to the nucleus.
In human cells there are four closely related Ras proteins: HRas, N-Ras, K-RasA and K-RasB. Although these proteins
interact with the same set of effectors in vitro, they activate
them with different efficiency (Voice et al., 1999). They also
show a variable ability to induce cell transformation and cell
motility (Voice et al., 1999). In addition, it has become clear
that K-Ras and H-Ras operate in different microdomains at the
plasma membrane (Prior et al., 2001; Prior and Hancock,
2001). H-Ras is found in lipid rafts and segregates into the bulk
membrane after activation, whereas K-Ras is located in the
bulk membrane, irrespective of bound nucleotide. The
difference in location is at least partly due to the hypervariable
region, where less than 10-15% of the residues are identical
among the four Ras proteins (Prior et al., 2001).
R-Ras shows 55% identity with Ras, and its minimal effector
region is identical to that of Ras, but R-Ras contains a 26 amino
acid (aa) extension in its N-terminus. Many Ras effectors and
some exchange factors interact with R-Ras, but R-Ras shows
a much lower ability to transform cells compared with Ras
(Cox et al., 1994). However, R-Ras regulates cell adhesion,
spreading and phagocytosis by activating integrins (Zhang et
al., 1996; Berrier et al., 2000; Self et al., 2001), and it has also
been shown to antagonize Ras/Raf-initiated integrin
suppression (Sethi et al., 1999). How R-Ras activates integrins
is not known, but its effector loop and prenylation site, as well
as the proline-rich sequence in the hypervariable region of RRas, are essential for this activation process (Oertli et al., 2000;
Wang et al., 2000). The proline-rich region has been shown to
bind the adaptor protein Nck, which is known to interact with
proteins accumulating in focal adhesions (Wang et al., 2000).
Furthermore, the Eph receptor tyrosine kinase, EphB2,
phosphorylates a tyrosine residue in the effector region of RRas, leading to suppression of R-Ras-mediated adhesion (Zou
et al., 1999). A similar relationship has also been found to exist
between activated Src and R-Ras (Zou et al., 2002).
Focal adhesions (FA) are specialized signalling platforms on
the cell surface that mediate cell-matrix interactions via
integrins, which are associated with different cytoskeletal
proteins (Sastry and Burridge, 2000). FAs are dynamic
structures that assemble and disassemble when cells migrate or
divide. This assembly/disassembly process is regulated by the
Rho GTPases (Kaibuchi et al., 1999). Activation of RhoA
promotes the assembly of large focal adhesions through
increased contractility, whereas Rac1 induces the formation of
small adhesions called focal complexes at the leading edge of
migrating cells (Nobes and Hall, 1995). The turnover of FAs
is regulated by Ras (Nobes and Hall, 1999). However, FAs may
not be the only platforms that mediate cell signalling. Recent
studies indicate that microdomains called lipid rafts may also
Key words: GTPase, R-Ras, Targeting, Focal adhesion
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Journal of Cell Science 116 (18)
participate in signal transduction (Simons and Toomre, 2000).
Whether the lipid rafts have a direct role in processes mediating
cell adhesion is still uncertain (Pande, 2000).
To better understand the role of R-Ras in cell adhesion we
decided to study its targeting to the cell surface. We show here
that R-Ras is preferentially targeted to focal adhesions and that
this targeting process is dependent on the nucleotide state of
R-Ras. Only GTP-bound R-Ras is associated with focal
adhesions, whereas the GDP form is excluded from these
structures. The hypervariable region of R-Ras was sufficient in
targeting another -Ras protein to focal adhesions, indicating
that this region contains the targeting signal. Finally, we show
that the targeting of R-Ras and the integrity of focal adhesions
is dependent on the cholesterol content of the plasma
membrane. Our data underscore the importance of specific
localization of Ras molecules on the plasma membrane in
mediating cell signalling.
Cells transfection, labelling and cholesterol depletion
Hela cells were grown overnight on collagen-coated (30 µg/ml)
coverslips and transiently transfected with constructs expressing RRas, Rac and their corresponding mutants by Fugene 6 according to
the manufacturer (Roche) (Peränen and Furuhjelm, 2001). Equal
molarity of constructs was used in double transfection studies.
Labelling of pEGFP-H-Ras61L-, pEGFP-R-Ras38V- and pEGFP-
Materials and Methods
Constructs
Plasmid pEXV-Ras-wt (a gift from A. Hall) was used to prepare RRas-38V and R-Ras-43N mutants by PCR mutagenesis, and these
were cloned into pGEM-3 (Promega, Madison, WI) (Peränen et al.,
1996). Two mutations, P202A and P203A, were made in the
hypervariable region of R-Ras-38V in the pGEM-R-Ras-38V plasmid.
A similar approach was used to create the S172P and C213A
mutations. The open reading frames (ORFs) from the pGEM
constructs of R-Raswt, R-Ras38V, R-Ras-43N, R-Ras38V/C213A and
R-Ras-38V/S172P were cut out and cloned into either pcDNA4/TO
(Invitrogen, Carlsbad, CA) or pEGFP-C1A (Clontech, Palo Alto, CA)
(Peränen and Furuhjelm, 2001). A fragment corresponding to the
hypervariable region of R-Ras (191-218 aa) was cloned into pEGFPC1A to obtain pEGFP-HVR. Plasmid pEGFP-R-Ras38V-deltaNT was
created by cloning a PCR fragment, corresponding to the 29-218 aa
in R-Ras, into pEGFP-C1A. Plasmid pEGFP-R-Ras38V-dC contained
R-Ras38V ORF missing the last six amino acids from the C-termini
(213-218). Plasmid pEGFP-R-Ras38V contained R-Ras38 that was
deleted of its 176-212 amino acids but retained the last six amino acids
(213-218). H-Ras and K-Ras were obtained by PCR from human
HeLa cDNA, and H-Ras61L and K-Ras12V were created by PCRbased mutagenesis. H-Ras61L/R-Ras38V, R-Ras38V/H-Ras61L and
R-Ras38V/K-Ras12V chimeras were constructed by splice overlap
mutagenesis. These genes were ultimately cloned into pEGFP-C1A.
Plasmids pEXV-Rac12V and pEXV-Rac17N (gifts from A. Hall) were
used to excise the corresponding Rac1 mutants to reclone them to
pEGFP-C1A and pEGFP-N1 (Clontech). The human Arf6 reading
frame was amplified by PCR from HeLa cDNA and cloned into
pEGFP-N1 or pcDNA4/TO; the Arf6-27N and Arf6-67L mutants
were made by site-specific PCR mutagenesis as described earlier
(Peränen et al., 1996). All constructs based on PCR were verified by
DNA sequencing. Details of the constructs are available on request.
Western blot
For western blot analysis HeLa cells were grown overnight on two 6
cm plates and transiently transfected with pEGFP-R-Raswt, pEGFPR-Ras38V or pEGFP-R-Ras43N using Fugene 6 according to the
manufacturer (Roche Diagnostics, Mannheim, Germany). After 20
hours the cells, which were about 80% confluent, were lysed by SDSPAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis)
sample buffer and the DNA was disrupted by passing it through a
needle. The presence of R-Ras was detected by western blot using
anti-R-Ras (Santa Cruz Biotechnology, Santa Cruz, CA) as previously
described (Peränen et al., 1996).
Fig. 1. R-Ras uses endomembranes to reach the plasma membrane.
Hela (A-C) and Cos-7 (D-F) cells were transfected with vectors
pEGFP-R-Raswt (A,B) and pEGFP-R-Ras43N (C,D), or double
transfected with pEGFP-R-Ras43N and pcDNA4/TO-Arf6-27N
(E,F). EGFP-R-Raswt colocalized with a p115 Golgi marker (arrow)
and was also seen on the nuclear envelope (arrowhead). In addition
to these organelles, EGFP-R-Ras43N was found on vesicular (C) and
vacuolar (D) structures, which colocalized with Arf6-27N
(arrowheads) (E,F). HeLa cells were transfected with pEGFP-RRaswt (wt), pEGFP-R-Ras38V (38V), pEGFP-R-Ras43N (43N) and
pEGFP-C1A, and total cell extracts were analysed by western blot
(G). The positions of molecular size markers are indicated.
Targeting of R-Ras to focal adhesions
Rras38V/213A-transfected HeLa cells with [9,10(n)-3H]palmitic acid
(Amersham Biosciences Europe) at 200 µCi/ml in modified Eagle’s
medium plus 5% dialysed bovine serum for 4 hours.
Immunoprecipitation of the indicated Ras molecules from the labelled
cells was done by H-Ras- and R-Ras-specific antibodies as described
earlier (Peränen and Furuhjelm, 2001). The immunoprecipates were
analysed by SDS-PAGE and fluorography. Cholesterol depletion of
HeLa cells with 0.5-1% β-methylcyclodextrin (MβCD) for 30-60
minutes was carried out as described previously (Parton and Hancock,
2001). Cholesterol/MβCD inclusion complexes were added to
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cholesterol-depleted cells or to cells grown in serum (Parton and
Hancock, 2001).
Cell spreading
Subconfluent Hela cells were transfected overnight with indicated
EGFP-R-Ras constructs. They were then harvested with 0.5 mM
EDTA in phosphate-buffered saline (PBS), resuspended in MEM
containing fatty-acid-free bovine serum albumin (5 mg/ml), and the
cells were counted. An appropriate number of cells were then plated
onto collagen- (30 µg/ml) coated cover slips. After 60 minutes at 37oC
cells were fixed with paraformaldehyde and stained for vinculin.
Spread versus unspread EGFP-positive cells were counted.
Immunocytochemistry
Transfected C2C12 or HeLa cells were processed for
immunofluorescence or confocal microscopy as previously described
(Peränen et al., 1996; Peränen and Furuhjelm, 2001). Antibodies used
were anti-Arf6 (NeoMarkers, Fremont, CA), anti-caveolin (BD
Biosciences, San Diego, CA), anti-β1 integrin (BRL/GIBCO,
Gaitersburg, MD), anti-paxillin (Trans lab), anti-phospho-caveolin
(Trans lab), anti-p115 (Trans lab), anti-R-Ras (Santa Cruz), anti-talin
(Sigma), anti-vinculin (Sigma). Goat anti-rabbit IgG-lissamine and
goat anti-mouse IgG lissamine were from Jackson Immunoresearch.
Results
R-Ras uses the endomembrane transport route
To investigate the targeting of R-Ras, we constructed
constitutive active (38V) and dominant-negative (43N)
mutants of R-Ras and tagged them to EGFP. Western blot
analysis of Hela cell extracts harbouring these constructs
showed the presence of a 55 kDa protein, as well as
endogenous R-Ras (Fig. 1G). The constructs, and
corresponding constructs of the original wild-type R-Ras, were
then transfected into HeLa cells and processed for
immunofluorescence and confocal microscopy. Each of the RRas proteins localized to a perinuclear region, to the Golgi and
to the plasma membrane (Figs 1, 2). Identical results were
obtained for untagged Ras constructs (not shown). The
subcellular localization of R-Ras was very similar to that found
for H-Ras and N-Ras, suggesting that R-Ras uses an analogous
endomembrane trafficking route (Choy et al., 1999). In
addition, we found EGFP-R-Ras43N on numerous vesicles in
HeLa cells, and on vacuoles in COS cells (Fig. 1C,D). The
EGFP-R-Ras43N vesicles were not positive for such known
organelle markers as Rab4, Rab5, Rab6 and Rab11 (not
shown). However, we observed that EGFP-R-Ras43N
Fig. 2. Plasma membrane localization of R-Ras molecules. HeLa
cells transiently expressing EGFP-R-Raswt (A), EGFP-R-Ras38V
(B), EGFP-R-Ras43N (C) and EGFP (D) were fixed and analysed by
fluorescence microscopy. Note that EGFP-R-Raswt and EGFP-RRas38V are found in adhesion-like structures, whereas EGFP-RRas43N and EGFP are not. Localization of endogenous R-Ras (E) to
vinculin (F) containing focal adhesions in untransfected HeLa were
seen using specific antibodies against R-Ras and vinculin. EGFP-RRas38V was also detected in focal adhesions in C2C12 cells (G,H).
Arrowheads indicate focal adhesion-like localization. Bars, 10 µm.
(I) The percentage of cells containing R-Ras in focal adhesions was
calculated for R-Raswt, R-Ras38V and R-Ras43N. Values are means
± s.d. from three independent experiments in which >100 cells were
scored per condition.
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Journal of Cell Science 116 (18)
Fig. 3. R-Ras in focal adhesions. Cells were
transfected with pEGFP-R-Ras38V (R-Ras38V),
and pEGFP-R-Ras43N (R-Ras43N) fixed, stained
for vinculin and analysed by confocal microscopy.
Note that R-Ras43N does not colocalize with
vinculin, whereas R-Ras38V shows a strong
colocalization. In R-Ras-38V/Rac1-12V the
pCDNA4-TO-R-Ras38V vector was cotransfected
with pEGFP-Rac12V and stained with an antibody
against R-Ras. Arrowheads indicate colocalization
of R-Ras and EGFP-Rac1 to focal adhesions. Bars,
10 µm.
colocalized with Arf6, especially with the Arf6-27N mutant on
vesicular structures (Fig. 1E,F), indicating that R-Ras could be
internalized to a recycle compartment (Brown et al., 2001).
GTP-dependent targeting of R-Ras to focal adhesions
The plasma membrane is considered to be the main platform
for Ras-mediated signalling, where R-Ras is thought to
regulate adhesion and integrin activation (Zhang et al., 1996;
Sethi et al., 1999). Interestingly, both EGFP-R-Raswt and
EGFP-R-Ras38V were localized to focal adhesion-like
structures in HeLa cells, whereas EGFP-R-Ras43N was evenly
distributed on the plasma membrane (Fig. 2A-F). EGFP-RRas38V showed the strongest staining intensity in focal
adhesions. Quantification of cells harbouring the abovementioned constructs showed that 84% of the R-Ras38Vexpressing cells contained R-Ras38V in focal adhesions,
whereas the value for EGFP-R-Raswt was 40% (Fig. 2I). By
contrast, only 0.5% of cells expressing EGFP-R-Ras43N
contained R-Ras-positive focal adhesions, suggesting that the
GTP-bound form of R-Ras is preferentially targeted to focal
adhesions. When R-Ras was expressed in excess its
localization to the focal adhesions was more difficult to
observe due to strong staining of the surrounding plasma
membrane, possibly indicating that the binding of R-Ras to
focal adhesions was saturated (data not shown). Identical
results were obtained with untagged R-Ras constructs in
combination with anti-R-Ras antibodies (Fig. 3). R-Ras-38V
was also observed in C2C12 cells (Fig. 2G,H), therefore its
localization to focal adhesions was not restricted to HeLa cells.
However, in the HT1080 fibrosarcoma cell line and COS-7
cells EGFP-R-Ras38V was not associated with focal adhesions
(not shown). HT1080 cells contain a mutant N-ras allele that
mediates a transformed phenotype, including disorganized
actin and poor adherence, and these features may counteract
the targeting of R-Ras to focal adhesions (Marshall et al.,
1982). This indicates that the localization of R-Ras to focal
adhesions is typical for strongly adherent cells, like HeLa and
C2C12. Furthermore, we also showed that endogenous R-Ras
in HeLa cells is localized to focal adhesions that are positive
for vinculin (Fig. 2E,F). EGFP alone did not localize to focal
adhesions, but was preferentially found in the nucleus (Fig.
2D).
To characterize the identity of R-Ras-containing focal
adhesions we stained EGFP-Ras38V-expressing HeLa cells
with known focal adhesion markers. Vinculin colocalized
nicely with EGFP-Ras38V when analysed by confocal
microscopy (Fig. 3). This was also true for β1-integrin, talin
and paxillin (data not shown). By contrast, EGFP-R-Ras43N
did not colocalize with these markers (Fig. 3). It has recently
been shown that Rac12V colocalizes with p21-activated
protein kinase, α-PAK, to focal adhesions of HeLa cells
(Manser et al., 1997). Likewise, we found that EGFP-Rac12V
localized with R-Ras38V in focal adhesions, indicating that
there may be a functional link between Rac1 and R-Ras
(Fig. 3).
Targeting of R-Ras to focal adhesions
3733
Fig. 4. R-Ras modulates focal adhesion
formation and cell spreading. Hela cells grown
on collagen-coated glass coverslips were
transfected overnight with pEGFP-Raswt
(A,D), pEGFP-R-Ras38V (B, E) and pEGFP-RRas43N (C,F). Note that the R-Raswt cell
contains distal focal adhesions, the R-Ras38V
cell contains numerous large focal adhesions and
the R-Ras43V cell contains few small adhesions.
R-Raswt cells were often elongated (D), RRas38V cells (E) well spread and R-Ras43N
cells (F) retracted with filopodia-like structures.
The number of focal adhesions was calculated
from above transfected cells, 15 cells/construct/
experiment (J). Results shown are the mean ±
s.e.m. of three experiments. Cells expressing
EGFP-Raswt, EGFP-R-Ras38V and pEGFP-RRas43N were also plated on collagen-coated
coverslips for 60 minutes for estimation of cell
spreading (K). 30 cells were counted for each
construct and experiment. Results shown are the
mean ± s.e.m. of three experiments. Typical
spread cells are shown for corresponding
construct in G, H and I. Note the numerous large
focal adhesions in EGFP-R-Ras38V cells and the
few small in EGFP-R-Ras43N cells. Bars,
10 µm.
Focal adhesions formation is linked to R-Ras activity
R-Ras is known to regulate cell adhesion. Thus, we next
studied whether R-Ras influences the structure and number of
focal adhesions in HeLa cells. Cells were transfected overnight
with constructs encoding EGFP-R-Raswt, EGFP-R-Ras38V or
EGFP-R-Ras43N (Fig 4). Cells expressing EGFP-R-Raswt
were often elongated and contained focal adhesions in the
distal regions of the cell. When cells expressed EGFP-R-Ras38
they were symmetrical and spread, and they contained large
focal adhesions that were located nearer the center of the cell.
By contrast, cells expressing EGFP-R-Ras43N were contracted
and often contained filopodia-like structures. Moreover, the
focal adhesions located distally and were very small compared
with those found in EGFP-R-Ras38-expressing cells. We next
quantified the number of focal adhesions in cells expressing
these EGFP-R-Ras constructs (Fig. 4J). We found that RRas38V-expressing cells contained more focal adhesions than
did R-Raswt, whereas R-Ras43-expressing cells had fewer
adhesions than R-Raswt. This suggests that active R-Ras
promotes the formation of focal adhesions, whereas R-R-Ras
inhibits their formation. The same EGFP-R-Ras constructs
were also used to test cell spreading (Fig. 4K). EGFP-R-Raswt
and EGFP-R-Ras38V cells spread with an equal efficiency,
whereas EGFP-R-Ras43N expression led to fewer spread cells.
The difference in cell spreading correlated with the formation
of focal adhesions. EGFP-R-Ras38N cells were symmetrical
with large focal adhesions, whereas EGFP-R-Raswt cells had
adhesions more peripherally. By contrast, EGFP-R-Ras43Nexpressing cells contained very few and tiny focal adhesions.
In summary, our results show that R-Ras is essential for the
formation of focal adhesions, and confirm previous studies that
R-Ras controls cell spreading (Berrier et al., 2000; Zhang et
al., 1996).
Potential targeting signals in R-Ras
R-Ras contains an extended N-terminus compared with other
Ras molecules. We deleted this N-terminal region (1-28 aa) and
expressed the molecule, EGFP-R-Ras38V-NT, in HeLa cells.
Because EGFP-R-Ras38V-NT was nicely localized to focal
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Journal of Cell Science 116 (18)
Fig. 5. R-Ras constructs and R-Ras palmitoylation. (A) GFP
constructs used were wild-type R-Ras (pEGFP-R-Raswt),
constitutively active R-Ras38V (pEGFP-R-Ras38V), dominantnegative R-Ras43 (pEGFP-R-Ras43N), constitutively active R-Ras
deleted of the first 28 amino acids (pEGFP-R-Ras38V-NT),
constitutively active R-Ras38V deleted of its last six amino acids
(pEGFP-R-Ras38VdC), constitutively active R-Ras38V deleted of its
HVR (175-212) but containing the last six amino acids (212-218)
(pEGFP-R-Ras38V-dHVR), constitutively active R-Ras38V-2PA
with two proline substituted on P202A and P203A (pEGFP-RRas38V-2PA), constitutively active R-Ras38V with a mutated
palmitoylation site C213A (pEGFP-R-Ras38V-CA), constitutively
active R-Ras38V with both palmitoylation and nucleotide
destabilizing mutations C213A/S172P (pEGFP-R-Ras38V-SP) and
the hypervariable region encoding 191-218aa of R-Ras (pEGFPHVR). (B) HeLa cells were transfected overnight with the following
constructs: pEGFP-H-Ras61L (1), pEGFP-R-Ras38V-CA (2) and
pEGFP-R-Ras38V (3). The cells were then labelled with
[3H]palmitic acid for 4 hours, immunoprecipitated and analysed by
SDS-PAGE. Note that the construct encoding EGFP-R-Ras with a
mutated palmitoylation site (C213A; lane 2) is not labelled with
palmitate, whereas EGFP-H-Ras61L (lane 1) is strongly labelled,
and EGFP-R-Ras38V (lane 3) to a lesser extent.
targeting R-Ras to focal adhesions. To test this hypothesis we
fused the hypervariable region (HVR: 191-218 aa) of R-Ras to
EGFP (Fig. 5A). This EGFP-HVR was efficiently transported
to the plasma membrane via Golgi. However, it was not
targeted to focal adhesions; instead, it showed a uniform
distribution at the plasma membrane, indicating that this part
of the hypervariable region is essential for membrane targeting
but insufficient for localization of R-Ras to focal adhesions
(Fig. 6C,D). We also made a construct encoding a protein
(EGFP-R-Ras38V-dHVR) that lacked the whole HVR (175212 aa) but which contained the six outermost amino acids
needed for lipid modification. This protein was trapped in the
Golgi and on small vesicles, indicating that HVR is essential
for proper transport to the plasma membrane (Fig. 6E,F). The
proline-rich sequence in the hypervariable region binds a SH3
domain of Nck, indicating that it might contribute to the
targeting mechanism of R-Ras (Wang et al., 2000). However,
the introduction of mutations (P202A, P203A) in the prolinerich sequence of R-Ras38V did not inhibit targeting of RRas38V to focal adhesions (Fig. 5A; Fig. 6G,H).
R-Ras is palmitoylated and contains a potential
palmitoylation site (C213) upstream of the CAAX box (Lowe
and Goeddel, 1987; Schmittberger and Waldmann, 1999). We
showed that EGFP-R-Ras38V is labelled with palmitate,
whereas EGFP-R-Ras38V/C213A, which contains a mutation
in the potential palmitoylation site did not incorporate
palmitate, suggesting that cysteine 213 is the target for
palmitoylation in R-Ras (Fig. 5A,B). Moreover, EGFP-RRas38V/C213A showed an increased accumulation in Golgi
and consequently a decreased appearance on the plasma
membrane when compared with the other R-Ras mutants (Fig.
6I,J). Such a Golgi retention has also been observed when the
palmitoylation sites of H-Ras and N-Ras are mutated (Choy et
al., 1999). EGFP-R-Ras38V incorporates far less palmitate
than EGFP-H-Ras61L; this can not be explained simply by the
fact that H-Ras contains two palmitoylation sites but R-Ras
contains only one (Fig. 5B). Both are labelled equally well by
methionine when analysed by immunoprecipitation (not
shown). One possibility is that the turnover rate of palmitate is
different for these two Ras molecules. The C213A mutation
did not inhibit R-Ras38V association with focal adhesions (not
shown). The introduction of an additional mutation (S173P),
which probably destabilizes nucleotide binding, into EGFP-RRas38V/C213A led to retention of R-Ras in the endoplasmic
reticulum (ER), suggesting that proper nucleotide binding or
protein folding is needed for transport from the ER (Fig. 5A;
Fig. 6K,L). Finally, deletion of both the CAAX box and the
palmitoylation site resulted in the accumulation of the
molecule in the cytoplasm and nucleus, suggesting that R-Ras
can not associate with focal adhesions when it is not bound to
the membrane (Fig. 5A; Fig. 6M,N). Taken together, our results
show that the hypervariable region of R-Ras is crucial for
membrane targeting and transport.
adhesions it is unlikely that this region is important for
targeting (Fig. 6A,B). The hypervariable region of small
GTPases has been implicated in membrane-specific targeting
processes (Choy et al., 1999; Chavrier et al., 1991; Michaelson
et al., 2001). This raised the possibility that the hypervariable
region, including the outermost C-terminus, is responsible for
Dissecting the R-Ras-specific targeting signal by using
R-Ras/H-ras/K-Ras hybrid molecules
H-Ras and K-Ras are known to localize to different
subdomains on the plasma membrane (Prior and Hancock,
2001). We fused H-Ras61L and K-Ras12V to EGFP, and
expressed them in HeLa cells. In contrast to EGFP-R-Ras,
Targeting of R-Ras to focal adhesions
neither EGFP-H-Ras61L nor EGFP-K-Ras12V localized to
focal adhesions (Fig. 7B). However, both proteins induced the
3735
formation of lamellipodia and ruffles, which are typical for
transformed cells. Moreover, their expression resulted in a
decrease in the number and size of the focal adhesions. To see
whether the HVR of R-Ras is essential for the targeting
process, we replaced the 175-218 aa region from R-Ras38V
with the corresponding regions (148-189 aa) from H-Ras or
from K-Ras (147-188 aa) (Fig. 7A). The hybrid proteins
EGFP-R-Ras38V/H-RasC and EGFP-R-Ras38V were
expressed in HeLa cells. Neither proteins were localized to
focal adhesions but were uniformly distributed at the plasma
membrane (Fig. 7B). In addition, both had a phenotype that
more resembled H-Ras61L and K-Ras12V. Because the 175218 aa region of R-Ras seemed to be essential for focal
adhesion targeting we reasoned that this region could also
function in H-Ras as a targeting signal. Thus, we made an HRas61L/R-RasC chimera, which was expressed in HeLa cells
(Fig. 7A). This chimera, which is mainly composed of H-Ras,
was localized to vinculin-containing focal adhesions,
indicating that the 175-218 aa of R-Ras contains the essential
elements for focal adhesion targeting (Fig. 7B).
Focal adhesions are sensitive to cholesterol depletion
It has been shown recently that H-Ras resides in cholesterolrich lipid rafts and caveolae on the plasma membrane, and that
activation of H-Ras leads to its segregation from rafts (Prior et
al., 2001; Prior and Hancock, 2001). K-Ras, by contrast, is
localized predominantly to the disordered plasma membrane
(Prior et al., 2001). Caveolin, the main component of caveolae,
is known to participate in integrin-mediated adhesion and Ras
signalling (Parton and Hancock, 2001; Wei et al., 1999; Roy
et al., 1999). Thus, we stained EGFP-R-Ras38V-expressing
cells with anti-caveolin and anti-phospho-caveolin recognizing
caveolin phosphorylated on tyrosine 14 (Lee et al., 2000).
Caveolin was found in patches over the cell surface and along
the cell margins, but there was no colocalization with EGFPR-Ras38V (data not shown). By contrast, phospho-caveolin
colocalized nicely with EGFP-R-Ras38V in focal adhesions
(Fig. 8A,B). To investigate whether the localization of RRas38V to focal adhesions is dependent on cholesterol-rich
subdomains, we depleted serum-starved cells expressing
EGFP-R-Ras-38V with 0.5-1% β-methylcyclodextrin for 30
minutes. This resulted in the smooth distribution of EGFP-RRas-38V on the plasma membrane and redistribution of
phospho-caveolin to small dot-like structures that no longer colocalized with EGFP-R-Ras38V (Fig. 8E,F). This was
associated with a simultaneous decrease in the size and number
of focal adhesions. Repletion of cholesterol depleted cells with
cholesterol/CD inclusion complexes for 30-60 minutes resulted
in reformation of focal adhesions and retargeting of both
EGFP-R-Ras38V and phospho-caveolin (Fig. 8G,H). Finally,
cholesterol replenishment on cells that had not been serum
Fig. 6. Determinants affecting R-Ras targeting. HeLa cells were
transiently transfected with pEGFP-R-Ras38V-NT (A,B), pEGFPHVR (C,D), pEGFP-R-Ras38V-dHVR (E,F), pEGFP-R-Ras38V-2PA
(G,H), pEGFP-R-Ras38V-CA (I,J), pEGFP-R-Ras38V-SP (K,L) and
pEGFP-R-Ras38V-dC (M,N), fixed and stained with anti-vinculin.
Observe the absence of EGFP-HVR from vinculin-containing
adhesions. Note the accumulation of R-Ras38V-dHVR, R-Ras38VCA in Golgi and R-Ras38V-SP in ER. EGFP-R-Ras38V-dC that
lacks signals for lipid modifications is found in the nucleus.
3736
Journal of Cell Science 116 (18)
starved showed normal focal localization for both EGFP-RRas-38V and phospho-caveolin (Fig. 8C,D). We conclude that
the integrity of focal adhesions and the localization of EGFP-
A
G38V
218
174
pEGFP-R-Ras38V
GFP
Q61L
147
189
147
188
pEGFP-H-Ras61L
GFP
G12V
pEGFP-K-Ras12V
GFP
G38V
174
GFP
pEGFP-R-Ras38V/H-RasHVR
148
Q61L
189
147
pEGFP-H-Ras61L/R-RasHVR
GFP
175
G38V
218
174
pEGFP-R-Ras38V/K-RasHVR
GFP
148
188
R-Ras38V and phospo-caveolin to adhesions are dependent on
the cholesterol content of the plasma membrane.
Discussion
We showed here that R-Ras localizes to focal adhesions on the
plasma membrane. The localization is determined by the
GTP/GDP state, so that only R-Ras-GTP is recruited to focal
adhesions. R-Ras-GTP has been shown to enhance both cell
adhesion and cell spreading (Zhang et al., 1996; Berrier et al.,
2000), and this is supported by our study. We showed that RRas also regulates the formation, number and size of focal
adhesions. Thus, it is likely that the targeting of R-Ras-GTP to
focal adhesions is functionally linked to an increase in
adhesion and spreading.
To unravel the targeting signal of R-Ras we constructed
different R-Ras mutants. We showed that the N-terminus
present in R-Ras is not necessary for the targeting process.
Although mutations in the proline-rich sequence of R-Ras38V
suppresses cell attachment, the mutants are still more potent
than R-Raswt in inducing cell attachment (Wang et al., 2000).
Because the mutations in this sequence did not affect R-Ras
targeting, the sequence must have other functions that are
related to the binding of Nck (Wang et al., 2000). One
possibility is that Nck could mediate a cross-talk between RRas and Rac1, because it binds PAK, which is known to
interact with Rac1 (Manser et al., 1994; Manser et al., 1997).
Moreover, we showed that R-Ras and Rac1 colocalize in focal
adhesions. Such a cross-talk could be important in amplifying
signals that mediate cell adhesion and spreading.
We showed that lipid modification is essential for R-Rasspecific targeting, because R-Ras lacking these signals is found
in the cytoplasma and nucleus. In addition, we showed for the
first time that amino acid C213 of R-Ras is the most probable
attachment site for palmitic acid. When this site was mutated,
R-Ras accumulated in the Golgi. This was also the case when
the hypervariable region was deleted. Together, this suggests
that palmitoylation and the HVR region are important for the
transport of R-Ras by endomembranes to the plasma
membrane.
Fig. 7. Chimeric Ras constructs unravelled the R-Ras-specific focal
adhesion targeting signal. (A) Activated forms of H-Ras (61L),
K-Ras (12V) and R-Ras (38V), as well as indicated hybrids, were all
fused to EGFP. The R-Ras38V/H-RasHVR contained the first 1-174
amino acids from R-Ras38V and the 148-189 amino acids from the
C-termini of H-Ras12V. The H-Ras12V/R-RasHVR contained the Ntermini 1-147 amino acids from H-Ras61L and the 175-218 amino
acids from the C-termini of R-Ras38V. The R-Ras38V/K-RasHVR
hybrid contained the 1-174 amino acids from the N-termini of RRas38V and the 148-188 amino acids from the C-termini of KRas12V. (B) Hela cells were transfected overnight with pEGFP-HRas61L (a,b), pEGFP-K-Ras12V (c,d), pEGFP-R-Ras38V/HRasHVR (e,f) pEGFP-R-Ras38V/K-RasHVR (g,h), and pEGFP-HRas61L/R-RasHVR (i,j), fixed and stained for vinculin. Note that
neither K-Ras12V nor H-Ras61L was found in focal adhesions.
However, both showed typical ruffle and lamellipodia structures and
usually a reduced number of focal adhesions. Replacing the R-Ras
specific HVR with corresponding regions from H-Ras61L and KRas12V inhibited R-Ras targeting (e-h). By contrast, adding the
HVR from R-Ras to H-Ras61L led to focal adhesion targeting (i,j).
Arrowheads show H-Ras61L/R-RasHVR in focal adhesions.
Targeting of R-Ras to focal adhesions
Fig. 8. Cholesterol-dependent localisation of R-Ras to focal
adhesion. Hela cells transfected with pEGFP-R-Ras38V were grown
over night in serum-free (K) medium (A,B). Cholesterol
replenishment (CD/Chol) was carried out for 30 minutes on EGFPR-Ras38V expressing cells grown overnight in the presence of serum
(C,D). Some serum-starved cells were depleted of cholesterol by
adding 0.5% β-methylcyclodextrin (MβCD) for 30 minutes (E,F).
(G,H) Cholesterol replenishment of cholesterol depleted cells (CDCD/Chol) for 30 minutes. EGFP-R-Ras38V expressing cells
(A,C,E,G) were fixed with paraformaldehyde, permeabilized with
0.1% Triton-X-100 and stained with an antibody against phosphocaveolin (P-Caveolin) (B,D,F,H). Bars, 10 µm.
Deletions and mutations might have pronounced effects on
the function of different proteins, making it difficult for us to
find targeting signals. R-Ras is closely related to H-Ras and
K-Ras, which makes it possible to switch regions between
these molecules without making gross changes in the protein
architecture. However, H-Ras and R-Ras have opposing
effects on integrin activation. R-Ras promotes integrin
activation, whereas H-Ras suppresses integrin activation
(Zhang et al., 1996; Hughes et al., 1997). When the
hypervariable region of R-Ras (aa 175-218) was replaced by
the corresponding region (aa 147-189) of H-Ras the targeting
of R-Ras to focal adhesions was inhibited. Interestingly, an
identical replacement between R-Ras and H-Ras was recently
shown to suppress R-Ras-mediated integrin activation
(Hughes et al., 2002). The hypervariable region of K-Ras also
3737
inhibited the targeting of R-Ras. Furthermore, when the
hypervariable region of H-Ras (aa 147-189) was replaced by
the corresponding region (aa 175-218) of R-Ras, the H-Ras
molecule was targeted to focal adhesions, showeing that the
hypervariable region of R-Ras contains a focal adhesionspecific targeting signal. Recently, a similar construct was
shown to confer R-Ras specificity to H-Ras (Hansen et al.,
2002). Taken together, this suggests that the hypervariable
region of R-Ras is important for both focal adhesion targeting
and integrin activation, and that these two processes are
closely linked to each other.
What would be the advantages of GTP-dependent targeting
of R-Ras to focal adhesions? First, the coupling of R-Ras
activation to targeting would localize the function of R-Ras to
a defined region on the plasma membrane, eliminating
randomized signalling. Second, a localized high concentration
of R-Ras molecules could amplify the signal that mediates cell
adhesion. This could lead to the recruitment of other signal
molecules and scaffolding proteins, thereby building up the
focal adhesion. This is supported by the fact that R-Ras
enhances the phosphorylation of focal adhesion kinase (FAK)
and p130cas (Kwong et al., 2003). Conversely, deactivation of
R-Ras would lead to exclusion of R-Ras from focal adhesions,
making it free for a new round of targeting. Deactivation could
be mediated by GTPase-activating proteins (GAPs), or through
phosphorylation. Interestingly, the effector domain of R-Ras is
phosphorylated by an Eph receptor kinase and by the activated
Src, leading to suppression of R-Ras-mediated cell adhesion
(Zou et al., 1999; Zou et al., 2002). Finally, a specific lipid
raft-like composition of the adhesion structure might support
protein-protein-based targeting of R-Ras to focal adhesions.
The cholesterol content especially may have an important role
in modulating the rigidity of focal adhesions (Gopalakrishna
et al., 2000), as we show here.
Why is H-Ras not localized to focal adhesions? One
possibility is that the time during which H-Ras resides in focal
adhesions is more transient. This is supported by the fact that
H-Ras12V has been found to be associated with focal
adhesions at early times of expression and when expressed at
relatively low levels (Nobes and Hall, 1999). In addition,
activated H-Ras has a more suppressive function on integrins,
inducing loss and enhanced turnover of adhesions (Nobes and
Hall, 1999; Hughes et al., 1997), which might explain why RRas is excluded from HT1080 cells that possess a mutant Nras allele.
Both H-Ras and N-Ras use the endomembrane system to
reach the plasma membrane (Choy et al., 1999). We showed
that this is also true for R-Ras. Likewise, palmitoylation is
crucial for the efficient transport of R-Ras from Golgi onwards.
Whether R-Ras proteins are internalized from the plasma
membrane is unclear. However, we observed the GDP form of
R-Ras on vesicular structures colocalizing with Arf6,
suggesting that R-Ras undergoes internalization and recycling
(Brown et al., 2001). An interesting possibility is that R-Ras
deactivation versus activation is coupled to a membrane-based
recycling route that is important for the regulation of adhesion
turnover. Future studies in this direction could be important in
understanding the functions of focal adhesions.
In conclusion, our data on R-Ras further strengthens the
importance of specific micro-domains on the plasma
membrane as platforms for signalling by small GTPases.
3738
Journal of Cell Science 116 (18)
We thank Drs P. Auvinen, P. Lappalainen and Sandra Falck for
comments on the manuscript. The Academy of Finland, Helsinki
University Foundation, Biocentrum Helsinki, and Nylands Nation
supported this research.
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