2004, 24(8):3485. DOI:
Mol. Cell. Biol.
and Angel Pellicer
Ignacio Perez de Castro, Trever G. Bivona, Mark R. Philips
Only on the Golgi Apparatus
Receptor Is Specific to N-Ras and Occurs
Low-Grade Stimulation of the T-Cell
Ras Activation in Jurkat T cells following
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on September 18, 2012 by CNIO
MOLECULAR AND CELLULAR BIOLOGY, Apr. 2004, p. 3485–3496
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 8
Ras Activation in Jurkat T cells following Low-Grade Stimulation of
the T-Cell Receptor Is Specific to N-Ras and Occurs Only on the
Ignacio Perez de Castro,1Trever G. Bivona,2Mark R. Philips,2and Angel Pellicer1*
Department of Pathology and New York University Cancer Institute1and Departments of Medicine, Pharmacology
and Cell Biology,2New York University School of Medicine, New York, New York 10016
Received 28 January 2003/Returned for modification 1 April 2003/Accepted 17 December 2003
Ras activation is critical for T-cell development and function, but the specific roles of the different Ras
isoforms in T-lymphocyte function are poorly understood. We recently reported T-cell receptor (TCR) activa-
tion of ectopically expressed H-Ras on the the Golgi apparatus of T cells. Here we studied the isoform and
subcellular compartment specificity of Ras signaling in Jurkat T cells. H-Ras was expressed at much lower
levels than the other Ras isoforms in Jurkat and several other T-cell lines. Glutathione S-transferase–Ras-
binding domain (RBD) pulldown assays revealed that, although high-grade TCR stimulation and phorbol ester
activated both N-Ras and K-Ras, low-grade stimulation of the TCR resulted in specific activation of N-Ras.
Surprisingly, whereas ectopically expressed H-Ras cocapped with the TCRs in lipid microdomains of the
Jurkat plasma membrane, N-Ras did not. Live-cell imaging of Jurkat cells expressing green fluorescent
protein-RBD, a fluorescent reporter of GTP-bound Ras, revealed that N-Ras activation occurs exclusively on
the Golgi apparatus in a phospholipase C?- and RasGRP1-dependent fashion. The specificity of N-Ras
signaling downstream of low-grade TCR stimulation was dependent on the monoacylation of the hypervariable
membrane targeting sequence. Our data show that, in contrast to fibroblasts stimulated with growth factors
in which all three Ras isoforms become activated and signaling occurs at both the plasma membrane and Golgi
apparatus, Golgi-associated N-Ras is the critical Ras isoform and intracellular pool for low-grade TCR
signaling in Jurkat T cells.
Ras proteins are small, guanine-nucleotide-binding proteins
that are implicated in the regulation of multiple cellular func-
tions, including proliferation, differentiation, and apoptosis
(26). Mutations that render Ras constitutively active occur in
many human malignancies (5, 33). Ras proteins cycle between
inactive GDP-bound and active GTP-bound states. The two
main groups of molecules involved in the regulation of Ras
proteins are the guanine nucleotide exchange factors that pro-
mote the transition from the inactive GDP-bound to the active
GTP-bound state and the GTPase-activating proteins (GAPs)
that inhibit Ras by stimulating its GTPase activity.
Mammals have three functional ras genes, H-ras, K-ras, and
N-ras, the products of which have very similar structures (26).
The K-ras gene contains two alternative fourth coding exons,
giving rise to two splice variants, K-Ras4A and K-Ras4B. Since
the K-Ras4A alternative accounts for less than 10% of total
K-ras mRNA, we will refer to K-Ras4B as the K-Ras isoform.
At the amino acid level, Ras isoforms are identical for the first
80 amino acids, exhibit 85% identity for the next 80 residues,
and display only 15% amino acid conservation within the C-
terminal 25 amino acids (3, 6). The C-terminal hypervariable
region directs the posttranslational modifications of the pri-
mary ras gene products that determine their subcellular local-
ization (18, 19).
Ras proteins play an important role in the signaling path-
ways that activate cytokine gene induction and in the control of
T-cell development (17). Since the activation of Ras upon
T-cell stimulation was first demonstrated (12), a critical role of
Ras in antigen receptor signaling in lymphocytes has been
appreciated (1, 16, 20). In fact, the loss of Ras function pre-
vents the proliferation, cytokine production, and lymphocyte
development induced by the recognition of the antigen (39,
A number of functional differences between the Ras iso-
forms have been reported (26). For example, different ras
genes have been found mutated in different tumor types (5,
33), and mice deficient in the different Ras isoforms exhibit
different developmental phenotypes (14, 21, 24, 44). Despite
these differences, the specific function(s), if any, of the various
Ras isoforms is poorly understood. However, a number of
studies have pointed out the importance of N-Ras in T-cell
function. Firstly, activating mutations of N-Ras are frequently
found in human and mouse hematopoietic tumors (5, 27, 33,
38, 42). More recently, by using an N-Ras-deficient mouse
model, we have shown that N-Ras is an important component
of the T-cell signaling network and its function (29). The func-
tional consequences of the absence of N-Ras in T cells include
deficient CD8?selection, a decreased thymocyte proliferation,
a significant reduction in the production of interleukin-2 upon
thymocyte activation, and an increased sensitivity to influenza
infection in vivo.
The purpose of this work was to determine the mecha-
nism(s) underlying the specific role of N-Ras in T-cell function.
Our results show that, although all three Ras isoforms are
expressed in human T cells, N-Ras is the only isoform activated
* Corresponding author. Mailing address: Department of Pathology,
New York University School of Medicine, 550 First Ave., New York,
NY 10016. Phone: (212) 263-5342. Fax: (212) 263-8211. E-mail:
on September 18, 2012 by CNIO
following low-grade stimulation of the T-cell receptors (TCR)
in Jurkat T cells. Moreover, N-Ras activation takes place ex-
clusively on the Golgi apparatus as a consequence of signaling
through phospholipase C?1 (PLC?1) and RasGRP1.
MATERIALS AND METHODS
Cells and transfection assays. Jurkat T leukemia cells (clone E6-1) and the
PLC?1-deficient mutant (J gamma 1) are derived from a human acute T-cell
leukemia and were obtained from the American Type Culture Collection. CEM
(CCRF-CEM) and Karpas (KARPAS-299) cell lines are derived from a human
T-cell acute lymphoblastic leukemia and a human T-cell non-Hodgkin lym-
phoma, respectively. HEK293 cells, which are a permanent line of primary
human embryonic kidney cells, were also obtained from the American Type
Culture Collection. All the cells were kept at logarithmic growth in RPMI 1640
medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM
sodium pyruvate, and 100 U of penicillin G and streptomycin each per ml.
The majority of the transfection assays were performed by lipofection with
DMRIE-C (Gibco BRL) (for Jurkat) or Superfect (Gibco BRL) (for COS-1) and
the conditions recommended by the manufacturer. In the indicated cases, Amaxa
technology was used to transfect Jurkat T cells according to the manufacturer’s
Plasmids, antibodies, and reagents. Yellow fluorescent protein (YFP)–Ras-
binding domain (RBD), untagged Ras, green fluorescent protein (GFP)-Ras,
and cyan fluorescent protein (CFP)-Ras vectors were previously described (8, 9).
CFP–H-RasC184L and CFP–N-RasL184C palmitoylation mutants were gener-
ated by using a QuikChange site-directed mutagenesis kit (Stratagene). Human
RasGRP cDNAs were amplified by PCR (primer sequences available upon
request) and cloned in frame into the mammalian expression vectors pYFP-N1
(Clontech) and pcDNA3.1(?)/Neo (Invitrogen). All plasmids were verified by
bidirectional sequencing. Antibodies used for Ras detection included agarose-
conjugated anti-pan-Ras Y13-259 (Oncogene Research) and monoclonal anti-
bodies for mouse N-Ras (F155), H-Ras (F235), and K-Ras (F234) (Santa Cruz
Biotechnology). Mouse anti-human CD3 (UCHT1) and CD28 (5D10) (Ancell)
were used for TCR-dependent activation, whereas phorbol 12-myristate 13-
acetate (PMA) plus ionomycin (Sigma-Aldrich) was used for TCR-independent
activation of Jurkat cells.
Cell stimulation and imaging. For TCR-dependent stimulation, Jurkat cells
were incubated with high (5 ?g/ml) or low (1 ?g/ml) doses of both mouse
anti-human CD3 plus anti-CD28 antibodies. PMA (100 ng/ml) plus ionomycin
(500 ng/ml) was used for TCR-independent stimulation. For the specific micro-
localization of Ras proteins in the plasma membranes of T cells, Jurkat cells were
first incubated with anti-CD3 and anti-CD28, washed with phosphate-buffered
saline, and then incubated with Texas red-conjugated donkey anti-mouse immu-
noglobulin G (heavy plus light chains) (Jackson ImmunoResearch Laboratories,
Inc). The stimulation of COS-1 cells was performed by adding 40 ng of epidermal
growth factor (EGF)/ml to the media. For examination by fluorescence micros-
copy, the cells were plated in 35-mm dishes containing a glass coverslip-covered
15-mm cutout (MatTek). By using a Harvard Apparatus microincubator and dual
confocal microscopy, the images were captured with a Zeiss 510 laser-scanning
confocal microscope (LSM) with the manufacturer-specified filter sets for single
and dual emissions. TIFF images were processed with Adobe Photoshop 6.0.
Protein expression and Ras activation. For the detection of Ras variants, the
proteins were extracted in 200 ?l of lysis buffer (10% glycerol, 1% Nonidet P-40,
50 mM Tris-HCl [pH 7.4], 200 mM NaCl, 2.5 mM MgCl2, 1 mM phenylmethyl-
sulfonyl fluoride, 1 ?M leupeptin, 10 ?g of soybean trypsin inhibitor per ml, 0.1
?M aprotinin). Lysates were cleared by centrifugation, and the protein concen-
tration was quantified by the Bradford method. Ras proteins from Jurkat cells (1
mg per sample) were also immunopurified by using an anti-pan-Ras immunoaf-
finity column. In all cases, the proteins were fractionated in triplicate in sodium
dodecyl sulfate–15% polyacrylamide gels, and Ras variants were detected by
using antibodies specific for each isoform.
For Ras activation experiments, 2 ? 107Jurkat T cells were serum starved at
37°C for 2 h and then incubated in the presence or absence of anti-CD3 plus
anti-CD28 or PMA plus ionomycin for 10 min. To detect GTP-bound Ras, 75%
of the cells were quickly sedimented and lysed in 400 ?l of lysis buffer. Lysates
were cleared by centrifugation, incubated for 2 h at 4°C with glutathione S-
transferase (GST)–Raf–RBD fusion protein (a gift of J. L. Bos, Utrech Univer-
sity, Utrech, The Netherlands) coupled to glutathione agarose beads, and
washed four times with lysis buffer. To detect total Ras, the remaining cells
(25%) were lysed in 100 ?l of Laemmli buffer. Finally, GTP-bound and total Ras
proteins were detected by Western blotting with isoform-specific antibodies.
Active ERK1/2 proteins were detected in the primary lysates by using phos-
pho-specific antibodies according to the manufacturer’s recommendations (Pro-
mega). To normalize the activation of endogenous ERK1/2, lysates were also
probed with anti-ERK1/2.
In all cases, the immunoblots were revealed by using Supersignal West Femto
maximum sensitivity substrate (Pierce). Scanned TIFF images were processed
and bands were quantified with the Quantity One software (Bio-Rad).
Indirect immunofluorescence assays. Jurkat T cells were fixed with 4% para-
formaldehyde and permeabilized and blocked with 0.5% Triton X-100–5% bo-
vine serum albumin in phosphate-buffered saline. The cells were dual stained
with monoclonal antibodies to N-Ras (F155; Santa Cruz Biotechnology) or
RasGRP1 (M133; a gift of James C. Stone, University of Alberta, Alberta,
Canada) and a polyclonal antiserum against human giantin (PRB-114; Covance
Research) (each diluted 1:250), followed by Texas red-conjugated horse anti-
mouse combined with fluorescein isothiocyanate-conjugated goat anti-rabbit an-
tisera (Jackson ImmunoResearch). The cells were mounted with photobleach
retardant medium (DAKO Corp.), and they were imaged with a Zeiss 510
N-Ras mediates TCR signaling in Jurkat T cells upon low-
grade stimulation. To determine the isoform specificity of Ras
activation downstream of the TCR, we stimulated Jurkat T
cells by cross-linking the TCR with anti-CD3 and anti-CD28
antisera, and we analyzed cell lysates for GTP-bound Ras by
using the GST-RBD pulldown method (10) combined with
isoform-specific immunoblotting. As shown in Fig. 1A, K-Ras
and N-Ras, but not H-Ras, were activated following TCR
cross-linking. Interestingly, whereas the stimulation of Jurkat T
cells with 5 ?g of anti-CD3 plus anti-CD28/ml showed similar
activation of both N-Ras and K-Ras, low-grade TCR stimula-
tion (1 ?g of anti-CD3 plus anti-CD28/ml) induced activation
only on N-Ras (Fig. 1B). Since the majority of evidence sup-
ports that a weak-moderate signal results in T-cell positive
selection (22, 36), our data indicate that N-Ras should be the
Ras isoform involved in transducing the TCR-dependent sig-
nals that mediate cell survival and differentiation into mature T
Since our results showed no activation of endogenous H-Ras
upon TCR stimulation, we tested the capacity of the GST-
RBD pulldown method to detect GTP-bound H-Ras. As ex-
pected, when a constitutively active H-Ras mutant (H-RasL61)
was transiently overexpressed in Jurkat T cells, GTP-bound
H-Ras was affinity purified from Jurkat cell lysates by using
GST-RBD (data not shown), validating the ability of this assay
to detect GTP-bound H-Ras.
The apparent differential activation of N-Ras and K-Ras
upon low-grade TCR stimulation may be due to different sen-
sitivities of the method toward the different isoforms. How-
ever, two factors argue against this possibility. First, there is no
evidence that GST-RBD shows any Ras isoform preference.
Second, the K-Ras- versus N-Ras-specific antibodies used to
analyze the pulldown were equally efficient at recognizing their
cognate proteins (data not shown). Nevertheless, we directly
tested the possibility that the difference was due to different
sensitivities of detection by activating Jurkat T cells with either
a high concentration (10 ?g/ml) of anti-CD3 and anti-CD28 or
a strong TCR-independent stimulus (PMA plus ionomycin). In
both cases, K-Ras activation was readily detected in this cell
type, and no differences were detected between its activation
levels and those of N-Ras (Fig. 1C). Thus, the preferential
activation of N-Ras upon low-grade TCR stimulation is not
3486PEREZ DE CASTRO ET AL.MOL. CELL. BIOL.
on September 18, 2012 by CNIO
due to differential sensitivities of the assay but rather to an
intrinsic difference among the isoforms that is likely to have
Although mammalian ras genes are expressed in all cell
lineages and organs, some differences have been detected in
the levels of expression of each of these genes in embryonic
development and in various adult tissues (15, 25). For example,
human leukemia cells and mouse primary thymocytes express
substantially more N-Ras and K-Ras than H-Ras (25, 37).
Since differential expression of Ras isoforms could explain why
only N-Ras was activated in Jurkat cells upon TCR engage-
ment, we determined the levels of expression of N-, K-, and
H-Ras in human T cells. First, we determined the relative
affinities of the isoform-specific anti-Ras antisera and discov-
ered that, whereas the anti-K-Ras and anti-N-Ras reagents had
very similar affinities for their cognate antigens, the anti-H-Ras
antiserum was 10-fold more efficient (data not shown). Ras
expression in Jurkat cells, two other human T-cell lines (Kar-
pas and CEM), and a human epithelial cell line (HEK293) was
analyzed by immunoblotting with these isoform-specific anti-
bodies (40). HEK293 cells expressed all three Ras isoforms
(Fig. 2A). In contrast, whereas all three T-cell lines expressed
relatively high levels of K- and N-Ras, we did not detect H-Ras
expression in these cells, despite the higher sensitivity afforded
by the anti-H-Ras antibody (Fig. 2A). To increase the sensi-
tivity of the assay, we immunoprecipitated all cellular Ras with
a pan-Ras antibody and then probed the immunoprecipitates
with isoform-specific antisera. By using this method, H-Ras
was detected in Jurkat cells, although at a much lower level
than were K-Ras and N-Ras (Fig. 2B).
N-Ras does not colocalize with TCR complexes upon CD3-
plus-CD28 stimulation. The distribution of Ras isoforms
within subdomains of the plasma membrane may influence the
activation and signaling of the GTPase (31). The TCR is
thought to partition into lipid rafts. Indeed, cross-linking of
TCR complexes in T cells induces the coalescence of lipid raft
microdomains thought to organize signaling components (23).
To examine the microlocalization at the plasma membrane of
each Ras isoform during TCR activation, we transfected Jurkat
cells with GFP-tagged Ras proteins and observed their subcel-
lular localization in living cells by using LSM before and after
activating Jurkat cells by cross-linking TCR complexes. To
augment patching and to directly visualize patched lipid rafts,
we followed anti-CD3 and anti-CD28 antibodies with Texas
red-conjugated goat anti-mouse antiserum (Fig. 3). As a pos-
itive control, we utilized GFP-tagged CD8, which is known to
FIG. 1. N-Ras-specific activation in Jurkat cells following low-
grade TCR stimulation. (A) Jurkat cells (2 ? 107per point) were
serum starved for 2 h and incubated with or without the indicated
amounts of anti-CD3 plus anti-CD28. Proteins from stimulated and
unstimulated cells were used to collect GTP-bound and total Ras as
described in Materials and Methods. (B) To quantify Ras activation,
GDP-bound and GTP-bound bands were quantified by using Quantity
One software. The graph shows GTP/GDP ratios relative to the non-
treated cells (NT). (C) Both N-Ras and K-Ras are equally activated by
strong TCR-dependent and TCR-independent stimuli. Cells were kept
untreated or activated with the indicated mitogens and processed as
described for panel A. ION, ionomycin.
VOL. 24, 2004TCR ACTIVATE N-Ras EXCLUSIVELY ON GOLGI APPARATUS 3487
on September 18, 2012 by CNIO
associate with the TCR complexes in lipid rafts (2). As ex-
pected, CD8-GFP was localized homogeneously along the
plasma membrane in untreated cells but was redistributed
along with TCR to membrane patches after cross-linking. The
steady-state localization of GFP–H-Ras, GFP–K-Ras, and
GFP–N-Ras in untreated Jurkat cells paralleled that observed
in other cell types (9). Whereas GFP–K-Ras was expressed
predominantly on the plasma membrane, GFP–N-Ras and
GFP–H-Ras were expressed on both the plasma membrane
and Golgi apparatus. GFP–H-Ras behaved like CD8-GFP,
colocalizing in patches with the TCR after cross-linking. In
contrast, neither GFP–K-Ras nor GFP–N-Ras was enriched in
these membrane patches following TCR cross-linking. Para-
doxically, N-Ras, the isoform preferentially activated down-
stream of the TCR upon low-grade stimulation, was not en-
riched along with the TCR in lipid rafts.
Low-grade TCR stimulation in Jurkat cells activates N-Ras
on Golgi apparatus. By using GFP-RBD, an in vivo probe for
GTP-bound Ras, we have recently demonstrated that, al-
though overexpressed H-Ras is present on both the plasma
membrane and Golgi apparatus of Jurkat cells, high-level TCR
stimulation activated only the pool on the Golgi (4). To extend
this analysis to the other Ras isoforms and to study low-level
TCR stimulation, we cotransfected Jurkat cells with H-Ras,
N-Ras, or K-Ras together with the YFP-RBD. As in fibro-
blasts, Jurkat cells transfected only with YFP-RBD and then
stimulated by cross-linking the TCR showed no recruitment of
the probe to any membrane compartment (data not shown),
demonstrating that endogenous levels of Ras are insufficient to
generate a signal and confirming that the assay, as utilized,
reports the activation state of only the ectopically expressed
GTPase, permitting isoform-specific analysis. Cells expressing
YFP-RBD and CFP-tagged forms of the three Ras isoforms
were incubated with different amounts of anti-CD3 plus anti-
CD28 to generate either low- or high-grade TCR stimulation,
and the cells were imaged alive every 30 s for 20 min (Fig. 4A).
Consistent with previous observations for COS-1 cells (8), in
resting Jurkat cells, YFP-RBD was expressed homogenously in
both the nucleoplasm and cytosol (Fig. 4A, a to c). High-grade
stimulation of the TCR elicited recruitment of YFP-RBD by
both N-Ras and H-Ras exclusively to a paranuclear structure
consistent with the Golgi apparatus (Fig. 4A, d and e). This
pattern of activation was observed in the vast majority of co-
transfected cells. YFP-RBD recruitment to the plasma mem-
brane was never observed in cells transfected with N-Ras or
H-Ras. In contrast, low-grade stimulation of the TCR stimu-
lated activation of N-Ras but not H-Ras (Fig. 4A, g and h). As
with high-grade stimulation, the pool of N-Ras that was acti-
vated appeared exclusively on the Golgi apparatus (Fig. 4A, h).
The kinetics of N-Ras activation on the Golgi apparatus in
response to TCR stimulation was relatively rapid: 30, 50, and
90% of transfected Jurkat cells showed activation 2, 5, and 10
min after stimulation. Activation was transient: the number of
positive cells returned to unstimulated levels after 20 min (data
not shown). In contrast to the cells cotransfected with YFP-
RBD and H-Ras or N-Ras, the majority of cells cotransfected
with YFP-RBD and K-Ras showed little membrane recruit-
ment of the reporter upon high-grade TCR stimulation (Fig.
4A, c, f, and i). However, in those cells that showed a redistri-
bution of the probe (?20%), recruitment was exclusively to the
plasma membrane (Fig. 4A, f). Importantly, no membrane
recruitment to any compartment was observed in cells coex-
pressing YFP-RBD and K-Ras following low-grade TCR stim-
ulation (Fig. 4A, i). Thus, results with YFP-RBD membrane
recruitment (Fig. 4A) were concordant with those obtained by
GST-RBD pulldown (Fig. 1) and confirmed that, under con-
ditions of low-intensity TCR stimulation, N-Ras was preferen-
tially activated. Moreover, the latter assay demonstrated that
the pool of activated N-Ras was restricted to the Golgi appa-
Because the lack of Ras activation at the plasma membrane
of Jurkat cells was unexpected, we confirmed that our probe
was capable of reporting the activation of all Ras isoforms on
the plasma membranes of fibroblasts stimulated with growth
factors. As previously reported (8), in COS-1 cells overexpress-
ing H-Ras, YFP-RBD was transiently recruited in response to
EGF to the plasma membrane and subsequently to the Golgi
apparatus (Fig. 4B, j, m, and p). An identical pattern was
observed in COS-1 cells expressing N-Ras (Fig. 4B, k, n, and
q). Interestingly, whereas Ras activation on the Golgi appara-
tus of Jurkat cells was observed within 5 min of TCR stimula-
tion, activation on the Golgi apparatus of COS-1 cells in re-
sponse to EGF was relatively delayed (peak at 40 min). COS-1
cells expressing K-Ras showed only transient activation on the
FIG. 2. Expression levels of the three Ras isoforms in different
human cell lines. (A) Jurkat, Karpas, and CEM T cells and HEK293
epithelial cells were analyzed for Ras isoform levels by immunoblotting
as described in Materials and Methods (left panels). Recombinant
proteins for each Ras isoform were used as positive controls (right
panels). (B) For Jurkat cells, total Ras proteins were also immunopu-
rified (IP) by using an anti-pan-Ras immunoaffinity column, and Ras
isoforms were detected by using antibodies specific for each isoform.
As has been previously reported (40), a doublet was detected for
K-Ras and N-Ras.
3488 PEREZ DE CASTRO ET AL.MOL. CELL. BIOL.
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plasma membrane in response to growth factor (Fig. 4B, l, o,
and r). Thus, Ras activation in Jurkat T cells differs from that
in COS-1 cells in several respects. First, N-Ras is preferentially
activated in response to low-grade TCR stimulation. Second,
N-Ras activation takes place exclusively on the Golgi appara-
tus. Finally, activation on the Golgi apparatus downstream of
TCR stimulation is rapid relative to that on the Golgi appara-
tus of fibroblasts stimulated by growth factors.
To unambiguously identify the Golgi apparatus as the
paranuclear structure on which N-Ras and H-Ras are activated
upon TCR engagement, we used Golgi-specific markers in
both the live-cell imaging and indirect immunofluorescence of
fixed cells (Fig. 5). We cotransfected Jurkat cells with CFP-
tagged N-Ras or H-Ras plus the Golgi marker galactosyl trans-
ferase (GalT) tagged with YFP. The paranuclear pool of N-
Ras and H-Ras colocalized with GalT (Fig. 5A), confirming
the Golgi localization of both Ras isoforms. We also analyzed
the localization of endogenous N-Ras by indirect immunoflu-
orescence with an N-Ras isoform-specific antibody and anti-
giantin as a Golgi marker. Interestingly, the staining for en-
dogenous N-Ras was stronger on the Golgi apparatus than on
the plasma membrane (Fig. 5B). These data demonstrate that
the Golgi localization of CFP–N-Ras reflects the localization
of the endogenous protein in Jurkat cells.
Mono- versus dipalmitoylation of H-Ras and N-Ras affects
TCR-mediated activation on the Golgi apparatus of Jurkat
cells. To investigate the molecular basis for the specificity of
N-Ras over H-Ras signaling on the Golgi apparatus in re-
sponse to low-grade TCR stimulation (Fig. 4), we analyzed the
hypervariable regions of these isoforms. H-Ras differs from
N-Ras in that it contains two (rather than one) palmitoylation
sites upstream of its CAAX motif (Fig. 6A). We introduced a
FIG. 3. N-Ras does not colocalize with TCR complexes in TCR-dependent activation of Jurkat cells. Jurkat cells were transfected with
expression vectors encoding CD8-GFP, GFP–H-Ras, GFP–K-Ras, and GFP–N-Ras and were imaged alive 48 h later by LSM. Untreated,
transfected Jurkat cells (NT) revealed the intrinsic steady-state localization of each fusion protein that included the plasma membrane and, in the
case of GFP–N-Ras and GFP–H-Ras, the Golgi apparatus (arrowheads). Jurkat cells were incubated with anti-CD3 plus anti-CD28, and then TCR
complexes were visualized by adding a Texas red-conjugated goat anti-mouse antibody. Jurkat T cells activated in this fashion showed a
characteristic TCR patching revealed by red fluorescence. The overlay reveals areas of colocalization (yellow) between patched TCR (red) and
GFP-tagged molecules (green).
VOL. 24, 2004 TCR ACTIVATE N-Ras EXCLUSIVELY ON GOLGI APPARATUS3489
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FIG. 4. N-Ras is the only Ras isoform activated on the Golgi apparatus upon low-grade TCR engagement in Jurkat T cells. Jurkat (A) and
COS-1 (B) cells were cotransfected with YFP-Raf1-RBD plus either H-Ras, N-Ras, or K-Ras tagged with CFP. Forty-eight hours later, cells were
serum starved and either left untreated (a to c and j to l), stimulated with the indicated amounts of anti-CD3 plus anti-CD28 (d to i), or stimulated
with EGF (m to r) at the times indicated. Cells were imaged alive by LSM, and those expressing equivalent amounts of H-Ras, K-Ras, or N-Ras
were selected to analyze YFP-Raf1-RBD distribution. Arrowheads and arrows indicate Raf1-RBD redistribution to the Golgi apparatus and
plasma membrane, respectively. NT, nontreated cells.
on September 18, 2012 by CNIO
second palmitoylation site into N-Ras at codon 184 (N-
RasL184C). Conversely, we removed the second palmitoyl-
ation site in H-Ras (H-RasC184L). Each construct, when
tagged with CFP, localized like wild-type N-Ras and H-Ras on
the plasma membrane and the Golgi apparatus (Fig. 6B).
When these constructs were expressed along with YFP-RBD
in Jurkat cells, low-grade cross-linking of the TCR induced
N-RasL184C, on the Golgi apparatus (Fig. 6B). This result
suggests that monoacylation mediates the specificity of Ras
activation on the Golgi apparatus.
To extend these studies downstream of Ras activation, we
studied TCR-stimulated Erk1/2 activation in Jurkat cells ex-
pressing N-Ras, H-Ras, or the palmitoylation mutants de-
scribed above (Fig. 6C). Low-grade TCR stimulation induced
Erk1/2 activation in N-Ras-overexpressing cells to a much
higher degree than that observed in cells expressing similar
levels of H-Ras. The addition of a second palmitoylation site in
the hypervariable region of N-Ras (L184C) resulted in a mark-
edly diminished ability to support Erk1/2 activation. Con-
versely, removal of one of the two acylation sites in the hyper-
variable region of H-Ras (C184L) resulted in a more robust
activation of Erk1/2 in response to low-grade TCR stimulation.
Thus, the differential activation of N-Ras versus H-Ras on the
Golgi apparatus that is controlled by the acylation state of the
Ras protein translates into downstream signaling.
TCR stimulation in Jurkat cells activates N-Ras via PLC?
and RasGRP1. It has recently been demonstrated that H-Ras
activation on the Golgi apparatus of fibroblasts is mediated by
a pathway dependent on PLC? and RasGRP1 (4, 7). Similar
results were obtained in Jurkat cells overexpressing H-Ras (4).
Because the in vitro studies described above and previous in
vivo studies of lymphocytes from N-Ras-deficient mice (29)
suggest that Ras signaling in lymphocytes may preferentially
involve N-Ras, we sought to extend the studies of PLC? and
RasGRP1 to N-Ras. Whereas N-Ras was expressed on both
the plasma membrane and the Golgi apparatus in Jurkat cells
deficient in PLC?, stimulation of the TCR failed to induce the
activation of N-Ras on any compartment (Fig. 7A). Similar
results were obtained when wild-type Jurkat cells were pre-
treated with the PLC? inhibitor U73122 (4). In contrast, when
the TCR was bypassed by stimulation with PMA and ionomy-
cin, YFP-RBD was recruited to the Golgi apparatus in PLC?-
deficient cells (Fig. 7A). The spatially resolved results obtained
with YFP-RBD recruitment were recapitulated by GST-RBD
pulldown assays. Low-grade stimulation of the TCR activated
N-Ras in wild-type, but not PLC?-deficient, Jurkat cells (Fig.
7B). In contrast, bypassing the TCR by stimulation with PMA
plus ionomycin activated N-Ras in both types of cells (Fig. 7B).
to H-RasC184L, butnot
FIG. 5. N-Ras and H-Ras are expressed on the Golgi apparatus of
Jurkat cells. (A) CFP–H-Ras and CFP–N-Ras vectors were cotrans-
fected in Jurkat cells together with a vector expressing the Golgi
marker GalT tagged with YFP. CFP-Ras and YFP-GalT distributions
were determined by LSM in living cells. (B) The localization of en-
dogenous N-Ras in the Golgi apparatus was determined by immuno-
fluorescence. As described in Materials and Methods, endogenous
N-Ras and giantin (a Golgi-specific marker) were detected in fixed
Jurkat T cells.
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3492PEREZ DE CASTRO ET AL.MOL. CELL. BIOL.
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Thus, the activation of N-Ras on the Golgi apparatus of Jurkat
cells following TCR stimulation depends on PLC?.
RasGRP1 has been shown to be critical for T-cell activation
in vivo (11), and we have shown that it is required to activate
overexpressed H-Ras on the Golgi apparatus of both fibro-
blasts and Jurkat cells (4). To determine if RasGRP1, among
the four RasGRP family members, has a particular preference
for targeting to the Golgi apparatus in Jurkat cells, we tagged
each member of the family with GFP and studied their sub-
cellular localization before and after low-grade TCR stimula-
tion (Fig. 8A). In serum-starved, unstimulated cells, each Ras-
GRP family member was distributed homogeneously in the
cytosol. Upon TCR stimulation, RasGRP1 and RasGRP3
translocated to endomembranes but not to the plasma mem-
brane. No relocalization of RasGRP2 or RasGRP4 was ob-
served. Whereas RasGRP3 relocalized in stimulated cells to
both the endoplasmic reticulum and Golgi apparatus, Ras-
GRP1 translocation was specific for the Golgi apparatus. To
validate the translocation of GFP-RasGRP1, we studied the
translocation of the endogenous protein in Jurkat cells by
indirect immunofluorescence. Low-grade TCR stimulation in-
duced translocation of endogenous RasGRP1 to the Golgi
apparatus, as indicated by colocalization with giantin (Fig. 8C).
Because both RasGRP1 and RasGRP3 were observed to
translocate to the Golgi apparatus, we sought to determine
whether either or both could activate N-Ras on that compart-
ment. We coexpressed N-Ras with each RasGRP, along with
YFP-RBD, and observed recruitment of the reporter to the
Golgi apparatus as a consequence of the overexpression of
RasGRP1 but not RasGRP3 (Fig. 8B). These data are consis-
tent both with the RasGRP1 knockout studies (11) and with
RasGRP1 activation of H-Ras on the Golgi apparatus (4, 7).
Taken together, our data suggest that low-grade TCR stimu-
lation of Jurkat cells promotes the activation of N-Ras on the
Golgi apparatus via a PLC?/Ca2?plus diacylglycerol/Ras-
Ras activation is critical for T-cell function (17). Recently,
we analyzed the role of N-Ras in T-cell signaling by using mice
deficient in N-Ras (29). Compared to wild-type littermates,
mice lacking N-Ras were more sensitive to low-titer influenza
virus infection. Moreover, N-Ras-deficient mice exhibited a
defect in the selection of CD8-positive T cells, a process that is
regulated by low-grade TCR signaling (45). In the present
work, we have complemented these in vivo genetic experi-
ments with a cell biological analysis of Ras isoform expression
and signaling in T cells. Our study demonstrates that, under
conditions of low-grade TCR stimulation, N-Ras is preferen-
tially activated in Jurkat T cells. This observation explains why
other Ras isoforms cannot substitute for N-Ras in deficient
animals and demonstrates a critical role for N-Ras in T-cell
Until recently, the three Ras isoforms have been considered
redundant, and few biochemical differences have been de-
scribed. The embryonic lethality of K-Ras deficiency (24), but
not of N-Ras (44) or H-Ras (14) deficiencies, demonstrates
conclusively that the functions of all Ras proteins are not
entirely overlapping. Differential membrane trafficking of the
various Ras isoforms is firmly established (9) and has led to a
search for cell biological rather than biochemical differences
among the isoforms. Recent studies have focused on isoform
differences in localization in plasma membrane microdomains
(32, 34) and in endomembrane signaling (8) as possible expla-
TCR are believed to signal from plasma membrane micro-
domains known as lipid rafts that are enriched in signaling
molecules, including the adaptor protein LAT and enzymes
such as Lck and PLC? (23). The enrichment of Ras in lipid
rafts is somewhat controversial (31). In two recent studies of
nonlymphoid cells, H-Ras was enriched in lipid rafts but K-Ras
was excluded (32, 34). Our failure to observe K-Ras in clus-
tered rafts on Jurkat cells is consistent with these studies.
Interestingly, whereas inactive H-Ras was found to be enriched
in lipid rafts, activated, GTP-bound H-Ras was excluded, sug-
gesting a dynamic interaction with the microdomain (32). Our
observation that H-Ras is not activated by TCR stimulation
but that it is nevertheless colocalized with the receptor in lipid
rafts is consistent with the lipid raft association of inactive
H-Ras. In support of this interpretation, when GFP–H-
Ras61L, a constitutively active mutant, was substituted in our
system for GFP–H-Ras, the GTP-bound H-Ras protein failed
to cocap with the TCR (I. Pe ´rez de Castro, T. G. Bivona, A.
Pellicer, and M. R. Philips, unpublished observation). Al-
though in MDCK cells N-Ras has been colocalized with H-Ras
in lipid rafts (28), its localization had not been previously
analyzed in T cells. Our results demonstrate that N-Ras be-
haves like K-Ras in failing to partition into the T-cell mem-
brane microdomains defined by TCR and CD8. From these
data, we conclude that diacylation is required for Ras proteins
to partition into lipid rafts of Jurkat T cells. Since the diacy-
lated form of Ras, H-Ras, is expressed at very low levels in T
cells and, even when overexpressed, is not activated down-
FIG. 6. A single palmitoylation event in the C-terminal region of N-Ras is crucial for its specific role in TCR-dependent signaling. (A) Wild-
type and mutant H-Ras and N-Ras protein sequences corresponding to the membrane targeting domain. Cysteine palmitoylation sites are
underlined. (B) Jurkat cells were cotransfected with YFP-Raf1-RBD and either CFP–N-RasL184C or CFP–H-RasC184L mutants. Forty-eight
hours later, cells were serum starved and either left untreated (NT) or activated with 1 ?g of anti-CD3 plus anti-CD28/ml. Ras localization and
RBD redistribution were analyzed in live cells by LSM and compared with those found for wild-type H-Ras and N-Ras isoforms (Fig. 3 and Fig.
4, g to i). Arrowheads indicate the Golgi apparatus. (C) Jurkat cells were transfected by using Amaxa technology with either CFP–N-Ras,
CFP–N-RasL184C, CFP–H-Ras, or CFP–H-RasC184L vectors. Forty-eight hours later, cells were serum starved and either left untreated (?) or
activated with 1 ?g of anti-CD3 plus anti-CD28/ml (?). ERK1/2-activated proteins were detected with phospho-specific antibodies. Values
between the upper panels indicate ERK1/2 activation relative to the CFP–N-Ras transfected cells and were determined by quantification of
phospho-ERK1/2 bands (P-ERK1/2) and subsequent normalization with total ERK1/2 (middle panel). The bottom panel shows the levels of
CFP-Ras proteins (?52 kDa) detected for each construct. WT, wild type.
VOL. 24, 2004 TCR ACTIVATE N-Ras EXCLUSIVELY ON GOLGI APPARATUS3493
on September 18, 2012 by CNIO
stream of the TCR, we further conclude that the lipid rafts of
T-cell plasma membranes do not participate directly in Ras
activation and cannot explain the preference for N-Ras over
K-Ras in activation following low-grade TCR stimulation.
Having failed to explain the isoform preference of Ras sig-
naling in T cells on plasma membrane microdomains, we next
investigated subcellular compartment-specific signaling. It has
recently been demonstrated that, although H-Ras expressed
ectopically in Jurkat cells was present on both the plasma
membrane and Golgi apparatus, the signaling in response to
high-grade TCR activation was restricted to the Golgi appara-
tus and was dependent on PLC? and RasGRP1 (4). Moreover,
we showed that the Ca2?-activated Ras GAP CAPRI blocked
H-Ras activation on the plasma membrane (4). We have now
shown that, like H-Ras, N-Ras was activated only on the Golgi
apparatus following TCR stimulation. Unlike H-Ras, low-
FIG. 7. N-Ras activation upon TCR engagement in Jurkat cells is PLC?1-dependent. (A) J gamma 1 cells, which are deficient in PLC?1, were
cotransfected with CFP–N-Ras and YFP-Raf1-RBD and, 48 h later, stimulated as indicated. Colocalization of CFP–N-Ras (red) and YFP-Raf1-
RBD (green) is shown in yellow. (B) Wild-type and PLC?1-deficient Jurkat cells (2 ? 107per point) were serum starved for 2 h and left untreated
(NT) or incubated with 1 ?g of anti-CD3 plus anti-CD28/ml (?CD3??CD28) or PMA plus ionomycin (PMA?ION). Proteins from stimulated
and unstimulated cells were used to collect and detect GTP-bound and total N-Ras as described in Materials and Methods.
3494PEREZ DE CASTRO ET AL.MOL. CELL. BIOL.
on September 18, 2012 by CNIO
grade TCR stimulation was sufficient to activate N-Ras on the
Golgi apparatus. N-Ras activation on the Golgi apparatus was
also dependent on both PLC? and RasGRP1. These results
suggest that plasma membrane-associated Ras exchange fac-
tors such as Grb2/Sos are counterbalanced in T cells by CA-
PRI. These data are consistent with the finding that the mu-
tation of the phospho-tyrosine docking sites for Grb2/Sos on
LAT does not inhibit TCR-mediated Ras activation (46). How-
ever, since we observed the activation of K-Ras during high-
grade stimulation of TCR, our results are consistent with a role
for Grb2/Sos in the activation of K-Ras on the plasma mem-
brane. Thus, the intensity of TCR stimulation controls not only
the Ras isoform utilization but also the subcellular compart-
ment from which the Ras signal is propagated.
Our analysis of palmitoylation mutants of N-Ras and H-Ras
demonstrates that monoacylation regulates the ability of
Golgi-associated Ras to become activated in response to low-
grade TCR stimulation. One model that may explain this result
is that in which monoacylation is required for the relevant Ras
protein to partition into the proper microdomain of the trans-
Golgi network membrane to be acted upon by RasGRP1. Al-
ternatively, mono- versus diacylation may specify interactions
between Ras and various guanine nucleotide exchange factors
or GAPs. Indeed, the posttranslational modification of Ras
influences not only subcellular localization but also interaction
with regulators (35, 41).
Because working with primary lymphocytes presents obsta-
cles, several T-cell lines have been extensively used for study-
ing T-cell signaling and function. Although the results obtained
with these cell lines require validation in primary T cells, in-
creasing evidence supports the utility of Jurkat and other T-cell
lines in elucidating signaling pathways. For example, the re-
quirement for RasGRP1 in the activation of N-Ras on the
Golgi apparatus of Jurkat cells is consistent with the severe
impairment in Ras signaling observed in murine T cells defi-
cient in this exchange factor (11). Indeed, RasGRP1 has been
strongly associated with Ras activation in T cells, thymocyte
development, and TCR signaling (11, 13). Importantly, it was
FIG. 8. RasGRP1 mediates TCR-dependent activation of N-Ras on the Golgi apparatus of Jurkat cells. (A) Wild-type Jurkat cells were
transfected with YFP-tagged RasGRP proteins. Forty-eight hours posttransfection, cells were serum-starved for 2 h and stimulated as described
for Fig. 6B. Panels show the localization of RasGRP1 to RasGRP4 proteins before (left) and after (right) TCR stimulation. (B) To assess the role
of RasGRP1 and RasGRP3 in N-Ras activation on the Golgi apparatus, wild-type Jurkat cells were cotransfected with untagged expression vectors
of each RasGRP protein, CFP–N-Ras, and YFP-RBD. Forty-eight hours later, cells were serum starved, and RBD redistribution was analyzed by
LSM. Arrowheads indicate the Golgi apparatus. (C) Recruitment of endogenous RasGRP1 to the Golgi apparatus upon TCR engagement was
analyzed by immunofluorescence. As described in Materials and Methods, endogenous RasGRP1 and giantin (a Golgi-specific marker) were
detected in nontreated (NT) and TCR-stimulated (5 ?g of anti-CD3/ml plus 5 ?g of anti-CD28/ml) Jurkat T cells.
VOL. 24, 2004 TCR ACTIVATE N-Ras EXCLUSIVELY ON GOLGI APPARATUS3495
on September 18, 2012 by CNIO
recently reported that RasGRP1 plays a critical role in T-cell
development, homeostasis, and differentiation by transducing
low-grade TCR signals (30). Moreover, N-Ras-deficient mice
are also defective in some T-cell functions mediated by low-
grade stimuli (29). Thus, the striking similarities between the
T-cell phenotypes of RasGRP1- and N-Ras-deficient mice can
be explained by the elimination of elements of a common
pathway. These data strongly support the idea that the TCR/
PLC?/RasGRP1/N-Ras pathway plays a pivotal role in low-
grade TCR signaling.
We are grateful to David J. McKean, James C. Stone, Konstantina
Alexandropoulos, and Johannes Bos for providing plasmids.
This work was supported by grants AI36224 and GM55279 (to
M.R.P.), grants CA36327 and CA50434 (to A.P.) from the National
Institutes of Health, the New York State Breast Cancer Research
Program, and the Burroughs Welcome Fund (to M.R.P.), and by a
(M01RR00096) awarded to the New York University School of Med-
grant fromNIH NCRR
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