TCR-Induced Activation of Ras Proceeds at
the Plasma Membrane and Requires
Palmitoylation of N-Ras
This information is current as
of June 18, 2017.
Ignacio Rubio, Stefan Grund, Shu-Ping Song, Christoph
Biskup, Sabine Bandemer, Melanie Fricke, Martin Förster,
Andrea Graziani, Ute Wittig and Stefanie Kliche
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J Immunol 2010; 185:3536-3543; Prepublished online 16
August 2010;
doi: 10.4049/jimmunol.1000334
http://www.jimmunol.org/content/185/6/3536
The Journal of Immunology
TCR-Induced Activation of Ras Proceeds at the Plasma
Membrane and Requires Palmitoylation of N-Ras
Ignacio Rubio,* Stefan Grund,*,1 Shu-Ping Song,* Christoph Biskup,† Sabine Bandemer,*
Melanie Fricke,* Martin Förster,‡ Andrea Graziani,x Ute Wittig,* and Stefanie Kliche{
T
cells are versatile cells that react to varying environmental
challenges with sometimes strikingly opposed biological
outcomes. For example, during positive/negative selection
immature T cells either survive or succumb to apoptosis depending
on self-Ag recognition strength (1). Similarly, T cells that become
engaged by APCs can either buildup a full immunological response
or enter a hyporesponsive state known as T cell anergy, depending
on intensity, duration, and other characteristics of the trigger. These
and other cues conveyed by cognate Ags are translated into intracellular signals by the TCR.
Within seconds of activation, the TCR addresses a large array of
signal transduction pathways that ultimately induce changes in gene
expression that control cell proliferation or differentiation and cause
the shaping of T cell effector functions. Among the many signaling
mediators engaged, the Ras/Erk pathway is arguably one of the most
*Institute for Molecular Cell Biology, Centre for Molecular Biomedicine, †Biomolecular Photonics Group, and ‡Clinic of Internal Medicine I, University Hospital,
Friedrich-Schiller-University Jena, Jena; {Institute of Molecular and Clinical Immunology,
Otto-von-Guericke-University, Magdeburg, Germany; and xDepartment of Clinical and
Experimental Medicine, University of Piemonte Orientale A. Avogadro, Novara, Italy
1
Current address: Department of Pharmaceutical Technology, Friedrich-SchillerUniversity Jena, Jena, Germany.
Received for publication February 2, 2010. Accepted for publication July 18, 2010.
S.K. was supported by Deutsche Forschungsgemeinschaft Grants GRK1167 (TP11)
and SFB854 (TP10 and 12). S.-P.S. was supported by Deutsche Forschungsgemeinschaft Grant RU 860/3-1.
Address correspondence and reprint requests to Dr. Ignacio Rubio, Institute for
Molecular Cell Biology, Centre for Molecular Biomedicine, University Hospital,
Friedrich-Schiller-University Jena, Hans-Knöll-Strabe 2, 07745 Jena, Germany.
E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: DAG, diacylglycerol; DGK, diacylglycerolkinase;
GEF, guanine nucleotide exchange factor; IB, immunoblot; IS, immunological synapse;
Mw, calculated m.w. in kDa; PM, plasma membrane; SE, Staphylococcal enterotoxin.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000334
important signal transducers downstream of the TCR. Recent genetic and biochemical studies have shown that diacylglycerol (DAG)
liberated by TCR-activated PLCg stimulates Ras-GTP loading via
the direct binding and concomitant recruitment of the guanine
nucleotide exchange factor (GEF) RasGRP1 (2, 3), whose expression is largely restricted to leukocytes and neuronal cells. In
addition to the direct engagement, DAG feeds into RasGRP1 by
triggering PKC-dependent phosphorylation of RasGRP1 (4). These
and other studies have thus disclosed a pathway initiated by PLCgdependent Ca2+ and DAG generation, leading to RasGRP1 activation via both PKC dependent and independent means as a major
pathway of Ras activation downstream of the TCR (2, 5). In addition to RasGRP1, however, T cells express other GEFs, like Sos1,
Sos2, or RasGRF2, and full-blown activation of Ras is likely to
involve a complex interplay of various GEFs (6, 7).
Another level of complexity is posed by the subcellular distribution
of Ras. K-Ras, H-Ras, and N-Ras (collectively Ras) possess a conserved, yet distinct, C-terminal motif that targets them for posttranslational modifications at the endoplasmic reticulum and Golgi
apparatus (collectively endomembranes). These include farnesylation of all Ras variants and additional palmitoylation at cysteine
residues in H-Ras and N-Ras (8, 9). The latter modification is reversible and determines both membrane association strength and
localization of Ras. Although farnesylated and unpalmitoylated N/
H-Ras exhibits loose and reversible binding to membranes and is
largely confined to endomembranes (8, 10–12), palmitoylation traps
N/H-Ras on membranes, and tags Ras proteins for exocytic transport and residency at the plasma membrane (PM) (8, 13). Given
the many different localities and the nodal role of Ras in TCR signaling, it is crucial to know the subcellular sites of Ras activation
to understand whether spatial segregation of Ras activity plays
a role in TCR signaling. In support of this notion, imaging studies
have documented TCR-driven Ras-GTP formation at the PM and
at the Golgi of T cells (5, 14). In particular, Ras activation in endo-
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Ras transmits manifold signals from the TCR at various crossroads in the life of a T cell. For example, selection programs in the
thymus or the acquisition of a state of hypo-responsiveness known as anergy are just some of the T cell features known to be
controlled by TCR-sparked signals that are intracellularly propagated by Ras. These findings raise the question of how Ras can
transmit such a variety of signals leading to the shaping of equally many T cell traits. Because Ras proteins transit through
endomembrane compartments on their way to the plasma membrane (PM), compartmentalized Ras activation at distinct subcellular sites represents a potential mechanism for signal diversification in TCR signaling. This hypothesis has been nurtured
by studies in T cells engineered to overexpress Ras that reported distinct activation of Ras at the PM and Golgi. Contrary to this
scenario, we report in this study that activation of endogenous Ras, imaged in live Jurkat T cells using novel affinity probes for
Ras-GTP, proceeds only at the PM even upon enforced signal flux through the diacylglycerol/RasGRP1 pathway. Physiological
engagement of the TCR at the immunological synapse in primary T cells caused focalized Ras-GTP accumulation also only at
the PM. Analysis of palmitoylation-deficient Ras mutants, which are confined to endomembranes, confirmed that the TCR does
not activate Ras in that compartment and revealed a critical function for palmitoylation in N-Ras/H-Ras activation. These findings
identify the PM as the only site of TCR-driven Ras activation and document that endomembranes are not a signaling platform
for Ras in T cells. The Journal of Immunology, 2010, 185: 3536–3543.
The Journal of Immunology
metry (FACScalibur flow cytometer and CellquestPro software [BD Bioscience, Heidelberg, Germany]).
Plasmids
3xHA-N-Ras and 3xHA-H-Ras in pcDNA3 were obtained from the Missouri S&T cDNA Resource Center (http://www.cdna.org). GTPases to be
cloned in fusion with mCherry were excised from parental dsRed2-C1
constructs (18) by XhoI/HindIII (K-Ras, K-RasG12V), XhoI/BamHI
(M-RasQ71L, TC21Q72L), or BglII/EcoRI restriction (Rap1AG12V) and
subcloned into pmCherry C1 (Clontech, Mountain View, CA). pmCherryN-Ras was subcloned accordingly by BglII/KpnI restriction using HA-NRas in pCMV (kind gift of Ian Prior, University of Liverpool, Liverpool,
U.K.) as the parental plasmid. All other pmCherry-N-Ras and pmCherry-KRas mutants as well as 3xHA-N-RasC181S, 3xHA-H-RasC181/184S, and
3xHA-H-RasC181/184A in pcDNA3 were consequently generated by standard point mutagenesis approaches.
Rat RasGRP1 in fusion with mCherry was prepared by replacing EGFP
in EGFP-RasGRP1 (kind gift of Isabel Merida, Centro Nacional de Biotecnologia/CSIC and Universidad Autonoma de Madrid, Madrid, Spain)
with mCherry from pmCherry C2 via AgeI/BsrGI restriction cloning.
Trimeric EGFP (E3) reporter constructs were generated by adding two
EGFP modules to the 59 end of previously described E1 type reporter
versions (18) (Fig. 2A). The dimeric EGFP sequence was produced by
limited BsrGI digestion of a trimeric EGFPx3-plasmid (19) (kindly
Materials and Methods
Materials
DGKa inhibitor R59949, TRITC-phalloidin and fatty-acid-free/endotoxinlow BSA were purchased from Sigma-Aldrich, Taufkirchen, Germany. Bodipy-TR-C5-ceramide and Blue-CMAC (Molecular Probes, Eugene, OR) and
transfection reagent DMRIE-C were from Invitrogen, Carlsbad, CA. Staphylococcal enterotoxin (SE)E, SEB, and SEA were from Toxin Technology, Sarasota, FL. GST-c-Raf-RBD protein was produced in Escherichia coli
(17).
Abs
Abs used: Pan-Ras (Ab-4), Merck, Bad Soden, Germany. H-Ras (F235), N-Ras
(F155), K-Ras (F234), PKCu, GFP all from Santa Cruz Biotechnology, Santa
Cruz, CA. Phospho-T202/Y204- p44/42 Erk (E10) and anti-HA were from
Cell Signaling Technology, Danvers, MA. Anti-Erk1 from BD/Transduction
Laboratories, Heidelberg, Germany. Anti-CD3 (UCHT-1) and anti-CD28 were
purchased from BD/Pharmingen, Heidelberg, Germany. Anti-CD3 IgG (OKT3) hybridoma was acquired from the American Type Culture Collection
(Manassas, VA) cell bank. Anti-CD3 IgM (C305) and anti-CD28 IgM hybridomas were kindly provided by Arthur Weiss (Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA) and Ulrich
Moebius (Morphosys AG, Heidelberg, Germany), respectively.
Cell culture and transfection
Jurkat T cells (German Collection of Microorganisms and Cell Cultures cell
bank) and Raji B cells (American Type Culture Collection) were grown in
RPMI 1640 (Biowest, Nuaillé, France) supplemented with glutamine and
10% heat-inactivated FCS in a 5% CO2 atmosphere. For live-cell imaging
Jurkat T cells were transfected with DMRIE-C, following the manufacturer´s instructions. For imaging of cotransfected T cells (Fig. 2) and for
biochemical experiments cells were electroporated with the Microporator
device (Invitrogen, Carlsbad, CA) in the 10 ml (imaging) or 100 ml (biochemical assays) format, respectively, using the provided parameters for
Jurkat cells.
Primary human T cells were prepared from healthy donors using the pan
T cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) and
maintained in RPMI 1640 medium containing 10% FCS, stable L-glutamine, and 1000 U/ml penicillin/streptomycin. Approval for these studies
was obtained from the Ethics Committee of the Medical Faculty at the
Otto-von-Guericke University, Magdeburg, Germany. Informed consent
was obtained in accordance with the Declaration of Helsinki. For electroporation of cDNA constructs, human peripheral T cells (8 3 106) were
washed in PBS containing Ca2+/Mg2+ and resuspended in 200 ml OptiMEM (Invitrogen). Thirty micrograms of DNA was added and after 3
min, cells were transfected by electroporation (square-wave pulse, 1000V
0.5 ms, two pulses [pulse interval 5 s]; BioRad X-cell). The cells were then
added to prewarmed cell culture medium as described previously and
cultured for 24 h before use. GFP-expression was evaluated by flow cyto-
FIGURE 1. K-Ras and N-Ras are activated in response to TCR ligation.
A, Jurkat T cells were deprived of serum for 2 h and subjected to a RasGTP pulldown following cross-linking of the TCR. Samples were immunoblotted with Ras-isoform specific Abs to determine the activation pattern
of individual Ras species. Ras isoforms can also be discriminated by differences in electrophoretic mobility. The appearance of faint H-Ras immunoreactive bands upon long film exposures probably results from the
cross-reactivity of the Abs with N-Ras (data not shown). B, H-Ras is not
expressed in Jurkat T cells. Cell extracts from Jurkat cells electroporated
with untagged H-Ras, 3xHA-tagged H-Ras or empty vector were immunoblotted with an isoform-specific anti–H-Ras Ab. After Western blot
development, membranes were stained with Coomassie dye to ascertain
equal loading. Asterisk denotes a unspecific band. IB, immunoblot.
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membranes reportedly proceeds via the PLC/DAG/RasGRP1 pathway (5). These findings support a model by which Ras activation at
different subcellular locations enables T cells to diversify the signaling output of the TCR. In contrast, active PLCg and DAG, both
essential upstream activators of RasGRP1, localize exclusively to
the PM of activated T cells (15, 16), arguing against RasGRP1dependent Ras activation in endomembranes. Another aspect important to consider is that all reported Ras-GTP imaging data have
been recorded in T cells engineered to overexpress Ras. Because Ras
overexpression can distort finely tuned processes, such as Ras trafficking, posttranslational processing or the activation process itself
(9), it is difficult to judge whether the results from Ras overexpression studies reflect the true behavior of endogenous Ras.
These considerations evidence that the unambiguous identification of the subcellular sites of Ras activation and signaling in T cells
can only be accomplished by the visualization of native Ras-GTP in
the absence of overexpression. In the current study, we have imaged
endogenous Ras-GTP formation in life T cells using novel affinity
probes with increased fluorescence and avidity to Ras-GTP. Because the segregation of N/H-Ras to PM and endomembranes is
largely dictated by the palmitoylation status, we have also investigated
the role of palmitoylation in Ras activation downstream of the TCR.
3537
3538
provided by Alison L. Barth, Carnegie Mellon University, Pittsburgh,
PA) and inserted into BsrGI-cut E1-R3 or E1-R1 plasmids. The final E3constructs thus feature exactly the same linkers and overall architecture
as the parental EGFPx3 plasmid previously shown to exhibit 3-fold
higher fluorescence than EGFP (19).
Confocal life-cell microscopy
Live-cell imaging was carried out on a Zeiss LSM 510 axiovert confocal
microscope equipped with a thermostated stage chamber (IBIDI, München,
Germany). Confocal images (optical slice of #1 mm) were acquired using
a 633 water immersion objective lens. EGFP and Cherry/Bodipy were
excited with the Argon 488 nm and the HeNe 543 nm line in subsequent
tracks. Emitted fluorescence was collected with a 505–550 nm bandpass
and a 560 nm longpass filter, respectively. T cell preparation, including
staining with Bodipy-TR-C5-ceramide, was performed exactly as described
(18). Cells were plated on poly-L-lysine-coated self-made glass-bottom 35mm dishes and monitored for at least 5 min before stimulation. All images
of a series were exported as TIF files and subjected to the same processing
routine using Zeiss ZEN 2008 Light Edition software.
Fluorescence quantification
FIGURE 2. Triple-RBD probes report Ras-GTP distribution in T cells. A,
Overview of fluorescent reporter molecules. Asterisks denote R59A and N64D
point mutations in RBD. B, Jurkat
T cells were electroporated with the indicated constructs and protein expression was assessed 24 h later by anti-GFP
Western blotting. C, T cells were electroporated with E3-R3 in combination
with the indicated mCherry-GTPase
constructs. After 24 h, cells were imaged
confocally. The prominent, unaccounted
nuclear accumulation of EGFP-RBD
polypeptides has been noticed before
(12, 18). D, mCherry-N-RasG12V was
transfected into Jurkat T cells, along
with the indicated reporter probes and
protein distribution, was analyzed by
confocal microscopy. Cells shown in C
and D are representative of at least
70% of all viable cells. Scale bars, 10
mm. Original magnification 363. Mw,
calculated m.w. in kDa.
nucleus from the cytosol (Supplemental Fig. 1 for a flowchart of the
segmentation procedure). Based on the masks, mean fluorescence intensities were calculated for PM, Golgi, and cytoplasm. The nuclear and
immediate perinuclear region were excluded from the analysis. PM/
cytoplasma and Golgi/cytoplasma ratios were plotted as a function of
time and presented as normalized mean values (6 SEM). The segmentation algorithm was programmed in MatLab (MathWorks, Natick, MA).
Biochemical Ras activation assays
T cells grown to a density of 106 cells/ml were deprived of serum for 2 h in
RPMI 1640 supplemented with 0.2% fatty-acid-free/endotoxin-low BSA
and 50 mM HEPES pH 7.5, counted and resuspended at 107 cells/ml in the
same solution. Cell suspensions were kept in a warm water bath at 37˚C
and tubes were carefully flipped every 2–3 min. After appropriate stimulation 1 ml of the cell suspension was transferred to 1.5 ml reaction vials
and quickly spun in a table top centrifuge. Medium was aspirated off and
the cell pellet was lysed with 1 ml ice-cold lysis buffer (50 mM HEPES
pH 7.5, 140 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% NP-40, protease
inhibitors) supplemented with 25 mg GST-RBD protein and 100 mM GDP.
GDP, and GST-RBD were included at this point to quench postlytic GTPloading and GAP-dependent Ras-bound GTP hydrolysis, respectively. Cell
extracts were cleared by centrifugation and GST-RBD/Ras-GTP complexes were collected on Glutathione-Sepharose. Precipitates were washed
once with 500 ml lysis buffer and processed for SDS-PAGE analysis.
T cell-APC conjugate formation
Mock pulsed or superantigen (SEE, SEB, and SEA) pulsed and Blue-CMAC
loaded Raji B cells were incubated for 15 or 30 min at 37˚C with E3-R3(A/D),
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To quantify organelle-associated fluorescence, we have programmed an
automatic cell image segmentation algorithm that yields masks for
four cellular regions: PM, nucleus, Golgi, and cytosol. The Golgi compartment is identified by the fluorescence signal of the Golgi tracker
Bodipy-TR-C5-ceramide. GFP fluorescence is used to distinguish PM and
TCR ACTIVATES Ras AT THE PLASMA MEMBRANE
The Journal of Immunology
E3-R1(A/D), or E1-R3(A/D) expressing Jurkat or human primary T cells on
poly-L-lysine-coated coverslips and fixed with 3.5% paraformaldehyde in PBS
for 10 min. Cells were permeabilized with 0.1% Triton-X100 in PBS, blocked
with 5% horse serum in PBS, and incubated with the indicated Abs (anti-GFP
mAb, anti-CD3 IgG, or anti-PKCu) and TRITC-phalloidin in combination
with Cy3-conjugated secondary Abs (Dianova, Hamburg, Germany). Coverslips were mounted in Mowiol 488 and imaged with a LEICA TCS SP2 laser
scanning confocal system (Leica Microsystems, Wetzlar, Germany) using
a plan apochromatic oil emerging 633 objective (NA 1.4). For each experiment, a minimum of 30 conjugates were analyzed for polarization of E3-R3
(A/D) or E1-R3(A/D) to the immunological synapse (IS) in T cells that featured enrichment of F-actin at the T cell/B cell interface. Figure construction
of images was performed in COREL Photopaint.
Results
FIGURE 3. E3-R3 reports TCRdriven endogenous Ras activation. A,
Jurkat cells transfected with E3-R3
were deprived of serum, stained with
the Golgi marker Bodipy-TR-C5ceramide, challenged with 5 mg/ml
each anti-CD3/anti-CD28 and imaged confocally. E3-R3 fluorescence
is shown in green or white pseudocolor for better observation. A total of
20% of monitored cells reacted as illustrated. Bottom lane shows computed cell masks for quantification
of organelle-associated fluorescence.
Yellow, PM; Red, Golgi; Green, Cytosol. B, Quantification of fluorescence associated with PM (yellow
curve) and Golgi (red curve) for the
experiment shown in (A) (mean 6
SEM; n = 10). C, T cells expressing
E3-R3(A/D) and C3-R1(A/D) were
challenged with 5 mg/ml each antiCD3/anti-CD28 and imaged alive.
E3-R3(A/D) and C3-R1(A/D) fluorescence is depicted in white pseudocolor. In all five monitored cells
that reacted with movement of E3-R3
(A/D) to the PM, C3-R1(A/D) did
not redistribute. Scale bars, 10 mm.
Original magnification 363.
To visualize Ras-GTP, we used affinity probes derived from the
Ras binding domain of the Ras-effector c-Raf (RBD). A fusionprotein of EGFP and RBD (EGFP-RBD or E1-R1, Fig. 2A) reports activation of overexpressed Ras (5, 12, 14). However, E1-R1 is
incapable of visualizing endogenous Ras-GTP because probe redistribution must be near quantitative to be visible, which requires
imaging cells with probe expression levels in the range of endogenous Ras levels that are arduous to detect. To accomplish visualization of endogenous Ras we have previously oligomerized RBD to
generate a multivalent probe (E1-R3) with increased affinity/avidity
for Ras-GTP (18). In a complementary approach to raise detection
sensitivity we have now augmented probe fluorescence by appending
two EGFP modules to E1-R3. The resulting construct E3-R3 features
a 3-fold increase in fluorescence (19), which allows to reduce probe
concentration and thus achieve higher fractional probe distribution
without suffering a loss in fluorescence. Expression of E3-R3 in
T cells resulted in a polypeptide of the correct size (Fig. 2B). To
assess the usefulness of E3-R3 as a live-cell reporter of Ras-GTP in
T cells, we studied its recruitment by Ras proteins. As shown in Fig.
2C constitutively active G12V mutants of N-Ras and K-Ras colocalized with E3-R3. Colocalization was specific because the effector
loop point mutation D38A, which reduces affinity to RBD by 72fold (22), impeded colocalization. The endomembrane resident palmitoylation mutant N-RasG12V/C181S (12) also recruited E3-R3
and the D38A mutation, again, abolished colocalization, showing
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As a first step we determined which Ras species are activated in
response to TCR ligation. In agreement with previous reports (14,
20), cross-linking of the TCR with any of three anti-CD3 Abs caused
GTP-loading of N-Ras and K-Ras (Fig. 1A). In contrast, H-Ras was
not detectable in Tcell extracts (Fig. 1B) (14). This expression
pattern is reminiscent of findings in other lymphoid leukemia cell
lines (21) and peripheral T cells (20). Importantly, T cell costimulation via CD28, SLAM, or LFA-1 did not lead to significantly
increased Ras-GTP formation as triggered by 5 mg/ml anti-CD3
(data not shown). These experiments evidenced that K-Ras and NRas are the relevant Ras isoforms in TCR signaling and that costimulation was dispensable for full-blown activation of Ras.
3539
3540
TCR ACTIVATES Ras AT THE PLASMA MEMBRANE
that E3-R3, specifically, decorated Ras-GTP also in endomembranes. Constitutively, active Rap1A, which possesses 66-fold
lower affinity for RBD (23), did not recruit E3-R3. E3-R3, however, cross-reacted with the more closely related GTPases TC21
and M-Ras.
The 33RBD polypeptides are potent scavengers of Ras-GTP and
exhibit cytotoxic effects (18). In Jurkat cells, E3-R3 expression
resulted in up to 40% cell death (data not shown). To deal with probe
toxicity, we have previously introduced well characterized attenuating point mutations into RBD (18, 24). Accordingly, incorporation
of the tandem mutation R59A/N64D into E3-R3 resulted in a nontoxic probe, E3-R3(A/D) (Fig. 2A), that showed the same robust,
effector site dependent recruitment by N-RasG12V as E3-R3 (Fig.
2D; data not shown). Colocalization was contingent on the high
avidity provided by RBD oligomerization, because the monomeric
version E3-R1(A/D), which possesses 100-fold lower affinity for
Ras-GTP than RBD (24), did not redistribute (Fig. 2D). E3-RG3,
a probe composed of triple RalGDS-RA, a domain of comparable
affinity for Ras-GTP as R1(A/D) [KDs of 1 mM and 3.8 mM, respectively (24)] was also recruited by active Ras. In sum, E3-R3 and
its attenuated derivative E3-R3(A/D) were specific, high-affinity/
avidity probes for Ras-GTP, which featured 3-fold higher fluorescence than the E1-R3 precursor probes.
To visualize endogenous Ras activation, we expressed E3-R3 in
T cells and used a vital dye to label the Golgi, the major site of
residency of N-Ras besides the PM (8). TCR activation with either
of three anti-CD3 Abs alone or in combination with anti-CD28
Abs caused rapid translocation of E3-R3 to the PM but not to the
Golgi (Fig. 3A; data not shown). The same response was observed
with E3-R3(A/D) (Fig. 3C; data not shown). To quantify PM and
Golgi associated E3-R3 fluorescence, masks identifying PM and
Golgi were created by an automated segmentation routine (bottom
lane in Fig. 3A; see Supplemental Fig.1 for a further description of
the segmentation process). E3-R3 association with the PM was
still apparent 10 min poststimulation (Fig. 3A, 3B) even though
biochemically assayed Ras-GTP accumulation had largely vanished by that time (Fig. 1). Differences in the experimental protocols are likely to account for this variation in activation time
courses. For example, immobilization on poly-lysine, an assumedly inert matrix used to fix T cells to microscopy slides, prolonged Ras activation in biochemical assays (Supplemental Fig.
2). Moreover, RBD-probes facilitate Ras-GTP formation by protecting Ras against GAP action (18, 25), likely resulting in extended activation kinetics. To subject the specificity of the probes
to further scrutiny, we transfected E3-R3(A/D) along with its redfluorescent monomeric counterpart C3-R1(A/D). The latter is not
recruited by Ras-GTP (18, 24) (Fig. 2D) and thus served as internal control of signal specificity. TCR ligation caused translocation of E3-R3(A/D) to the PM, whereas C3-R1(A/D) did not
reallocate in the same cell (Fig. 3C), indicating that PM illumination
by E3-R3(A/D) truly reflected Ras activation rather than the incidental association of RBD-probes with the PM. Because TC21
and M-Ras are negligibly or not at all activated by the TCR, respectively (26, 27), a contribution of these GTPases to probe redistribution could be excluded. We conclude that activation of
endogenous K-Ras and N-Ras by the TCR occurs at the PM.
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FIGURE 4. Endogenous Ras activation via the PLCg/
DAG/RasGRP1 pathway proceeds at the PM. A, Jurkat
T cells were treated with 10 mM R59949 for 15 min or
left untreated, challenged with 5 mg/ml anti-CD3 and
subjected to a Ras-GTP pulldown. Immunodetection was
performed with pan-Ras Ab. B, T cells transfected with
E3-R3 were stained with the vital Golgi marker and
treated with 10 mM R59949 for 15 min. Cells were
placed in the microscope stage, challenged with 5 mg/ml
anti-CD3 and imaged confocally. C, T cells cotransfected with E3-R3 and mCherry-RasGRP1 were deprived of serum for 2 h, challenged with 5 mg/ml each
anti-CD3 and anti-CD28, and imaged confocally. Note
that RasGRP1 redistributes to the PM and to internal structures, whereas Ras-GTP accumulation is only detected at
the PM. Scale bars, 10 mm. Original magnification 363.
The Journal of Immunology
3541
PLCg/DAG-dependent engagement of RasGRP1 is a major pathway of Ras activation downstream of the TCR (2, 5). Diacylglycerolkinases (DGKs) convert DAG to phosphatidic acid and thus
negatively regulate this pathway, a scenario well documented for
DGKa and DGKz (28, 29). To avoid missing sites of low-intensity
Ras-GTP formation, we manipulated the PLC/DGK/RasGRP1 pathway to rev up Ras activation. Inhibition of DGKa with R59949
markedly increased TCR-dependent accumulation of Ras-GTP (Fig.
4A), which occurred only at the PM (Fig. 4B). As a second approach
to force Ras-GTP formation, we overexpressed RasGRP1, a measure
known to enhance TCR-dependent Ras activation (2). As before,
TCR ligation triggered E3-R3 accumulation only at the PM (Fig. 4C),
confirming that TCR-driven Ras activation takes place at the PM.
Physiological activation of the TCR proceeds at the IS, an intercellular contact area between APC and T cell. To visualize Ras
activation in the context of the IS, E3-R3(A/D) redistribution was
imaged upon conjugation of T cells and superantigen pulsed Raji
B cells (Fig. 5A). Upon 15 min of conjugate formation Ras-GTP
reporter probes redistributed to the IS in over 50% of Jurkat cells
or 90% of primary T cells that had undergone efficient conjugation, as judged by the enrichment of F-actin at the T cell/APC
interface (Fig. 5A, 5B). At 30 min postconjugation, this number
increased to 92% in Jurkat cells and remained high in primary
T cells (Fig. 5B). Accumulation of the RBD probes at endomembranes was not observed in either cell background. As opposed to
E3-R3(A/D) or E1-R3(A/D), the monovalent probe E3-R1(A/D) did
not redistribute at any time point of the conjugation process (data not
shown). In parallel experiments, we labeled the TCR (Fig. 5A) or
PKCu (data not shown), two IS constituents that accumulate in the
central core of the IS known as central supramolecular activation
cluster (30). In 92% of conjugates active Ras localization did not
strictly overlap with the TCR or PKCu label but extended to the area
surrounding the central supramolecular activation cluster, indicating that Ras-GTP accumulates preferentially in the peripheral
supramolecular activation cluster. This pattern matches well the re-
ported distribution of the two upstream regulatory molecules DAG
and RasGRP1 in the IS (15, 28, 31). In conclusion, signals elicited
by the TCR at the IS promote focalized Ras activation at the PM.
FIGURE 6. Endomembrane-resident Ras is not activated by the TCR. A,
Jurkat T cells transfected with 3xHA-N-Ras or 3xHA-N-RasC181S were
deprived of serum and subjected to a Ras-GTP pulldown after crosslinking of the TCR. Endogenous and overexpressed HA-tagged Ras were
simultaneously detected with pan-Ras Ab. Asterisk denotes L chain of
anti-CD3/CD28 Abs. Transfection efficiency ranged between 30 and 40%.
B, Densitometric quantification of data shown in A. Shown are mean 6
SEM of four independent experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 5. T cell stimulation with
APCs promotes Ras activation at the IS. A,
Jurkat T cells expressing E3-R3(A/D) or
human primary T cells expressing E1-R3
(A/D) were mixed for up to 30 min with
mock-pulsed or superantigen-pulsed Raji
B cells, labeled with TRITC-phalloidin to
label F-actin or anti-OKT3 to label the
TCR. To visualize GFP in transfected human primary T cells, lymphocytes were incubated with an anti-GFP mAb and F-actin
was stained as described previously. All
cells were imaged confocally. The identity
of B and T cells is indicated in the phase
contrast images. Original magnification 363.
B, Quantitative analysis of conjugates with
F-actin focalization at the IS showing E3-R3
(A/D) or E1-R3(A/D) polarization in Jurkat
and primary T cells, respectively. Thirty randomly selected conjugates were analyzed in
each of three independent experiments. Data
represent means 6 SD.
3542
Discussion
Imaging studies have documented that several established upstream
regulators of Ras are recruited and engaged at the PM of APCchallenged T cells. Thus, PKCu, which probably contributes to
Ras activation by phosphorylating RasGRP1 (4), and RasGRP1
itself are exclusively associated to the PM of APC-triggered
T cells (28, 31–34). DAG, which serves to recruit RasGRP1,
and the DAG metabolizing enzyme DGKa reside at the PM and
DAG further accumulates at the IS upon conjugation with APCs
(15, 35, 36). Finally, active PLCg (as evaluated by phosphorylation of Tyr783) specifically and exclusively localizes to the IS of
activated T cells (16). All these findings strongly indicate that
TCR-induced Ras activation, at least as mediated by the PLCg/
DAG/RasGRP1 pathway, is bound to proceed at the PM of activated T cells and, specifically, at the IS of T cells challenged with
APCs. Contrary to this line of evidence, visualization of overexpressed Ras activation has documented that Ras-GTP accumulates
both at the PM and at endomembranes upon TCR ligation (5, 14).
Remarkably, endomembrane-activation of Ras has been proposed
to be mediated precisely by the same PLCg/DAG/RasGRP1 pathway that is presumed and predicted to act at the T cell surface
based on the imaging data referred above. The images of endogenous Ras-GTP formation presented in the current manuscript
evidence that endogenous Ras activation does proceed only at the
PM of stimulated T cells, in accordance with the observation that
all required upstream modulators reside at the PM. Our findings
have far-reaching implications, because they exclude the endomembrane compartment as a signaling platform for Ras in T cells.
This notion further implies that residency of Ras at endomembranes
is most likely only for the purpose of posttranslational processing
and clearance to the PM, a concept supported by the finding that
palmitoylation mutants of Ras, which are locked on the endomembrane compartment owing to the interruption of posttranslational
processing, are not engaged by the TCR (Fig. 6A). However, it is
certainly impossible to rule out that other signals or extracellular
cues may under certain circumstances, and possibly via other
mechanisms, lead to the biologically relevant accumulation of RasGTP at endomembranes. Indeed, the fact that Golgi-activation of
overexpressed Ras is observed in nonhematopoietic cell lines, such
as COS fibroblasts (5, 12), which evidentially do not express
RasGRP1 (17), indicates that mechanisms other than the DAG/
RasGRP1 pathway may be in charge of spatial control of Ras activity. Recently, reallocation of GTP-loaded N/H-Ras to endomembranes following depalmitoylation at the PM has been reported to
sustain Ras-GTP accumulation at the Golgi (13). However, magnitude and relevance of this process are not precisely known because
all collected experimental evidence so far stems from Ras overexpression studies (11, 13, 37). Moreover, the data presented in this
study evidence that this process is unlikely to operate in T cells or, at
best, it will not affect a meaningful proportion of the Ras population,
because any accumulation of Ras-GTP at T cell endomembranes,
irrespective of the underlying mechanism, should have become
visible in our imaging experiments.
An alternative explanation for the reported accumulation of RasGTP in the Golgi apparatus of T cells overexpressing Ras is that this
phenomenon may be an incidental result of overexpression. Ras
overexpression might cause overstraining of the posttranslational
processing machinery of T cells leading to the aberrant accumulation of incompletely processed Ras proteins in endomembranes, and
this pool of mislocalized Ras could subsequently escape normal
regulation during TCR signaling. Ras overexpression alone, however, is unlikely to suffice as the cause of aberrant activation at
endomembranes because overexpressed endomembrane-resident
Ras is not significantly activated in response to TCR ligation (Fig.
6), at least at the 2- to 4-fold overexpression rate achieved in our
experiments. We presume therefore that it is the combination of Ras
overexpression and RBD-probe expression that may distort normal
Ras regulation leading to the aberrant accumulation of Ras-GTP at
T cell endomembranes. Indeed, Ras activation as a consequence of
RBD-polypeptide expression is not unprecedented (25) and is
likely to result from the ability of RBD to protect Ras from GAP
action. Thus, several mechanisms for the incidental accumulation
of overexpressed Ras-GTP at endomembranes can be envisaged.
In conclusion, our findings document that endogenous Ras activation by the TCR occurs only at the PM, implying that all signals
transmitted via the TCR/Ras pathway originate at the T cell surface. Consistent with this conclusion, our data also reveal a previously unnoticed requirement for palmitoylation in N-Ras/H-Ras
activation, which most likely reflects the role of palmitate as a PM
anchor for Ras. This raises the possibility of pharmacologically
blocking Ras palmitoylation as a new means of intervention in
immunosuppressive therapies.
Acknowledgments
We thank Alison L. Barth, Jonathan Lindquist, Stefan Lorkowski, Isabel
Merida, Ian Prior, and Jim Stone for the generous provision of reagents.
We also thank Frank Böhmer, Ian Prior, and Jim Stone for helpful discussions, Michael Grün and Susanne Köthe for technical help, and Stefan
Heinemann for providing access to LSM instrumentation.
Disclosures
The authors have no financial conflicts of interest.
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