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Departments of
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Medical Microbiology and Immunology and
Biochemistry, University of Alberta, Edmonton, Canada
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The best characterized Ras-MAPK activation pathway is that stimulated through the receptor protein tyrosine kinase family of growth factor receptors (7). When ligated, these receptors become autophosphorylated, allowing for the binding of the adaptor molecule Grb2. Grb2 associates with Sos, a RasGEF, and the recruitment of Grb2-Sos complexes to the membrane activates the membrane-associated Ras. In T lymphocytes, ligation of the TCR results in activation of Ras, Raf, and ERK (8). This pathway is required for T cell function, including IL-2 production (8, 9) and CTL degranulation (10). The mechanism linking TCR stimulation to activation of the Ras-Raf-MEK-ERK pathway in T cells is unclear although several mechanisms have been proposed.
Both TCR stimulation and addition of PMA induce accumulation of GTP-bound Ras in T cells (11). Because both treatments activate protein kinase C (PKC), it was originally thought that TCR stimulation might be linked to the Ras-MAPK pathway by PKC (11). Early work suggested that PKC could activate Ras by inhibiting RasGAPs (11, 12). However, studies in which PKC was inhibited by a pseudosubstrate peptide indicated that PKC-independent pathways for TCR-mediated activation of Ras also exist (12, 13). Other studies indicate that PKC can act downstream of Ras at the level of Raf. TCR-mediated activation of Raf-1 has been reported to be PKC dependent (14), and at least one isoform of PKC is capable of activating Raf-1 by direct phosphorylation (15), although mutation of the PKC phosphorylation site does not block activation of Raf by PMA (16).
A second model for activation of the Ras-MAPK pathway in T cells is that, analogous to the mechanism of Ras activation by growth factor receptor protein tyrosine kinases, Ras activation occurs when the Ras-GEF Sos is recruited to the cell membrane by protein-protein interactions (17). In T cells, this is thought to be mediated by the adapter protein, linker for the activation of T cells (LAT). LAT is a transmembrane protein containing multiple Grb2-binding sites that become phosphorylated in response to TCR stimulation and, therefore, could recruit Grb2-Sos complexes to the membrane (18, 19). However, it is not known whether Sos is the physiologically relevant RasGEF in T cells (17).
Ras guanylnucleotide-releasing protein (RasGRP), also known as
CalDag-GEFII, is a novel RasGEF expressed in neuronal cells and
lymphoid cells (20, 21, 22). RasGRP contains a diacylglycerol
(DAG) binding domain and can mediate Ras activation in response to PMA
(20, 21). In Jurkat T cells, TCR stimulation induces
RasGRP association with the membrane and RasGRP overexpression enhances
Ras-ERK activation in response to TCR stimulation (23).
Furthermore, RasGRP has been shown to be essential for T cell
development (24). Therefore, DAG production downstream of
TCR stimulation likely mediates effects through RasGRP as well as
through PKC. Additional mechanisms of MAPK regulation in T cells have
been suggested, including direct recruitment of Shc-Grb2-Sos complexes
to phosphorylated CD3
(17, 25), interactions between
Lck and MAPK (26), modulation by NO (27), and
regulation of phosphatases (28).
In the present study, we evaluate the role of PKC in the regulation of ERK1 and ERK2 in Ag and IL-2-dependent CTL clones. We demonstrate that dependence on PKC for ERK1/2 activation varies with stimulation conditions and varies over the time course of stimulation. Our results indicate that there are PKC-independent but DAG-dependent mechanisms, in addition to a PKC-dependent pathway, regulating MAPK in T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine CD8+ CTL clones, clone 11 and clone AB.1 (29) were maintained by weekly stimulation with irradiated splenocytes from C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) and IL-2, as described previously (29), and used 4–7 days later.
Antibodies
The hybridoma producing 145-2C11 (anti-CD3
) was obtained
from the American Type Culture Collection (Manassas, VA). The hybridoma
producing PY-72 (anti-phosphotyrosine) was obtained from Dr. B.
Sefton (The Salk Institute, La Jolla, CA). Anti-MAPK (ERK1 plus ERK2)
mAb was purchased from Zymed (San Francisco, CA). Phospho-p44/42 MAPK
(Thr202/Tyr204) Abs 9101S and 9105S were purchased from New England
Biolabs (Beverly, MA). Abs to PKC-
(H-7), PKC-ß1 (C-16), PKC-
(C-17), PKC- (E-5), and anti-PKC-
(C-18) were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). An additional PKC- Ab was
purchased from BD Biosciences (Mississauga, Ontario, Canada). RAS10 Ab
was obtained from Upstate Biotechnology (Lake Placid, NY). m199 is a
mAb generated in one of our laboratories (generated in the laboratory
of J.C.S.) against rat RasGRP and is available from Santa Cruz
Biotechnology. Rabbit anti-hamster Ig and
anti-mouseHRP Abs were purchased from Jackson
Immunoresearch (West Grove, PA).
Chemical reagents
PMA was purchased from Sigma (Mississauga, Ontario, Canada). U73343, U73122, and the PKC inhibitors bisindolylmaleimide (BIM) I, calphostin C, Gö-6983, and Ro-31-8220 were purchased from Calbiochem (San Diego, CA).
Ab immobilization
Ninety-six-well flat-bottom Falcon 3912 microtiter plates (Becton Dickinson, Oxnard, CA) were coated with 10 µg/ml 145-2C11 overnight at 4°C. Wells were washed twice with PBS, blocked with 2% BSA in PBS for 30–60 min at 37°C, and then washed twice with PBS before use.
MAPK (ERK1 and ERK2) analysis
CTL clones were harvested and washed in Dulbecco’s PBS (D-PBS) (Life Technologies, Rockville, MD). For some experiments, PMA was added to cell cultures at 10 ng/ml, 50 ng/ml, or 100 ng/ml 18 h before cells were used, to down-regulate PKC expression. Cells in D-PBS were preincubated with inhibitor or carrier control (DMSO or ethanol), at the indicated concentration, for 30 min at 37°C except for calphostin C, which was activated by exposure to light for 30 min, then incubated with cells for 5–10 min. Cells were stimulated in one of three ways. For acute PMA stimulation, 100 ng/ml PMA was added and cells were harvested after 10 min, or the time indicated in the figures, of incubation at 37°C. For immobilized anti-CD3 stimulation, cells were added to microtiter wells in which 145-2C11 had been bound. For soluble, cross-linked stimulation, cells were incubated with 10 µg/ml 145-2C11 for 15 min on ice, washed, and resuspended in D-PBS with drug or carrier control, then 5 µg/ml rabbit anti-hamster Ab was added to cross-link the 145-2C11. Cells were aliquoted into BSA-blocked microtiter plates at 1 x 105 cells/well and incubated at 37°C. Cells were lysed at the indicated times after stimulation by the addition of 2x Laemmli reducing sample buffer and boiled for 3 min. For studies with inhibitors, cell viability was checked at the end of the assay by trypan blue exclusion. None of the drugs used had a significant impact on cell viability over the duration of the assay. Whole cell lysates were subjected to SDS-PAGE. For MAPK mobility shift assays, a 15% low-N,N'-methylene-bis-acrylamide (175:1 acrylamide:Bis) gel was used. The activated forms of MAPK ERK1 (p44) and ERK2 (p42) experience reduced mobility under these conditions. For all other Western blot analyses, a standard 10% gel was used. Proteins were transferred to Immobilon P (Millipore Corporation, Bedford, MA). Immunoblotting was performed using anti-mouseHRP or protein-AHRP and visualized by enhanced chemiluminescence (NEN, Boston, MA). In all experiments, detection of activated ERKs was repeated using both the mobility shift assay and anti-phospho-ERK Western blotting with identical results.
Ras assay
Cells were treated with 2 µg/ml of BIM or DMSO carrier for 30 min at 2.5 x 107 cells/ml before stimulation. 107 cells in D-PBS were stimulated in 6-cm dishes coated with 10 µg/ml 145-2C11 and blocked with BSA or in dishes coated with BSA alone for 15 or 30 min at 37°C. At the end of the incubation the cells were lysed in 1 ml magnesium-containing lysis buffer (25 mM HEPES (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 2 mM NaF, 10 mM MgCl2, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM PMSF). Samples of postnuclear lysates were saved for assessment of total Ras and ERK1/2 protein and the remainder of the lysate was used for Raf-1 affinity precipitation. A total of 15 µl Raf-1 Ras binding domain fusion protein bound to glutathione agarose, obtained from Upstate Biotechnology, was added to each sample rotated at 4°C. After 30 min the beads were washed three times with magnesium-containing lysis buffer and resuspended in Laemmli sample buffer. The entire sample was loaded onto a 12% SDS-PAGE gel and probed with RAS10 Ab (Upstate Biotechnology).
Degranulation assay
Degranulation assays were performed as previously described (10). All samples were analyzed in triplicate and the SD is shown.
PKC assay
Cells that had been previously treated for 18 h with PMA or with carrier were lysed at 107/ml in extraction buffer containing 25 mM Tris (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 10 mM 2-ME, 1 mM PMSF. Postnuclear lysates were assayed for PKC activity using the SignaTECT PKC assay system from Promega (Madison, WI) as described in the product bulletin. This system uses the biotinylated peptide neurogranin as a substrate. The ratio of 32P cpm incorporation into the peptide from extract assayed in the presence of phosphatidylserine and DAG to the incorporation of extract assayed in the absence of activators was determined. In all experiments, the cpm incorporated into the peptide also decreased with increasing concentration of PMA in the overnight pretreatment.
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Results |
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1 (PLC-1) (30).
PLC-1 converts phosphatidylinositol 4,5-bisphosphate into inositol
1,4,5-trisphosphate and DAG. Inositol 1,4,5-trisphosphate activates
calcium-dependent pathways while DAG activates PKC. PKC has been
reported to promote MAPK activation in a number of cell types including
T cells. As expected, treatment of CTL clone AB.1 with the DAG analogue
PMA resulted in a rapid increase in the phosphorylation of ERK1 and
ERK2 and this response was completely inhibited by the PKC inhibitor
BIM (Fig. 1
A).
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B).
In dramatic contrast to the results obtained with cross-linked
anti-CD3 Ab, ERK phosphorylation resulting from immobilized
anti-CD3 stimulation comprised an initial phase from 15 to 25 min
after plating that was resistant to BIM, followed by a phase that was
BIM-sensitive (Fig. 2, B–D). The drug was effective in these cells as
BIM treatment inhibited the degranulation response in a dose-dependent
manner (data not shown). The delay in inhibition of ERK activation was
not due to slow uptake of BIM because this drug completely inhibited
the much higher level of ERK phosphorylation induced by PMA after only
10 min (Fig. 2A). The decline in ERK activation was not due
to cell death because BIM-treated cells exhibited no significant
decrease in viability as measured by trypan blue exclusion after 120
min of exposure, and remained viable when replated in growth media and
cultured overnight. Similar results were observed using CTL clone 11
and with Con A blasts generated using C57BL/6 spleen cells (data not
shown). The PKC inhibitors Gö-6983 or Ro-31-8220 exerted modest
or no inhibition, respectively (data not shown), of the ERK
phosphorylation induced by immobilized anti-CD3 mAb. Because the
PKC isoform selectivities of BIM, Gö-6983, and Ro-31-8220 differ,
this result suggests that only particular PKC isoforms are involved in
ERK activation.
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A, Ras
activation, as measured by the ability to bind a Raf-1 fusion protein,
is observed in the presence of BIM, particularly at the 15-min time
point. By 30 min there is a significant decrease in the level of Ras
activity in the 145-2C11-stimulated cells in the presence of BIM
compared with control cells. The BIM was effective in this experiment
as PMA stimulation of cells from the same pool of cells used for the
Ras assay was inhibited in the BIM-treated cells but not in the control
cells (Fig. 3B). These results suggest that there is
BIM-sensitive and BIM-resistant activation of Ras that mirrors the
results observed in Fig. 2 with ERK1/2 activation.
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). In
striking contrast with the effects of BIM, inhibition was evident at
all time points. This result suggests that all phases of ERK activation
following immobilized anti-CD3 stimulation require one or more DAG
binding proteins even though the early phase is BIM-resistant.
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1
inhibitor U73122. U73122, but not its inert analogue U73343, completely
inhibited ERK1 and ERK2 phosphorylation at all time points examined
(Fig. 5). Taken together, the effects of
these inhibitors suggested the existence of an activity upstream of
MAPK ERK1 and ERK2 that was PLC-1 dependent and DAG dependent, but
independent of BIM-inhibitable PKC activity.
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), the ERK
phosphorylation normally induced by acute PMA treatment did not occur
when cells were pretreated overnight with 100 ng/ml PMA (Fig. 6A). Also consistent with the
BIM inhibition data, ERK phosphorylation in response to immobilized
anti-CD3 stimulation was not significantly inhibited by this
treatment (Fig. 6A). When PMA-pretreated cells were
stimulated with immobilized anti-CD3 mAb, degranulation was
partially inhibited at 10 and 50 ng/ml and substantially inhibited at
100 ng/ml PMA in the overnight treatment (Fig. 6B). Abs to
CD45, which do not trigger degranulation were used as a control to show
that the overnight PMA treatment did not cause the cells to
nonspecifically degranulate. The incomplete inhibition in cells treated
with the higher concentrations of PMA is likely due to the
re-expression of PKC during the 4–5 h of incubation required for the
degranulation assay. These results also confirm that PKC activity is
required for optimal degranulation as was observed with the PKC
inhibitor studies.
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A demonstrate that
total PKC activity was diminished in the presence of PMA pretreatment,
particularly at the 50 and 100 ng/ml concentrations. To further confirm
that DAG-responsive PKC isozymes were down-regulated after overnight
PMA exhaustion, lysates from the PMA-pretreated and control cells were
probed with Abs to various PKC isozymes. As shown in Fig. 7B, PKC-, PKC-ßI, PKC-, PKC-, and PKC- were
all significantly down-regulated in response to PMA. We could not
detect PKC-ßII or PKC- in these cells (data not shown). Very
little of each isozyme remained in the cells after overnight treatment
with 50 or 100 ng/ml PMA. Taken together with the lack of
PMA-stimulated ERK1/2 activity, the decreased degranulation, and the
decreased enzymatic activity, our results strongly suggest that
overnight PMA treatment substantially depletes the cells of
DAG-responsive PKC isozymes. Despite the significant decrease in PKC
activity, there is still substantial anti-CD3-stimulated ERK1/2
activation (Fig. 6A).
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blot shown in Fig. 7B was stripped and reprobed with an Ab specific for RasGRP.
RasGRP expression was not decreased by PMA pretreatment (Fig. 7C). The anti-RasGRP mAb m199 detects a doublet, the
molecular basis for which is presently unknown. These results indicate
that unlike PKC, RasGRP is not down-regulated in response to chronic
PMA treatment. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CTL that are stimulated in suspension do not degranulate, whereas CTL degranulation is effectively stimulated by immobilized anti-CD3. These disparate outcomes correlate with the duration of signaling through various pathways including ERKs (10). The difference in duration of signaling between the two conditions might reflect quantitative differences in the duration of TCR stimulation, or qualitative differences in the pathways activated. Here we have found that each condition activates pathways upstream of ERK1 and ERK2 that have differential dependence on PKC, as indicated by the difference in BIM sensitivity. Our observation that BIM inhibits degranulation suggests that PKC is required either for sustained ERK1/2 activation or for some other aspect of degranulation.
An earlier study reported that PKC was not important for TCR-mediated
ERK2 activation based on the ineffectiveness of the PKC inhibitor
Ro-31-8425 (32). Indeed, when we used the similar drug
Ro-31-8220, little effect on ERK activation was seen. This suggests
that the Ro-sensitive PKC isoforms , ßI, ßII, , and are
not important for ERK activation. BIM is known to inhibit PKC isoforms
, ßI, , , and with varying efficiencies. Therefore, it
is highly likely that only certain PKC isoforms such as or are
involved in the TCR-mediated regulation of ERK. PKC- is unlikely to
play a major role because T cells deficient in PKC- have normal ERK
responses (33), although later time points after
stimulation were not specifically examined in this study. It is
important to note that PMA-stimulated ERK activation is highly
sensitive to all three inhibitors, indicating that this mode of
activation is not equivalent to the PKC-dependent phase of ERK
activation induced by immobilized anti-CD3.
The initial increase in ERK activity in response to immobilized anti-CD3 stimulation was relatively insensitive to the presence of BIM, whereas the sustained phase was inhibited by the drug. This pattern of regulation for the Raf-MEK-ERK pathway has also been found in other cell types. Howe and Juliano (34) found that only the late phase of integrin-mediated Raf activation is PKC dependent in fibroblasts, whereas the initial phase is dependent on Raf membrane localization, induced either artificially or by Ras-Raf interaction.
Ras activation in T cells has been proposed to be mediated by the
recruitment of Grb2-Sos complexes to the cell membrane via LAT
(18, 19). This process is solely dependent on
protein-protein interactions involving inducible protein tyrosine
phosphorylation. However, ERK activation is strongly inhibited by the
PLC-1 inhibitor U73122 in Jurkat T cells (23) and CTL
clones (Fig. 5). One of the functions of PLC-1 is the production of
DAG and we found that ERK activation in CTL is sensitive to the DAG
binding domain inhibitor calphostin C (Fig. 4). LAT is involved in the
recruitment and activation of PLC-1 following TCR stimulation
(18, 19, 35). Interestingly, a mutant of LAT that is
unable to bind PLC-1 but is still capable of binding Grb2 is unable
to support MAPK activation suggesting that a PLC-1-dependent pathway
is important for the regulation of MAPK activity in Jurkat cells
(36). In Slp-76-deficient T cells, which recruit Grb2 to
LAT, but do not activate PLC-1, Ras, and ERK2, are not activated
(35). Taken together, these data suggest that the major
function of LAT with respect to ERK activation is to activate PLC-1,
and that PLC-1 is essential for TCR-mediated ERK activation, most
likely due to a requirement for DAG. Our results do not necessarily
rule out a role for Grb2-Sos in TCR-induced signaling but suggest that
the bulk of MAPK activity is regulated by both DAG-dependent but
PKC-independent and PKC-dependent mechansisms.
Our data indicate that although DAG is essential for ERK activation in T cells, the major DAG receptor, PKC, is not required for the initial period of ERK activation as indicated by insensitivity to BIM and Ro-31-8220. These results suggest the existence of a DAG-responsive, but non-PKC, component upstream of ERK activation in T cells. The novel Ras-GEF, RasGRP contains a DAG binding domain and can mediate Ras and ERK activation in response to PMA (20, 21). We found that long-term PMA exposure did not down-regulate RasGRP expression, suggesting that it could be responsible for the CD3-stimulated ERK activation observed following chronic PMA treatment. RasGRP can mediate Ras activation in response to TCR cross-linking in Jurkat T cells and RasGRP overexpression enhances TCR-mediated activation of ERK (23). Therefore, RasGRP is likely to be one of the mediators of DAG-dependent, PKC-independent activation of ERK in T cells.
In summary, the extended period of ERK activation required for CTL
degranulation consists of an early PKC-independent phase and a late
PKC-dependent phase. The early phase, while PKC-independent, is
PLC-1-dependent and DAG-dependent and may involve non-PKC
DAG-responsive signaling elements such as RasGRP. Clearly, regulation
of the Ras-ERK pathway in T cells is complex. Further studies will be
required to identify all the relevant pathways and to distinguish their
relative roles in T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hanne L. Ostergaard, Department of Medical Microbiology and Immunology, 670 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta, Canada, T6G 2S2.
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; BIM, bisindolylmaleimide; DAG, diacylglycerol; D-PBS, Dulbecco’s PBS; ERK, extracellular signal-regulated kinase; GAP, GTPase-activating protein; GEF, guanylnucleotide exchange factor; RasGRP, Ras guanylnucleotide-releasing protein; PKC, protein kinase C; PLC, phospholipase C; LAT, linker for the activation of T cells.
Received for publication May 15, 2000. Accepted for publication September 25, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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