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with a Lipid Raft-Associated Inhibitor of 
B Factor Kinase (IKK) Complex Plays a Role in the Activation of the NF-B Cascade by TCR and CD281
Division of Clinical Immunology and Allergy, Department of Medicine, Center for Health Sciences, University of California, Los Angeles, CA 90095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(PKC) in the
activation of the NF-
B cascade in primary human CD4+
lymphocytes. Among six or so PKC isoforms expressed in T cells, only
PKC participates in the assembly of the supramolecular activation
clusters at the contact site of the TCR with Ag. Signaling via both the
TCR and CD28 is required for optimal activation of the multisubunit
IB kinase (IKK) complex in primary human T lymphocytes; this
activation could be inhibited by a Ca2+-independent PKC
isoform inhibitor, rottlerin. Moreover, endogenous PKC physically
associates with activated IKK complexes in CD3/CD28-costimulated
primary CD4+ T cells. The same set of stimuli also induced
relocation of endogenous PKC and IKKs to a GM1 ganglioside-enriched,
detergent-insoluble membrane compartment in primary T cells. IKKs
recruited to these lipid rafts were capable of phosphorylating a
recombinant IB
sustrate. Confocal microscopy further demonstrated
that exogenously expressed PKC and IKKß colocalize in the membrane
of CD3/CD28-costimulated Jurkat T cells. Constitutively active but not
kinase-inactive PKC activated IKKß in Jurkat T cells. Expression
of dominant-active PKC also had stimulatory effects on the CD28
response element of the IL-2 promoter. Taken together, these data show
that the activation of PKC by the TCR and CD28 plays an important
role in the assembly and activation of IKK complexes in the T cell
membrane. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B pathway (7, 8). These cascades appear to be
functionally linked through the action of a mitogen-activated protein
kinase kinase kinase (MAPKKK), MEKK1, which induces JNK as well as
inhibitor of B factor (IB) kinase (IKK)ß activity during CD28
coligation (7, 8, 9, 10). Both cascades are critical for the
activation of the IL-2 gene, including activation of the CD28 response
element (RE) in the promoter of that gene (7, 11, 12).
This composite RE, which includes c-Rel and AP-1 binding sites, is
activated in a synergistic fashion by the NF-B and JNK cascades
during TCR/CD28 coligation (7, 13). Although the mechanism
of NF-B activation by the TCR/CD28 is unclear, we have recently
shown that CD28 synergizes with TCR in the activation of homo- and
heterodimeric IKK complexes in primary human T cells (8).
Activated IKKs phosphorylate the inhibitory proteins, IB and
IBß, leading to their degradation and release of NF-B
transcription factors into the nucleus (8, 14, 15).
IKK, IKKß, and IKK
are the core components of a 700- to 900-kDa
multimolecular kinase complex, which is responsible for
stimulus-induced phosphorylation of IBs (16, 17, 18, 19, 20, 21, 22).
Although proteins like MEKK1 and the NF-B-inducing kinase have been
shown to associate with the IKKs, there is considerable debate as to
whether these are functionally involved in the regulation of IKK
activity (18, 23, 24). In addition, two other
serine-threonine kinases, namely Cot kinase and protein kinase C
(PKC), have now been implicated in IKK activation by CD3/CD28
(25, 26, 27, 28). Whether these kinases are involved in individual
signaling pathways or act in hierarchical fashion is still uncertain.
In this communication, we will focus on PKC because this is the only
PKC isoform that is recruited to the TCR synapse and the SMAC (1, 5, 29, 30). Moreover, PKC has been shown to be involved in
NF-B activation in T lymphocytes, in addition to its ability to
induce the JNK/AP-1 pathway (6, 26, 27, 28, 31, 32, 33). This
suggests that PKC plays a key role in the regulation of signaling
cascades influenced by CD28 receptor.
Because activated PKC is recruited to the T cell membrane, it is
possible that IKK activation may commence in a signalsome that
assembles at the TCR synapse. We demonstrate that CD3/CD28 coligation
in primary human T lymphocytes induces relocation of endogenous PKC
as well as IKKs to a GM1 ganglioside-enriched, detergent-insoluble
membrane compartment that overlaps with lipid rafts. IKKs recruited to
lipid rafts were actively capable of phosphorylating the recombinant
IB in vitro. Although exogenously expressed PKC induced IKKß
activity in Jurkat cells, it was also possible to show that endogenous
PKC associated with activated IKK complexes in primary human
CD4+ T cells. Expression of dominant-active (DA)
and dominant-negative (DN) PKC had stimulatory and inhibitory
effects, respectively, on the CD28RE. Taken together, these data
suggest that activation of PKC by TCR and CD28 plays an important
role in the assembly and activation of IKK complexes in the T cell
membrane.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OKT3 (anti-CD3) was obtained from Ortho Pharmaceuticals
(Raritan, NJ), and the 9.3 (anti-CD28) and 187.1 mAb were
generously provided by Bristol-Meyer Squibb (Princeton, NJ). Polyclonal
anti-PKC and anti-Lck were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA), and anti-IKK, anti-IKKß,
and anti-IKK were purchased from PharMingen (San Diego, CA).
Anti-Flag (M2) and FITC-conjugated anti-Flag Abs were obtained from
Sigma (St. Louis, MO). Tosyl-activated magnetic beads and M-450
anti-CD4 beads were purchased from Dynal (Great Neck, NY). The PKC
inhibitors Gö6976 and rottlerin were purchased from Calbiochem
(San Diego, CA). Recombinant IL-2 was from Chiron (Emeryville,
CA).
Preparation of CD4+ primary T cells
Human CD4+ T cells were purified from PBLs
and were stimulated with anti-CD3 and anti-CD28 mAbs coupled to
tosyl-activated magnetic beads as previously described (8, 34). After stimulation for 
14 days, beads were removed and
cells returned to their resting state over a 48-h time period
(8). These rested cells were restimulated as described
below.
Cellular stimulation with mAbs and PMA (P) plus ionomycin (I)
The Jurkat T cell clone BMS2 and resting primary human CD4+ T lymphocytes were stimulated with 2 µg/ml each anti-CD3(OKT3) or OKT3 plus anti-CD28 (9.3) mAb, secondarily cross-linked with 10 µg/ml mAb 187.1 for 30 min. Stimulation with P (100 nM) plus I (1 µg/ml) was used as positive control. Alternatively, Jurkat T cells were stimulated by OKT3/9.3 Abs coupled to magnetic beads for 30 min before conducting confocal microscopy.
Gene constructs and cellular transfection
Flag-IKK and IKKß (wild-type and kinase-inactive mutants)
were provided by Tularik (San Francisco, CA) (19). PKC,
wild-type (wt), DA, and DN (kinase-inactive) were provided by Dr. Baier
(University of Innsbruck, Innsbruck, Austria) (31). The
CD28RE/AP-1 luciferase reporter was previously described (8, 13). For cellular transfection, we used the indicated amounts of
cDNA for electroporation (240 V, 950 µF) as previously described
(8, 35)
Immunoprecipitation and Western blot analysis
A total of 2 x 107 primary CD4+ or Jurkat T cells were stimulated with anti-CD3 (aCD3) plus anti-CD28 (aCD28) mAb (2 µg/ml each), secondarily cross-linked with 187.1 (10 µg/ml), or P + I (100 nM and 1 µg/ml, respectively). Cells were lysed in buffer A (50 mM HEPES (pH 7.6), 250 mM NaCl, 1% Triton X-100, 2 mM MgCl2, 2 mM DTT, 0.1 mM Na3VO4, 20 µM ß-glycerophosphate, and 20 µM p-nitrophenylphosphate) and cleared by centrifugation. Lysate (200 µg) was treated with 2 µg of the indicated Abs, bound to protein A-Sepharose, and rocked for 2 h at 4°C. Immunoprecipitated complexes were washed, separated by SDS-PAGE, and transferred to nitrocellulose membrane (8). Membranes were sequentially overlaid with the indicated concentrations of the primary Abs, followed by a 1:2000 dilution of the secondary HRP-conjugated Ab.
Cellular fractionation into detergent-soluble and detergent-insoluble material
Cellular fractionation was performed as previously described (36). Briefly, 1 x 108 primary CD4+ cells were suspended for 15 min in hypotonic buffer E (10 mM Tris (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 0.2 mM PMSF, 1 mM sodium vanadate, 10 µg/ml leupeptin, and 0.01 TIU/ml aprotonin), then lysed with 50 mechanical strokes in a Dounce homogenizer. Lysates were spun for 30 min at 100,000 x g, and the supernatant, designated cytosol, was collected. The pellet was rinsed twice in buffer E and resuspended in buffer E, containing 1% Nonidet P-40 for 30 min. Samples were recentrifuged at 100,000 x g, and the supernatant, designated detergent-soluble material (DSM), was collected (33). The remaining pellet was rinsed once in buffer E plus detergent, followed by sonication in RIPA buffer (20 mM Tris (pH 7.5), 250 mM NaCl, 10 mM DTT, 10 mM MgCl2, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate), and cleared by centrifugation as described above. This supernatant was designated detergent insoluble material (DIM) (36).
Sucrose gradient fractionation
Aliquots of 1 x 108 Jurkat or primary human CD4+ lymphocytes stimulated or unstimulated were lysed in 1 ml of ice-cold MBS (25 mM MES pH6.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM Na3VO4, 1 mM PMSF, and 10 µg/ml aprotinin). Lysates were homogenized using 15 strokes of a Dounce homogenizer, mixed with an equal volume of 85% sucrose (w/v) in MBS, and transferred to a SW41 centrifuge tube. The mixture was first overlaid with 6 ml of 35% sucrose, then 3 ml of 5% sucrose in MBS with 1 mM Na3VO4, and centrifuged at 200,000 x g for 16 h at 4°C. Following centrifugation, eleven 1-ml fractions were sequentially collected from the top of the centrifuge tube. The low-density fractions (i.e., fractions 2–4) contained lipid raft material, whereas cytosolic material and soluble membranes were predominantly recovered in fractions 9–11. Primary human CD4+ T cells were similarly processed, except cells were stimulated with anti-CD3 + anti-CD28 mAb coupled to magnetic beads.
GM1 ganglioside staining
To determine the efficiency of the isolation of lipid rafts via sucrose gradient centrifugation, 2 µl of each fraction was dot blotted onto Immobilon-P Nitrocellulose Transfer membrane (Millipore, Bedford, MA). After drying, the membrane was blocked in 6% BSA for 1 h at room temperature and washed once in PBS with 0.1% Tween 20 (PBS-T). HRP-linked cholera toxin ß subunit (Sigma) was diluted in PBS-T to 4.2 µg/µl and was used to overlay the membrane for 1 h. The membrane was washed three times, incubated with SuperSignal Chemiluminescent Substrate (Pierce, Rockford, IL), and subjected to autoradiography.
Immune complex kinase assays
A total of 107 Jurkat or resting
CD4+ T cells in 1 ml of RPMI were either
unstimulated or stimulated with anti-CD3 + CD28 mAb as described
above (8). For the effects of PKC inhibitors on IKK
activity, CD4+ T cells were incubated with the
indicated concentrations of these drugs for 1 h before
stimulation. Two hundred microgram lysate was incubated with 2 µg
anti-IKK Ab, adsorbed onto protein A-Sepharose for 2 h. In
vitro kinase assays using GST-IB1–55(1–55) as substrate were performed
as previously described (8, 17).
Luciferase assays
A total of 107 Jurkat cells were
transfected with 10 µg of the consensus NF-B or CD28RE luciferase
(Luc) reporters in the absence or presence of one of the following
constructs: wt-PKC, DA-PKC, or DN-PKC. In separate
experiments, CD28RE Luc reporter was coexpressed with DA-PKC and
either DN-IKKß or DN-IKK. Cells were rested for 24 h and then
stimulated for 6 h with anti-CD3 + CD28 mAb or P + I.
Luciferase assays were performed as previously described
(8). Transfection efficiency was monitored by
cotransfection of a ß-galactosidase plasmid (CMV-ß-gal).
Confocal microscopy
Jurkat T cells were transfected with 10 µg of Flag-IKKß plus
10 µg of wt PKC by electroporation as described.
(35). Cells were rested for 24 h and stimulated for
1 h with anti-CD3 + CD28 mAb coupled to magnetic beads, at a
ratio of 4 beads/cell (34). Cells were vigorously
resuspended and the magnetic beads were removed. In a separate
experiment, Jurkat T cells transfected with Flag-IKKß and DA-PKC
were used without any stimulations. Cells spun onto a glass slide were
fixed in 4% glutaraldehyde for 30 min and permeabilized in 70%
methanol for 1 h. Slides were overlaid with 5 µg/ml goat
anti-PKC for 2 h. After washing in PBS (three times),
slides were treated with 5 µg/ml of PE-conjugated anti-goat plus
5 µg/ml FITC-conjugated anti-Flag for 2 h. Appropriate
controls consisted of omitting the primary Abs to rule out nonspecific
binding of the secondary reagent. Samples were washed extensively and
examined under a LEICA inverted TCS-SP confocal microscope, using a
x100 objective lens.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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plays a role in CD3/CD28-induced activation of IKKs in
primary human CD4+ T lymphocytes as well as Jurkat cells
We have previously shown that CD28 costimulation is essential for
the activation of heterogeneous IKK complexes, as well as activation of
the IL-2 promoter (8). We focused on the role of PKC,
because this is the only PKC isoform that is recruited to the TCR
(29, 30). Moreover, it was recently demonstrated by the
cellular transfection approach that PKC plays a role in the
activation of the IKKs in Jurkat cells (26, 27). To
demonstrate the relevance of these findings in NF-B activation in
primary human CD4+ T cells, we examined the
effect of PKC inhibitors on IKK activation during CD28 costimulation
(Fig. 1
). As previously shown, CD3/CD28
coligation but not anti-CD3 alone, induced IKK activation (Fig. 1, lanes 1–3). Rottlerin, a
Ca2+-independent PKC isoform inhibitor,
interfered in IKK activation (lanes 4–6), whereas
Gö6976, a Ca2+-dependent PKC isoform
inhibitor, had no effect (lanes 7–9). Similar
findings were made in Jurkat cells (data not shown). These data suggest
that Ca2+-independent PKCs participate in the
activation of IKK complexes by the TCR. These data do not rule out the
role of other PKC isoforms in NF-B pathway activation, e.g.,
stimulation by cytokines.
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has been implicated in CD28 costimulatory events
(29, 32), we examined whether this PKC isoform physically
associates with the IKK signalsome. PKC immune precipitates were
analyzed for the presence of IKK subunits by Western blotting. Compared
with resting CD4+ T cells, which did not show any
association between these proteins, a small amount of IKKß
coprecipitated with PKC in anti-CD3-treated cells (Fig. 2A). The amount of IKKß
coprecipitaiton was dramatically increased by CD28 costimulation
(lane 4), which is in agreement with the key role of
this receptor in IKK activation (Fig. 1
). To show the specificity of
PKC in this costimulatory event, we also performed an analysis in
which we compared PKC with PKC immune complexes. As shown in Fig. 2B, both IKK and IKKß coprecipitated with PKC in
anti-CD3 + CD28 or P + I-stimulated CD4+ T
cells. In contrast, PKC immune complexes did not contain any IKKs
under resting or stimulated conditions (Fig. 2A), showing a
specific role for PKC in CD3/CD28-induced NF-B activation
(26, 27). In a reverse protocol, anti-IKK immune
complexes could be seen to coprecipitate with PKC in CD3 + CD28 or P
+ I-treated cells (Fig. 2C). Parallel in vitro kinase assays
showed that PKC was associated with active IKK complexes capable of
phosphorylating the substrate, IB (Fig. 2D). In
contrast, anti-PKC precipitates were not associated with active
IKK complexes (Fig. 2D). Taken together, these data show
that PKC associates with a complex that also includes activated IKKs
in CD3/CD28-stimulated T cells. Although we do not know whether this is
a direct interaction or an indirect association via intermediary
components in a macromolecular complex, other workers have failed to
show a direct interaction of PKC with IKKs (27).
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with epitope-tagged IKKs could
induce IKK activity. Although DA-PKC induced IKKß activity, no
activation was obtained with wt or DN-PKC (Fig. 3, top). HA-IKKß was equally
expressed in these samples (Fig. 3, middle). Blotting for
PKC confirmed overexpression of wt, DA-, and DN-PKC (Fig. 3, bottom). In contrast, a parallel experiment using
Flag-IKK cotransfection failed to show an effect of PKC on IKK
activity (data not shown). This is in agreement with the findings of
Lin et al. (27) who showed that DA-PKC induced the
phosphorylation of coexpressed IKKß but not IKK. Taken together,
the above data show that PKC plays a key role in IKK activation in
primary human CD4+ lymphocytes as well as in
overexpressing Jurkat cells.
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is involved in the activation of the CD28RE
A biologically relevant target for the CD28 accessory receptor is
the IL-2 gene, in particular the CD28RE in the promoter of that gene
(7, 13). We have recently shown that IKKß, but not
IKK, contributes to the activation of that RE during CD3/CD28
costimulation (7, 8). The importance of PKC in the
activation of that RE was confirmed by the use of wt, DA-, or
DN-PKC, which were equally expressed in Jurkat cells together with a
CD28RE-Luc reporter (Fig. 4A).
Although the response of this reporter was minimally affected by
wt-PKC, DA-PKC itself was sufficient in CD28RE activation (Fig. 4B). This activity was further enhanced by CD3 + CD28 or P +
I costimulation (Fig. 4B). In contrast, DN-PKC interfered
in CD28RE activation (Fig. 4B). Similar inhibitory effects
on CD28RE activation were obtained with rottlerin, but not Gö6976
(Fig. 4C). To demonstrate that the IKKs function downstream
of PKC in the activation of that RE, we used DN-IKK and DN IKKß
cotransfection with DA-PKC. Although DN-IKKß interfered in the
DA-PKC-induced response, DN-IKK had minimal effects on CD28RE-Luc
activity (Fig. 4D). These findings are in agreement with the
ability of DA-PKC to induce IKKß, but not IKK, activation under
cotransfection conditions (Fig. 3).
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relocates to a detergent-resistant membrane compartment
together with talin and IKKs
Because PKC is recruited to the signal assembly complex at the
TCR (29, 30), it raised the question whether IKKs and
their associated components are also recruited to that membrane site.
We have previously shown that TCR ligation leads to the translocation
of several signaling components, including the 
-chain of the TCR,
cytoskeletal components, p56lck, p36(LAT),
PLC-1, and ZAP-70 to DSM and DIM
(36). Interestingly, the same signaling molecules are
recruited to the SMAC, where PKC localizes in the center cluster,
whereas talin appears in the peripheral zone (29, 30).
Talin is a member of the cytoskeletal complex that assembles at the TCR
synapse (37). To determine whether PKC and talin are
corecruited to the DIM, we fractionated primary human
CD4+ T cells into cytosolic DSM and DIM
components. Western blot analysis showed talin relocation from the
cytosol to the DIM during CD3 + CD28 or P + I, and, to a lesser extent,
anti-CD3 stimulation (Fig. 5A). Although endogenous
PKC underwent similar redistribution to the DIM during CD3 + CD28 or
P + I stimulation (Fig. 3), this kinase also relocated to the DSM under
these stimulatory conditions (Fig. 5A). In a related set of
experiments, we also asked whether IKK components relocate to these
cellular fractions. IKK, IKKß, and IKK were recruited to the
DIM, and to a variable degree the DSM, during cellular stimulation with
anti-CD3 + CD28 or P + I (Fig. 5B).
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A).
Immunoblotting showed PKC and talin recruitment to the hypodense
fractions during cellular stimulation (Fig. 6B). Although
PKC and talin did not appear in the low density fractions from
unstimulated cells, these fractions did show the constitutive presence
of Lck, which was further increased by CD3 + CD28 costimulation (Fig. 6B). Moreover, Western blotting for IKKß and IKK showed
their relocation to lipid rafts in CD3 + CD28-treated cells (Fig. 6B). Taken together, these data indicate that IKK components
are recruited to lipid rafts in parallel with PKC and
talin.
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Although for logistic reasons the entire experiment in Fig. 6B could not be repeated in primary
CD4+ T cells, we did perform sucrose gradient
fractionation on CD3 + CD28-stimulated primary T cells. As for Jurkat
cells, the lipid rafts were identified by cholera toxin staining and
shown to reside in fractions 3–5. Immunoblotting showed the presence
of IKKs in these low density fractions, in addition to their presence
in the DSM (fractions 10 and 11; Fig. 6C). Moreover, immune
precipitation with anti-IKK and performance of in vitro kinase
assays showed the presence of activated IKK complexes in fractions 2–4
as well as 10 and 11 (Fig. 6C, lower panel).
These findings indicate that lipid rafts from CD3/CD28 costimulated T
cells contain activated IKK complexes. Similar results were obtained in
Jurkat T cells.
Exogenously expressed PKC and IKKß relocate and colocalize in
the membranes of Jurkat T cells
To confirm the recruitment of PKC and IKKs to the membranes, we
used confocal microscopy. Exogenously transfected wt-PKC and
Flag-IKKß were expressed in a diffuse manner in unstimulated cells
(Fig. 7). However, upon stimulation with
anti-CD3 + CD28-conjugated beads, both PKC and IKKß were
redistributed to membrane cap sites (Fig. 7). Overlay of the green
(IKKß) and red (PKC) images yielded a composite yellow fluorescent
pattern, which indicates colocalization of these proteins in the
membrane cap sites of stimulated cells (Fig. 7). When cotransfected
with DA-PKC, Flag-IKKß colocalized in the membrane in a diffuse
distribution without the need for cellular stimulation (Fig. 7, right panel). These data suggest that PKC activity is
involved in the membrane recruitment of the IKK signalsome, which is
also consistent with coimmunoprecipitation studies shown in Fig. 2. In
accordance with this notion, DN-PKC resides in the cytosol of
unstimulated cells, similar to wt-PKC (data not shown). Although
endogenous PKC undergoes a similar redistribution as exogenously
expressed PKC during CD28 costimulation (data not shown), it was not
possible with the available anti-IKK Abs to obtain a strong enough
signal to study endogenous IKK redistribution. This was, however,
achieved by the biochemical and coimmunoprecipitation studies shown in
Figs. 2 and 6.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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participates in
CD3/CD28-induced activation of IKKs in primary human
CD4+ lymphocytes. DN-PKC and a
Ca2+-independent PKC inhibitor interfered in the
activation of the IKK complexes as well as the CD28RE of the IL-2
promoter. Moreover, IKKs and PKC were recruited from the cytosol to
a talin-enriched, detergent-resistant cellular compartment during CD28
costimulation. Similarly, PKC, talin, and IKKs were recruited to
GM1-enriched lipid rafts, which represent a detergent resistant
membrane compartment. IKKs recruited to the lipid rafts were active. It
was also possible to show that the activated IKK signalsome
coimmunoprecipitate with PKC from CD3/CD28 costimulated primary
CD4+ lymphocytes. Moreover, active PKC and
IKKß were recruited to and colocalized in the membrane of stimulated
Jurkat T cells as determined by confocal microscopy. It was also
demonstrated that exogenously expressed DA-PKC activated IKKß, but
not IKK, in Jurkat cells. Similarly, DN-IKKß, but not DN-IKK,
interfered in the activation of the CD28RE by DA-PKC. Taken
together, these findings demonstrate stimulus-dependent interaction
between PKC and IKK components, and present a novel mechanism to
explain synergistic activation of IKKs by TCR and CD28.
Although some components of the IKK signalsome have been identified,
its overall composition and regulation have not been fully
characterized (17, 18, 19, 20, 21, 22, 39). Indeed, the evolving scenario
points to heterogeneous IKK complexes responding to different stimuli
(24, 39, 40), including TCR/CD28 costimulation in primary
T cells (8). Stimulus-dependent assembly and activation of
IKK complexes is an attractive model, considering the multitude of
stimuli involved in NF-B activation (24, 40). Four
different serine-threonine kinases have now been implicated in the
activation of the IKKs by CD3/CD28, namely MEKK1, NF-B-inducing
kinase, Cot, and PKC (7, 25, 26, 27, 28). Although our
overexpression studies in Jurkat cells confirm the recently published
data about the role of PKC in IKK activation (Fig. 3) (26, 27), the current communication extends those findings by showing
the recruitment and colocalization of PKC and IKKs in the membrane
of T cells (
Figs. 5–7). Moreover, we demonstrate that PKC and IKKs
are physically associated with a macromolecular complex that can be
reversibly precipitated with Abs to PKC or IKKs from lysates
prepared from CD3 + CD28-stimulated cells (Fig. 2). Coudronniere et al.
(26) recently reported that rottlerin, an inhibitor of
novel PKC isoforms, interfered in the activation of the NF-B pathway
and the CD28RE by the TCR. We show here that treatment of primary human
CD4+ with rottlerin inhibits the
CD3/CD28-mediated activation of endogenous IKK complexes (Fig. 1).
Gö6976, an inhibitor of classical PKC isoforms, had no effects on
IKK activity and transcriptional activation of the CD28RE (Figs. 1 and 4). Although the role of other PKC isoforms in the activation of the
NF-B pathway has been demonstrated, our findings implicate PKC in
IKK activation and recruitment during CD3/CD28 costimulation T cells.
We found that endogenous PKC physically associated with the IKK
signalsome in the cell membrane (Figs. 2 and 7). Although this
interaction was minimal affected by of CD3 ligation, the association
was markedly augmented by CD28 costimulation (Fig. 2A).
Although the importance of PKC in IKK activation was recently
reported (26, 27), this is the first evidence for
CD3/CD28-induced association of PKC with the IKK complex in a
physiologically relevant system. Although a previous study could not
demonstrate that PKC directly interacts with IKK components
(27), it is possible that this interaction involve
additional components of a macromolecular complex, e.g., cytoskeletal
or scaffolding proteins. This aspect requires further study.
We propose that PKC/IKK association takes place in the immunological
synapse. PKC along with IKK, IKKß, and IKK were recruited to
the membrane fractions and lipid rafts in primary human
CD4+ lymphocytes (Figs. 5 and 6). Lipid rafts,
which are cholesterol- and sphingolipid-enriched membrane domains, are
docking sites for the attachment of costimulatory receptors and
signaling molecules in the cell membrane (1, 29, 30, 38).
Formation of the TCR synapse and the assembly of productive signaling
complexes in the lipid rafts require reorganization of the cortical
cytoskeleton (30, 35), and may explain our finding that
the IKKs and PKC are recruited to a detergent-insoluble cellular
compartment that includes talin and spectrin (Figs. 5 and 6). CD28
participates in the organization of lipid rafts and recruitment of the
associated signaling molecules to the TCR synapse (1, 5, 38). Recruitment of the IKK components to the lipid rafts
represents a novel aspect of CD28 function, which may also be
responsible for activation of the IKK complex (Fig. 6C) and
the NF-B pathway. The mechanism of CD28-induced recruitment of
signaling molecules to the lipid rafts is poorly understood. However,
recent studies have demonstrated that CD28 contributes to the
rearrangement of the cytoskeletal complex at the TCR contact site via
the activation of Vav (41). Vav has been shown to
participate in the recruitment of PKC to the T cell membrane and the
cytoskeleton during TCR engagement (41) We hypothesize
that this process is essential for the recruitment of IKKs to the lipid
rafts. Although ectopically expressed wt PKC and IKKß colocalized
in the membrane after CD3/CD28 costimulation, DA-PKC and IKKß
spontaneously localized in the membrane in the absence of any stimuli
(Fig. 7). This suggests that, in addition to its role in IKK activation
(26, 27), PKC may also play a role in the recruitment
of IKKs to the T cell membrane. The idea that the IKKs are activated in
the vicinity of the TCR is further strengthened by the demonstration
that TNF- induces the IKK holoenzyme to associate with components of
the p55 TNF receptor site in the cytoplasmic membrane
(42).
In conclusion, we have shown that, during CD28 costimulation,
PKC-induced IKK activation in primary human
CD4+ T cells involves their physical interaction
with an activation complex that localizes in the T cell membrane. This
may represent an early event in the activation of the NF-B cascade
during CD3/CD28 costimulation. The signaling molecules that attract
IKKs to the lipid rafts remain to be identified. However, because
DA-PKC facilitated the relocation of IKKß to the membranes (Fig. 7), it is tempting to speculate that PKC plays an active role in
this process. PKC activity has also been linked to JNK activation,
and this pathway synergizes with the NF-B cascade in the activation
of the IL-2 promoter (7, 31, 32, 33). In this regard, PKC
may act as a master switch responsible for the regulation of key
signaling pathways involved in the delivery of signal two.
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Acknowledgments |
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Footnotes |
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2 D.B. and C.A.T. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Andre Nel, Department of Medicine, Division of Clinical Immunology and Allergy, 10833 Le Conte Avenue 52-175 CHS, University of California, Los Angeles, CA 90095.
4 Abbreviations used in this paper: SMAC, supramolecular activation cluster; PKC, protein kinase C; JNK, c-Jun N-terminal kinase; IB, inhibitor of B factor; IKK, IB kinase; RE, response element; P, PMA; I, ionomycin; Luc, luciferase; DIM, detergent-insoluble material; DSM, detergent-soluble material; wt, wild type; DN, dominant negative; DA, dominant active; ß-gal, ß-galactosidase.
Received for publication June 8, 2000. Accepted for publication September 18, 2000.
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