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*
John P. Robarts Research Institute,
The Department of Microbiology and Immunology, and
The Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario, Canada
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß or 
heterodimer (1). By noncovalently
associating with TCR, the CD3 complex transduces the Ag signal to
intracellular signaling pathways (2). When T cells are activated, they
initiate cell cycle progression and produce cytokines (e.g., IL-2) (3).
Accumulating evidence indicates that T cell activation is aborted by
TCR signaling alone, and that additional costimulatory signals are
required to complement the TCR signal and activate the T cell. CD28 is
a 44-kDa homodimeric glycoprotein constitutively expressed on most
mature T cells (4). Intensive research in the last several years has
characterized this molecule as the receptor that generates
costimulatory signals in T cells. Cross-linking CD28 with specific Abs
does not trigger T cell activation; however, if CD28 is synergized with
TCR stimulation, cell proliferation and cytokine production are greatly
induced.
Despite a powerful influence on T cell activation, the mechanism of
signaling by CD28 remains uncertain. One candidate that may possibly be
involved in directing the CD28 costimulatory signal is
phosphatidylinositol-3-phosphate kinase
(PI3K).3 The p85
subunit of PI3K can associate with the cytoplasmic domain of CD28 via a
pYMNM motif, and CD28 ligation may activate this lipid kinase (5, 6, 7).
In addition to PI3K, an adapter protein, growth factor receptor-bound
protein 2, and a T cell-specific protein tyrosine kinase, inducible T
cell kinase, may also bind to the same pYMNM motif. A few reports,
however, dispute the importance of PI3K for CD28 costimulatory
signaling (8, 9, 10). The activation of p21ras in a
CD28-stimulated human T cell line was reported, and a growth factor
receptor-bound protein 2-complexed nucleotide exchange factor, Sos, may
trigger this response (11). However, this action was observed only as a
result of stimulation with CD28-specific Ab. The physiologic ligand of
CD28, B7-1, did not cause p21ras activation.
Therefore, the issue of stoichiometry appeared to limit further study
of the proximal events that trigger CD28 costimulatory signaling. Two
reports, including our own, have shown that CD28 stimulation couples to
the sphingomyelin-ceramide pathway (12, 13), which overlaps with the
signaling pathways triggered by the TNF-R, IL-1R, and Fas (14).
Since all of these receptors, under particular circumstances, generate
a costimulatory signal in T cells, and since the cell-permeable
ceramide analogues and sphingomyelinase can directly generate
costimulatory signals in T cells (13, 15), we have proposed that
sphingomyelin-ceramide turnover-mediated signaling is a major pathway
of CD28 costimulatory signaling. Ceramide alone activates c-Jun
NH2-terminal kinase (JNK) (16, 17), while a concomitant stimulation of
TCR and CD28 results in an increased activation of this kinase (18).
Activation of Rac1 and p38-K was also observed in a human T cell line
treated with C6-ceramide (a cell-permeable ceramide analogue), although
exactly how the ceramide pathway reacts with these molecules was not
demonstrated (19). JNK is a c-Jun transcription factor/activation
kinase that is stimulated by various stress signals; it is also an
anchor kinase of the Rac1/CDC42 small G protein/p21-activated kinase
(PAK)/MEK kinase (MEKK)1/stress-activated protein kinase/extracellular
signal-related protein kinase (SEK) pathway (20). The stimulation of
MEKK1, another kinase of this pathway, was also observed in a TCR- and
CD28-costimulated T cell line (21). Furthermore, the induced deficiency
of SEK1 in mice inhibited CD28-mediated T cell costimulation (22). Do
these data indicate that the JNK pathway is a potent costimulatory
pathway?
In this report, we show that CD28 stimulation causes an activation of the signaling cascade that includes PAK and MEKK1. The treatment of T cells with C2-ceramide mimics CD28 stimulation by PAK and MEKK1 activation. CD3 stimulation also activates this kinase cascade; however, coligation of CD3 and CD28 greatly increases the activation of the PAK cascade, while the same stimulation promotes c-Jun phosphorylation in the nucleus. C2-ceramide also augments the activation of the PAK and MEKK1 cascades after CD3 stimulation. These data demonstrate that CD28 costimulation involves coupling of the sphingomyelin/ceramide turnover-mediated signaling and the PAK/MEKK1/SEK/JNK pathways. The data also suggest that the TCR signal and the CD28 signal merge primarily at the level of a cytoskeletal signaling process which is mediated by Rho family G proteins.
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Materials and Methods |
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The Abs used in this work were as follows: anti-human CD3
(UCHT1) from PharMingen (San Diego, CA); anti-human CD28 (YTH913.2)
from Serotec (Oxford, U.K.); and anti-PAK (C-19 and N-20),
anti-MEKK1 (43Y and C-20), anti-c-Jun (KM-1), FITC-goat
F(ab')2 anti-rabbit IgG, and horseradish
peroxidase-conjugated anti-mouse IgG from Santa Cruz Biotechnology
(Santa Cruz, CA). The Abs to mouse CD3 (2C11) and mouse CD28 (37.51)
were purified from culture supernatants. Glutathione
S-transferase (GST)-I
B and kinase-deficient MEKK1,
MEKK1(1–301), were both purchased from Santa Cruz Biotechnology.
Myelin basic protein (MBP) was obtained from Sigma (St. Louis, MO).
Wortmannin, C2-ceramide, and dehydro-C2-ceramide were obtained from
Biomol (Plymouth Meeting, PA). Immunizing peptides (blocking peptides)
for PAK-specific Abs were purchased from Santa Cruz Biotechnology.
Cell culture, cell stimulation, and immunoprecipitation
Jurkat and EL4 cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in RPMI 1640 with 5% FCS and 40 µg/ml gentamicin. Jurkat cells or EL4 cells were either stimulated with anti-CD3 (at a final concentration of 10 µg/ml) and/or anti-CD28 (at a final concentration of 15 µg/ml) without cross-linking by secondary Abs or treated with C2-ceramide or dehydro-C2-ceramide in FCS-free RPMI 1640 as indicated. Following stimulation, cells were lysed in RIPA buffer (20 mM Tris (pH 7.4), 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 0.5% sodium deoxycholate, 2 mM EGTA, 1 mM sodium molybdate, 1 mM sodium orthovanadate, and 20 µg/ml aprotinin). After preclearing with protein A/G agarose (Santa Cruz Biotechnology), immunoprecipitates were prepared using both Abs to PAK or MEKK1 and protein A/G agarose.
To demonstrate the specificity of immunoprecipitation, Ab was neutralized by incubating with an excess amount of immunizing peptide for 2 h at room temperature before use.
Protein kinase assays
Immune complexes were washed four times in RIPA buffer and twice
in kinase buffer (50 mM HEPES (pH 7.5), 10 mM MnCl2,
10 mM MgCl2, and 1 mM DTT). Autokinase reactions for PAK
and MEKK1 were performed in kinase buffer containing 2 µM ATP (15
µCi/µM). For PAK activity toward MBP, kinase-deficient MEKK1
(MEKK1(1–301)), or GST-IB, immunoprecipitates were incubated in
kinase buffer containing 10 µg of MBP and 5 µM ATP (4 µCi/µM),
1 µg of MEKK1(1–301) and 5 µM ATP (4 µCi/µM), or 1 µg of
GST-IB and 5 µM ATP (4 µCi/µM), respectively. After a 15-min
incubation at 30°C, the reactions were stopped by the addition of
denaturing SDS-PAGE buffer. 32P-labeled proteins were
resolved by SDS-PAGE and analyzed using a Molecular Imager (Bio-Rad,
Hercules, CA).
Fluorescence microscopy study of phosphorylated c-Jun
To measure the phosphorylation of c-Jun in vivo, EL4 cells were cultured overnight on 22-mm square microscope cover slips. Cells on the cover slips were stimulated for 30 min with Abs (2 µg/ml for each Ab), washed, permeabilized, and fixed using standard methods (23). Cells were stained with mouse antiphosphorylated c-Jun mAb (IgG1) at a 1:20 dilution followed by FITC-goat F(ab')2 anti-mouse IgG (1:20) as described previously (24).
Samples were examined using a Zeiss Axioscope microscope (Stuttgart, Germany) equipped with epifluorescence filters.
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Results |
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Our recent experiments have demonstrated that CD28 stimulates
focal adhesion, such as molecular changes in T cells, which is
apparently controlled by the Rho family G proteins CDC42 and Rac1 (24).
The data indicated the possibility that the CD28 signal activates a
Ser/Thr kinase, PAK, which is known to associate with and be activated
by GTP-bound Rac1 or CDC42. Therefore, we extended our investigation to
measure the activity of PAK in CD28-stimulated T cells. Both an
autokinase assay (Fig. 1
A) and an in vitro
kinase assay against MBP (Fig. 1B) clearly
demonstrated a transient activation of PAK in anti-CD28-treated
human T cell line Jurkat cells. The immunizing peptide successfully
inhibited that immunoprecipitation of PAK.
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B used as the control substrate (Fig. 1
, C and D). Thus MEKK1 appeared to be a
substrate of PAK in CD28-stimulated T cells. Similar results were
obtained using the EL4 mouse T cell line (data not shown).
Recently, MEKK1 has been cloned in its full length as a 195-kDa protein
(26). In determining if CD28 stimulation activates MEKK1, the Jurkat
cell was not suitable, since it expresses MEKK2 and not MEKK1; only
anti-MEKK1 Ab is currently available for immunoprecipitation
assays. Therefore, we used EL4 cells for all MEKK1 assays. When we
assayed the expression of MEKK1, both anti-MEKK1(43Y) Ab to 1–301
amino acids of originally cloned MEKK1 and anti-MEKK1(C-22) Ab to
C-terminal 22 amino acids immunoprecipitated a 
190-kDa protein (Fig. 1E). This protein band demonstrated
autophosphorylation activity, and the stimulation of these cells with
anti-CD28 clearly increased this autophosphorylation activity (Fig. 1F).
The cell-permeable ceramide analogue can activate PAK
Next, we hypothesized that the signaling pathway stimulated by
ceramide overlaps with that activated by PAK. In vitro kinase assays
were used to test the effect of a cell-permeable ceramide analogue on
PAK activation. C2-ceramide activated PAK, while dehydro-C2-ceramide, a
biologically inactive form of C2-ceramide (27), did not (Fig. 2, A and B).
The activation of PAK occurred in a dose-dependent fashion (Fig. 2B). The activity of PAK induced by C2-ceramide was
confirmed using MEKK1(1–301) as the substrate (Fig. 2C).
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Given that JNK activity is under the control of PAK/MEKK1, CD28
and CD3 signals may merge at the level of PAK/MEKK1 activation. To
investigate this possibility, PAK and MEKK1 activity were studied in
Jurkat or EL4 cells stimulated with anti-CD3 plus anti-CD28.
Interestingly, CD3 activation significantly stimulated PAK; however,
when cells were stimulated with both CD28 and CD3, the activity of PAK
was dramatically increased (Fig. 3,
A and B). We then examined whether MEKK1
activity parallels that of PAK (Fig. 3, C and
D). The increase in phosphorylation resulting from
autokinase activity was lower than that observed in the PAK assay, but
CD3 plus CD28-stimulated cells evidently had a higher activity of
MEKK1. Next, we extended the study to determine whether C2-ceramide
mimics the costimulatory effect of CD28 stimulation by augmenting PAK
activity. As shown in Figure 3, E and F,
C2-ceramide greatly increased the activation of PAK when combined with
CD3 stimulation.
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The results described above strongly suggested that the CD3 signal
and the CD28 signal merge at PAK/MEKK1, possibly resulting in the
augmentation of JNK activity. Indeed, previous data showed that CD28
and CD3 in synergism stimulate JNK to a significant level as assayed by
measuring the kinase activity in vitro. Thus, both signals were
necessary to cause optimal T cell activation (Ref. 18 and our
unpublished observations). Following activation in the cytoplasm, JNK
translocates to the nucleus to phosphorylate the transcription factor
c-Jun at Ser63 and Ser72 (reviewed in 28 . To investigate definitively if the concomitant stimulation of CD3
and CD28 does indeed increase the active form of the c-Jun
transcription factor in cells, as was expected from the increased
activity of JNK, we examined the level of c-Jun phosphorylation in
cells after CD3 stimulation with or without CD28 costimulation. EL4
cells were stimulated with anti-CD3 and/or anti-CD28, and the
phosphorylated c-Jun was detected using a mAb against the
Ser63-phosphorylated form of c-Jun. In comparison with the
control or with stimulation with anti-CD28 or anti-CD3 alone,
the phosphorylation of c-Jun in the nucleus was significantly increased
when CD28 and CD3 were costimulated (Fig. 4). Thus, the augmentation of PAK and
MEKK1 activity resulting from concomitant stimulation of CD3 and CD28
appeared to facilitate the phosphorylation of c-Jun most likely by the
nuclear translocation of active JNK.
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Discussion |
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Previous studies have shown that the interaction among low m.w. G proteins, arranged in a cascade, is involved in cell surface receptor-mediated cell activation (29). In budding yeast, the BUD1 G protein cycle controls bud site selection; BUD1 controls CDC42, which in turn probably controls Rho. In mammalian cells, a similar G protein cascade consisting of Ras, CDC42, Rac, and Rho appears to exist. According to the results of experiments in which activated forms of each G protein have been microinjected into fibroblasts, CDC42, Rac, and Rho behave as a linear cascade, with CDC42 activating Rac, and Rac, in turn, activating Rho (30). Ras may be upstream of these Rho family G proteins, since the negative, dominant form of Rac blocked both the induction of membrane ruffling caused by activated Ras (31) and the transforming activity of oncogenic Ras (32). Different extracellular signals are known to activate the G protein cascade at different points. For instance, in fibroblasts, platelet-derived growth factor activates Ras, followed by Rac and Rho (23, 33, 34), while other signals, such as lysophosphatidic acid, activate Rho only (31). It is probable that the same G protein cascade is conserved in T cells and integrates various activation signals. TCR stimulation also activates Ras (35); therefore, it is likely that TCR stimulates those G proteins that are regulated by Ras, namely CDC42, Rac, and Rho. Strikingly, we have also observed that CD28 stimulation initiates GDP/GTP Rac1 turnover and that combined activation of CD28 and CD3 increased this turnover by 50% (H. Hanawa and A. Ochi, manuscript in preparation). Our results indicate that CD3 or CD28 stimulation alone activates CDC42 and/or Rac; however, coligation of CD3 with CD28 greatly potentiates the activation of these G proteins. Furthermore, we found that one of the kinases downstream of Rho is activated by CD3 (data not shown). This finding seems to support the role of the Ras/CDC42/Rac/Rho pathway in T cell activation.
Is MEKK1 the kinase that is downstream of PAK?
Our data indicated that immunoprecipitated PAK derived from CD28-stimulated T cells phosphorylates the N-terminal domain (1–301) of MEKK1. These data seem to contradict a recent publication which denies the direct kinase activity of PAK against MEKK1 (36). In that study, cloned PAK3 gene is expressed in insect cells and protein is affinity-purified using GST-CDC42Hs-conjugated beads. The study also described a negative result on the kinase reaction against MEKK1 by the active form of PAK. However, it should be noted that the experimental system used by those researchers was largely different from ours; the following are likely the reasons for the discrepancy: First, PAK activity is expected to be induced after contact with GTP-bound CDC42 or Rac1. Unless the interaction of PAK with GTP-bound CDC42 is allowed prior to kinase reaction, insect cell-expressed PAK may not be able to phosphorylate MEKK1. Second, it is our experience that PAK kinase assay against MBP often has high basal activity, while the activity against the MEKK1(1–301) is largely dependent upon stimulation by CD28. Thus, PAK activity against MEKK1 may be regulated in more specific manner than that against MBP by external stimulation. These two points may explain the failure to phosphorylate MEKK1 with PAK purified with GST-CDC42Hs. Finally, it is possible that the kinase reaction of PAK may require a protein that promotes binding with MEKK1, and that the PAK-specific Ab could allow us to immunoprecipitate PAK complexed with such a "docking protein". Another possibility may be that our PAK precipitate is contaminated with a kinase that intermediates between PAK and MEKK1. Fanger reported that MEKK1, like PAK, directly interacted with Rac/CDC42 to involve the SEK/JNK pathway of activation in COS cells (37). The data indicated that PAK and MEKK1 locate in parallel as the elements of Rac/CDC42-coupled signaling. We also realized that MEKK1(1–301) used as the substrate for PAK does not include the phosphorylation sites required for MEKK1 activation (36). Therefore, it is possible that phosphorylation by PAK does not activate MEKK1 but may regulate the caspase association and the cleavage of MEKK1 as reported recently (38). Further studies will be required to elucidate the possibility of involvement of other kinases and docking proteins between the PAK and MEKK1 cascade.
How does ceramide mediate the CD28 costimulatory signal?
We have demonstrated that cell-permeable ceramide augments PAK action in CD3-stimulated T cells. Although this result may imply that ceramide could be a mediator of the CD28 costimulatory signal, ceramide is also known as a mediator of apoptosis (39, 40). Therefore, a molecular mechanism that allows ceramide to cause programmed cell death or proliferation must exist. Accordingly, at this point, three G protein-coupled mitogen-activated protein kinase cascades that result in the activation of extracellular signal-related kinase (ERK), JNK, or p38-K have been identified in mammalian cells (reviewed in 20 . Accordingly, it is possible that the multiplicity of mitogen-activated protein kinase cascades contributes, through the integration of each signal, to the determination of death or proliferation in response to various stimuli. Indeed, the activation of JNK and p38-K and the concurrent inhibition of ERK appear to be critical in the induction of apoptosis in PC12 cells after nerve growth factor withdrawal (41). Alternatively, the duration of activation of each kinase may be critical in determining the outcome; for example, the transient activation of ERK by epidermal growth factor induces cell proliferation, whereas the sustained activation of ERK by nerve growth factor induces cell differentiation in PC12 cells (42). In the case of CD28 signal transduction, the activation of ERK by CD3 and the concomitant and transient activation of JNK by the PAK cascade through the effect of ceramide may be essential to the costimulatory activity.
How does PI3K fit into the G protein cascade model?
We attempted to verify the importance of PI3K in
PAK-mediated CD28 costimulatory signaling using the specific inhibitor
wortmannin. In our study, wortmannin inhibited the activation of PAK
following CD28, CD3, or CD3 plus CD28 stimulation (Fig. 5). Presumably PI3K activation by Ras
upon CD3 stimulation was blocked by wortmannin (43). This result
suggests that wortmannin inhibits PI3K activation downstream of both
the CD3 and CD28 pathways. Therefore, since PI3K is involved in CD28
and CD3 signal transduction, wortmannin is not suitable for dissecting
the role of PI3K in the CD28 costimulatory signaling pathway.
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The CD28 costimulatory signaling pathway that interplays with TCR
signaling may be summarized as shown in Figure 6. TCR stimulation causes the activation
of Ras, and then Ras induces the activation of the Raf/MEK/ERK module.
Ras activation may induce CDC42 and/or Rac activation through the PI3K
pathway. Phosphatidylinositol 3,4,5-triphosphate delivered by PI3K
causes dissociation of the Rho GDP-dissociation inhibitor from the
GDP-binding form of CDC42 and Rac (44), following which CDC42 and Rac
become accessible to the GDP/GTP dissociation stimulator. CD28
stimulation activates PI3K and sphingomyelinase, releasing the
metabolites phosphatidylinositol 3,4,5-triphosphate and ceramide which
activate CDC42 and Rac. In addition, ceramide may activate PAK
directly. CD3 and CD28 costimulation likely result in the synergistic
activation of CDC42 and/or Rac, followed by the activation of the
PAK/MEKK1/SEK/JNK module. Furthermore, the activation of Rac may result
in a cross-talk to the Rho-coupled signaling pathway that may also
transduce the signaling of another membrane receptor.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Atsuo Ochi, John P. Robarts Research Institute, 1400 Western Road, London, Ontario N6G 2V4, Canada. E-mail address:
3 Abbreviations used in this paper: PI3K, phosphatidylinositol-3-phosphate kinase; JNK, c-Jun NH2-terminal kinase; PAK, p21-activated kinase; MEKK, MEK kinase; SEK, stress-activated protein kinase/extracellular signal-related protein kinase; ERK, extracellular signal-related kinase; GST, glutathione S-transferase; MBP, myelin basic protein.
Received for publication September 22, 1997. Accepted for publication December 29, 1997.
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Z. M. Goeckeler, R. A. Masaracchia, Q. Zeng, T.-L. Chew, P. Gallagher, and R. B. Wysolmerski Phosphorylation of Myosin Light Chain Kinase by p21-activated Kinase PAK2 J. Biol. Chem., June 9, 2000; 275(24): 18366 - 18374. [Abstract] [Full Text] [PDF]
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G. Xiao and S.-C. Sun Negative Regulation of the Nuclear Factor kappa B-inducing Kinase by a cis-Acting Domain J. Biol. Chem., July 7, 2000; 275(28): 21081 - 21085. [Abstract] [Full Text] [PDF]
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R. A. Rebres, J. M. Green, M. I. Reinhold, M. Ticchioni, and E. J. Brown Membrane Raft Association of CD47 Is Necessary for Actin Polymerization and Protein Kinase C theta Translocation in Its Synergistic Activation of T Cells J. Biol. Chem., March 2, 2001; 276(10): 7672 - 7680. [Abstract] [Full Text] [PDF]
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T. A. Fischer, A. Palmetshofer, S. Gambaryan, E. Butt, C. Jassoy, U. Walter, S. Sopper, and S. M. Lohmann Activation of cGMP-dependent Protein Kinase Ibeta Inhibits Interleukin 2 Release and Proliferation of T Cell Receptor-stimulated Human Peripheral T Cells J. Biol. Chem., February 16, 2001; 276(8): 5967 - 5974. [Abstract] [Full Text] [PDF]
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B. Su, J. Cheng, J. Yang, and Z. Guo MEKK2 Is Required for T-cell Receptor Signals in JNK Activation and Interleukin-2 Gene Expression J. Biol. Chem., April 27, 2001; 276(18): 14784 - 14790. [Abstract] [Full Text] [PDF]
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