* Departments of Biochemistry and
Physiology and Biophysics,
Clinical Research Center and
Centre de Recherche en Gérontologie et Gériatrie, Faculty of Medicine, University of Sherbrooke, Quebec, Canada
Correspondence: Dr. Gilles Dupuis, Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, Quebec, Canada, J1H 5N4. E-mail: gdupui01{at}courrier.usherb.ca
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/
-component of the CD3 complex (mAb Leu
4 or OKT 3). The levels of expression of PKC 
, ßI, ßII, and
were reduced by 90% or more in PMA-treated cells, whereas the
expression of PKC
decreased by 
30%. In contrast, the chronic
treatment with PMA increased the expression of PKC and PKC
. There
was a lack of Ca2+ response and myo-inositol
trisphosphate (IP3) production in PMA-treated cells when they were
exposed to mAb Leu 4 but the cells responded to mAb C305. The treatment
with PMA did not affect the surface expression of Ti or CD3. The
overall levels of tyrosine-phosphorylated proteins were markedly
reduced in PMA-treated cells. We investigated whether these
observations were related to defects in signal transduction related to
protein tyrosine kinase (PTK) of the src and
syk families. The electrophoretic mobilities of
p59fyn or ZAP-70 were not changed in PMA-treated cells but
p56Ick migrated as a large band of
Mr 60–62 kDa. The decreased mobility of
p56Ick was related to a state of hyperphosphorylation. The
activity of modified p56Ick was not up-regulated in
activated Jurkat cells. Our data suggest that clonotypic Ti can trigger
Ca2+ mobilization independently of conventional PKC
isoforms. Our observations further suggest that conventional PKC
isoforms are involved early in the cascade of events associated with
Jurkat T lymphocyte activation.
Key Words: protein kinase C • down-regulation • phorbol myristyl acetate • signal transduction • protein tyrosine kinase
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ß (or 
/ in some cells) clonotypic heterodimer (Ti)
associated with the invariant , , and components of CD3 and
with the homodimeric protein or its alternatively spliced 
murine isoform [2
]. Sequence data have revealed that Ti
does not possess motifs characteristic of PTK and has not been reported
to be associated with cytoplasmic proteins [2
]. However,
it has been shown that the [3
, 4
]
component of CD3 and the protein [4
5
6
] can
transduce signaling when engineered as chimeric proteins, lending
support to the notion that the Ti-associated CD3 complex and the
homodimer provide the necessary link between the occupation Ti and
signal transduction [7
].
Occupation of the TcR induces, within seconds, the activation of
protein tyrosine kinase (PTK) of the src and syk
families [8
, 9
]. Two members of the
src family of PTK, the CD4-associated p56Ick and
the CD3-associated p59fyn, are largely responsible for the
initial steps of T cell signaling [2
, 8
,
10
11
12
]. A deletion of exon 7 in the sequence of
p56Ick in the J.CaM1 Jurkat cell mutant [13
]
or knocking out the gene of p59fyn in mice
[14
] leads to impaired Ca2+ mobilization and
tyrosine-phosphorylated proteins. Targets of p56Ick and
p59fyn include tyrosine residues located in specific
sequences, the ITAM motifs that are present in each component of CD3
and in the / protein [15
]. Phosphorylation of
these motifs serves to anchor SH2 domain-containing proteins that
participate in the assembly of the cellular machinery of signal
transduction [16
]. Occupation of the TcR also triggers
the tyrosine phosphorylation and activation of phospholipase C
(PLC)-1 that generates myo-inositol 1, 4, 5-trisphosphate
(InsP3) and diacylglycerol (DAG) [17
]. InsP3 binding to
its receptor Ca2+ channel [18
] initiates the
release of Ca2+ [19
] that is essential for
the activation of cytoplasmic and nuclear enzymes, some transcription
factors [20
21
22
23
24
25
], and some PKC isozymes
[26
, 27
].
Evidence has accumulated to support the view that PKC is essential to
the process of T cell activation [reviewed in 27]. A combination of
PKC activator and Ca2+ ionophores can bypass signal
transduction through the TcR/CD3 complex [28
]. In
addition, down-regulation of PKC or the use of PKC inhibitors impairs T
cell activation [29
30
31
]. PKC can exert a positive and a
negative effect on T cell signaling. The positive regulatory action of
PKC involves the activation of c-Raf-1 [32
,
33
] and transcription factors such as AP-1
[34
] and NF-
B [35
], whereas the
negative regulatory effect of PKC is characterized by a decrease in the
activity of PLC [36
] and an increase in the rate of
internalization of the TcR/CD3 complex [37 and references therein].
The site of action of PKC in the cascade of cell signaling is not fully
elucidated, although it has been reported to act upstream of Shc
[38
] or downstream of p21ras
[34
, 39
] and p56Ick
[40
].
In this report, we showed that treating Jurkat T cells with PMA under conditions that are known to down-regulate some PKC isoforms of the conventional and novel families [41 ] did not affect the production of IP3 and the Ca2+ response to Ti-directed (ß-subunit) mAb C305. In contrast, mAb directed against CD3 (Leu 4 and OKT 3) failed to induce the production of IP3 or a Ca2+ response. In addition, our results showed that the chronic treatment of the cells with PMA was associated with a marked decrease in the patterns of tyrosine-phosphorylated proteins, the hyperphosphorylation of p56Ick and a lack of up-regulation of its activity in activated Jurkat cells. Our data suggest that clonotypic Ti can trigger Ca2+ mobilization independently of conventional PKC isoforms. In contrast, these PKC isoforms appear to be essential for the TcR/CD3-dependent up-regulation of PTK activity and the Ca2+ signal initiated by ligation of the CD3 complex.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 (Western blotting) mAb were from PharMingen (San
Diego, CA). The anti-p56Ick (immunoassays) and
anti-p59fyn mAb were from Transduction Laboratories
(Lexington, KY). Anti-ZAP-70 pAb was the generous gift of Dr
André Veillette (McGill Cancer Center, McGill University,
Montreal). mAb C305 [42
] and OKT3 (American Type Culture
Collection, Rockville, MD) were obtained from pooled culture
supernatants of the respective hybridoma and partially purified by
ammonium sulfate precipitation, as published [43
]. mAb
Leu 4 was purchased from Becton Dickinson (Montreal). The following
anti-PKC pAb were from Life Technologies (Gaithersburg, MD; PKC),
Sigma (PKCßI and ßII) and Transduction Laboratories (PKC, ,
, and ). The peroxovanadium derivative bpV(pic) was a gift from
Dr. Barry Posner (Department of Medicine, McGill University, Montreal).
The chemiluminescence detection kit used in Western blotting was
purchased from Boehringer Mannheim (Montreal) or Amersham (Montreal).
Other chemicals were from Sigma or local suppliers.
Cells
Jurkat E6.1 cells and the mAb C305-producing murine hybridoma
(C305.2) were the generous gifts of Dr. Arthur Weiss (Howard Hughes
Medical Institute, Department of Medicine, University of California,
San Francisco, CA). All the cells used in this study were maintained in
complete culture medium that consisted of RPMI 1640 medium,
heat-inactivated (56°C, 30 min) fetal bovine serum (10%, v/v),
penicillin (100 units/mL), streptomycin (100 µg/mL), and garamycin
(40 ng/mL). The cells were passaged as described [43
].
Chronic treatment of Jurkat cells with PMA
These experiments were done as before [43
]
following the protocol of Tsutsumi et al. [41
]. Jurkat
cells were cultured in the presence of PMA (160 nM) or 4-PMA (160
nM) dissolved in freshly distilled DMSO (stock solution, 160 µM) or
DMSO (55 µL, controls), for a period of 24 h, washed, and used
in the experiments.
Quantification of [Ca2+]i and IP3
Jurkat cells (5 x 106 lymphocytes/mL) in
Gey’s balanced salt solution were loaded with a DMSO solution of Fura
2/AM (1 mM) at a final concentration of 3 µM [43
].
Changes in fluorescence were recorded with a spectrofluorimeter (SPF
500C; SLM Aminco, Urbana, IL) in the slow time-based mode.
[Ca2+]i was calculated from the equation,
[Ca2+]i = KD
[(F -
Fmin)/(Fmax -
F)], after calibration with Triton X-100 and EGTA
[43
], respectively, and using a
KD of 224 nM at 37°C [44
].
Inositol trisphosphate derivatives were measured as described
[45
], with some modifications. Jurkat E6.1 cells (3 x 106 lymphocytes) were cultured for 24 h in 1 mL of
myo-inositol-free RPMI 1640 medium, 10% FBS, and
myo-[3H]inositol (5 µCi/mL). The cells were
washed, incubated for an additional 30 min in RPMI 1640 medium, and
washed. They were transferred to Hanks’ balanced salt solution,
incubated for 15 min in the presence of LiCl (10 mM), and assays were
performed. The incubations were stopped by addition of
HClO4 (5%, v/v), and 200 µL of a solution of BSA (20
mg/mL) were added. The pH was adjusted to 7.0 and the various
myo-inositol phosphate derivatives were separated on Dowex
1-X8 columns (200–400 mesh, formate form).
Western blotting and quantification
Jurkat lymphocytes (4 to 5 x 106 cells)
cultured under various conditions were solubilized by treatment with
NP-40 (1% v/v) in 200 µL of a Tris (50 mM)-sodium chloride (125 mM)
buffer (pH 7.5) containing (mM), EDTA 5, EGTA 10, phenylmethylsulfonyl
fluoride (PMSF) 1, benzamidine 10, aprotinin 0.1 mg/mL; and protein
phosphatase inhibitors (mM), sodium orthovanadate 0.5, sodium
pyrophosphate 10, and sodium fluoride 10. The mixture was briefly mixed
and left on ice for 15 min, with occasional shaking. The extract was
centrifuged (15 min, 16,000 g) and protein concentration
determined with the Bio Rad Protein Assay kit (Bio Rad, Richmond, CA).
The proteins (50 µg/well) were separated by SDS-PAGE according to
Laemmli [46
] on 10% polyacrylamide gels using a
Mini-PROTEAN II apparatus (Bio Rad). The proteins were transferred to a
nitrocellulose membrane (Hybond-C Extra; Amersham Pharmacia Biotech,
Montreal, Quebec) using a Mini Trans-Blot apparatus (Bio Rad).
Efficiency of transfer was monitored with the Ponceau red stain
[47
]. After washing with a TBS buffer (Tris, 20 mM;
NaCl, 137 mM; pH 7.6) to remove the stain, the membrane was placed in a
heat-sealable plastic bag and treated with a TBS-T buffer [TBS buffer
containing 0.1% (v/v) Tween 80] and 5% (w/v) powdered skimmed milk
to block nonspecific binding sites. The mixture was incubated by rotary
mixing with a Roto-Torque apparatus (Cole Palmer, Niles, IL) for 1 h at room temperature or overnight at 4°C. The membrane was then
washed twice (5 min each) in a bath containing TBS-T buffer and then
incubated in the same buffer with the appropriate mAb. mAb were used at
the following concentrations: 40 ng/mL (anti-p56Ick), 1
µg/mL (anti-p59fyn), 50 ng/mL (anti-ZAP-70) and a
dilution of 1:2000 (anti-phosphotyrosine), for 1 h at room
temperature. The membrane was washed twice in TBS-T buffer and
incubated with the peroxidase-conjugated secondary Ab (dilution
1:10,000). After two washes in TBS-T buffer, the antigens were revealed
by chemiluminescence. Protein bands were scanned (Scanman, Logitech,
Fremont, CA) and quantitated by volume integration using the ImageQuant
software (Molecular Dynamics, Sunnyvale, CA). An exposed area of film
outside each lane was used as background and subtracted from
experimental values.
Western blotting of PKC isoforms was performed with Jurkat cell lysates obtained by solubilization with NP-40 (1% v/v), as above, except that the Tris buffer contained a commercial mixture of protease inhibitors (Boehringer Mannheim), PMSF, the mixture of EDTA and EGTA but did not contain protein phosphatase inhibitors. Protein sizing on SDS-PAGE gels, transfer, and detection (chemiluminescence) were as above. The primary antibodies were used at 1:1000–1:2000 dilutions and the peroxidase-conjugated secondary antibodies were used at a 1:2000 dilution.
Immunoprecipitations
Jurkat cells were lysed by treatment with NP-40 (1% v/v) as
above, the volume of cell extract (300 µg proteins) was completed to
250 µL with PBS, and the required dilution of primary antibody was
added. The mixture was incubated overnight at 4°C under rotary
mixing, then mixed with a suspension (25 µL) of PBS-washed Protein
A-Sepharose beads (Sigma) and incubated under rotary mixing for 3 h at 4°C. The beads were centrifuged, washed three times (10 min
each, 4°C) with a Tris buffer (pH 7.5) containing (mM), Tris 50, NaCl
150, and NP-40 (0.05% v/v), and beef gelatin (0.25%, w/v), and then
centrifuged. The immunoprecipitates were used for Western blotting or
PTK assays.
Determination of p56Ick activity
The pellet obtained as above was washed twice (10 min, 4°C)
with kinase assay buffer (MOPS, 20 mM, MgCl2, 10 mM, pH
7.0) and enzyme activity determined as described by Flint et al.
[48
] using acid-denatured enolase [49
].
The reaction mixture was separated on 12% polyacrylamide SDS-PAGE, the
gel was dried under vacuum, and exposed to an X-Omat RP XRP-1 (Kodak)
film. The relevant protein bands were cut out and counted.
Statistical analysis of data
Data were analyzed with the Sigma Stat computer program (Jandel
Scientific, San Rafael, CA) using Student’s t-test for
paired data. Significance was set at the 95% confidence level.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ß, , and in Jurkat lymphocytes
[41
] but not [41
, 50
].
In the present study, the levels of expression of PKC isozymes in
PMA-treated cells relative to control (untreated Jurkat cells) were
determined by densitometric analysis of Western blots. Data showed that
the treatment of Jurkat cells with PMA resulted in significantly
decreased levels of PKC isozymes of the conventional and novel families
(Fig. 1
). For instance, the relative level of expression of PKCßI was
approximately 20% of control, whereas this was 10% in the case of
PKC. There was a near absence of expression of PKC (1.6%) and
PKCßII (0.7%). The PMA treatment had less of an effect on the
relative level of PKC, which was reduced by 30%. In contrast,
the relative levels of PKC (133%) and (150%) were increased in
cells treated with PMA.
 View larger version (14K): [in a new window] |
Figure 1. Western blots of PKC isozymes in Jurkat cells. The cells were left
untreated (C) or were treated (T) for 24 h in the presence of PMA
(160 nM). They were lysed, the proteins resolved by SDS-PAGE (10%
polyacrylamide), electrotransfered to a membrane, and PKC isozymes were
revealed by chemiluminescence using isozyme-specific pAb and
appropriate peroxidase-conjugated secondary pAb. The X-ray films were
scanned and quantitated by densitometric analysis. Ratios refer to
values recorded in PMA-treated cells with respect to untreated cells.
Sizes were determined by calibration with colored marker proteins.
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-PMA- and
PMA-treated Jurkat cells exposed to mAb C305 and Leu 4-PMA
(not shown) also displayed the typical biphasic Ca2+
profile when challenged with mAb C305. When the external medium was
replaced with a low (EGTA-buffered) Ca2+-containing medium
(<100 nM), the response to mAb C305 was transient in each case (not
shown). mAb Leu 4 that is directed against the and components
of CD3 [51
, 52
] triggered a
Ca2+ response in untreated Jurkat lymphocytes (Fig. 2C)
.
The amplitude of the response was smaller than in the case of cells
challenged with mAb C305. There was a similar response in cells treated
with the inactive 4-PMA analog (not shown). When the experiments
were done with cells bathing in a low (EGTA-buffered)
Ca2+-containing medium (<100 nM), the Ca2+
profile was transient (not shown). Unexpectedly, Jurkat cells treated
with PMA failed to respond to mAb Leu 4. However, they responded to a
subsequent challenge with mAb C305 (Fig. 2D)
. An absence of
Ca2+ response was also observed when mAb OKT3 was used (not
shown), suggesting that the lack of response was related to the PMA
treatment rather than the mAb used. Furthermore, cross-linking anti-CD3
mAb with a secondary Ab did not generate a Ca2+ response
(not shown). The increases in [Ca2+]i are
summarized in Table 1
.
 View larger version (17K): [in a new window] |
Figure 2. Time-course of the Ca2+ responses of Jurkat E6.1
lymphocytes to anti-Ti (C305) or anti-CD3 (Leu 4) mAb. Fura 2-loaded
Jurkat T cells (5 x 106 lymphocytes/mL) were
incubated in a 1 mM Ca2+-containing medium. Control (A, C)
corresponds to experiments performed with untreated Jurkat cells.
PMA-treatment (B, D) refers to Jurkat cells treated with PMA (24 h, 160
nM). Changes in fluorescence were recorded in a slow time-base mode at
37°C. The point of addition of each mAb is indicated by the arrows.
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View this table: [in a new window] |
Table 1. Summary of the Increases in [Ca2+]i in
Untreated, 4-PMA- and PMA-Treated Jurkat T Lymphocytes
Exposed to mAb C305 and Leu 4
|
-PMA- and PMA-treated cells (Fig. 3
), respectively. Untreated Jurkat cells or cells treated with the
inactive 4-PMA analog responded to mAb Leu 4 with a 2.08 ±
0.05 and 1.74 ± 0.05-fold (with respect to unstimulated cells)
increase in IP3 but the response was absent (1.05 ± 0.01) in
cells chronically treated with PMA (Fig. 3)
.
 View larger version (16K): [in a new window] |
Figure 3. IP3 production by Jurkat E6.1 lymphocytes. Untreated and PMA-treated
cells were loaded with myo-[3H]inositol as
described in Materials and Methods and then exposed to mAb C305 (open
bars) or to mAb Leu 4 (filled bars) for 15 min. The content of IP3 was
determined by ion-exchange chromatography and is represented by
dividing the value (cpm) of labeled IP3 measured under experimental
conditions by the value determined in unstimulated (basal) samples
(basal values were arbitrarily set to 1.0). Ratio (average ±
SEM, vertical bars) are shown in the case of untreated
Jurkat cells (control) and cells treated for 24 h with the
inactive 4-PMA analog (160 nM) or with PMA (160 nM). The figure is
representative of two separate experiments done in triplicate.
|
and antigens were not available to mAb Leu 4 (or OKT
3). However, indirect immunofluorescence cytofluorimetric analysis
(FACScan instrument; Becton Dickinson, Montreal) revealed that the
level of expression of Ti (mAb C305) or CD3 (mAb Leu 4) were unchanged
in the PMA-treated cells in comparison to untreated lymphocytes
(Fig. 4
).
 View larger version (20K): [in a new window] |
Figure 4. Cytofluorimetric analysis of Ti and CD3 expression in untreated and
PMA-treated Jurkat cells. Jurkat cells were left untreated (A, B) or
were cultured for 24 h in the presence of PMA (160 nM; C, D). The
lymphocytes were exposed to the anti-TcR ß-subunit mAb C305 (A, C) or
to the anti-CD3 mAb Leu 4 (B, D) for 30 min at 4°C, washed, and
incubated with a FITC-labeled rabbit anti-mouse Ig polyclonal Ab. A
minimum of 10,000 cells were analyzed. Data are representative of two
separate experiments.
|
-PMA- and PMA-treated Jurkat cells exposed to mAb C305
and Leu 4-PMA analog (Fig. 5
, lanes 1–3 and 7–9). In contrast, the treatment with PMA
resulted in an overall decrease of 30–40% (densitometric scanning) of
the levels of tyrosine-phosphorylated proteins (Fig. 5
, lanes 4–6).
Densitometric analysis revealed that mAb C305 and Leu 4 increased the
levels of tyrosine phosphorylation in protein bands of
Mr 30–35, 40–43, 56, 60–62, 70, 77–80,
95–100, and 105–115 kDa in untreated and in 4-PMA-treated
lymphocytes (Fig. 5
, lanes 2, 3, 8, and 9). These results were similar
to those reported in Jurkat cells exposed to anti-Ti
[13
] or anti-CD3 [13
, 53
]
mAb. The effect of PMA treatment in unstimulated cells (basal
conditions) was assessed by dividing the values of densitometric data
of PMA-treated cells by the values of untreated cells and the results
are shown in Table 2
. In this case, the ratio of protein bands of
Mr 53, 60–62 and 70 kDa was not changed but it
was decreased in the other proteins. Ratios in the case of cells
exposed to mAb C305 or Leu 4 were corrected to account for the lower
levels of tyrosine phosphorylation in PMA-treated cells. This was done
by dividing the densitometric values for each protein band in
PMA-treated lymphocytes by the ratio value for the corresponding
protein band determined under basal conditions. Stimulation with mAb
C305 was found to trigger tyrosine phosphorylation to the same extent
as in untreated cells in most of the protein bands, except for those of
Mr 56, 70, and 105–115 kDa (Table 2)
. Similar
observations were made in the case of Jurkat cells exposed to mAb Leu
4, although the level of tyrosine phosphorylation was decreased in
protein bands of Mr 40–43, 90, and 95–100 kDa.
 View larger version (76K): [in a new window] |
Figure 5. Effect of PMA treatment on the pattern of tyrosine-phosphorylated
proteins in Jurkat cells. The cells were exposed to the anti-Ti
(ß-subunit) mAb C305 or to the anti-CD3 mAb Leu 4. Jurkat T cells
were left untreated (lanes 1–3) or were cultured for 24 h in the
presence of PMA (160 nM; lanes 4–6) or inactive 4-PMA (160 nM;
lanes 7–9). The cells (5 x 106 lymphocytes) were
left unstimulated (lanes 1, 4, and 7) or were exposed to mAb C305
(lanes 2, 5, and 8) or Leu 4 (lanes 3, 6, and 9) for 2 min at 37°C.
The cells were lysed, the proteins resolved by SDS-PAGE (10%
polyacrylamide), electrotransfered to a membrane, and the
phosphotyrosine-containing proteins revealed by Western blotting with
an anti-phosphotyrosine mAb and a chemiluminescence assay kit. The
Mr of reference proteins (kDa) are shown on the
left. Data are representative of five separate experiments.
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|
View this table: [in a new window] |
Table 2. Densitometric Ratios of Tyrosine-Phosphorylated Proteins in PMA-Treated
Jurkat Lymphocytes Relative to Untreated Cells
|
1 that results in its
activation [18
]. Based on the results of Figures 2
and 5
, we investigated whether the treatment with PMA was associated with a
decrease in the level of tyrosine phosphorylation of PLC-1. Jurkat
cells were cultured for 24 h in the absence (control)/presence of
PMA (160 nM), washed, and were then left untreated or exposed to mAb
C305 or Leu 4. Results of Western blots (anti-phosphotyrosine) of
immunoprecipitates (anti-PLC-1) revealed no significant differences
in the ratio of untreated cells to PMA-treated lymphocytes (not shown).
Electrophoretic behavior and immunoassays of p56Ick
Western blotting of untreated and PMA-treated cells using an
anti-p56Ick mAb revealed that the mAb recognized two
isoforms of the protein that migrated as a major entity of
Mr 56 kDa and a faint band of 60 kDa in
untreated cells (Fig. 6A
, lane 1) and in cells treated with 4-PMA (Fig. 6A
, lane 7),
independently of stimulus (Fig. 6A
, lanes 2, 3, 8, and 9). However, the
p60–62 isoform was the major protein band found in unstimulated (Fig. 6A , lane 4) and in stimulated (Fig. 6A
, lanes 5 and 6) PMA-treated
Jurkat cells. The ratio (densitometry, average ± SEM)
of p60–62 to p56 was 0.07 ± 0.01 (n = 3) in the
case of untreated and 4-PMA-treated cells and 3.09 ± 0.39
(n = 3) in PMA-treated Jurkat lymphocytes.
 View larger version (51K): [in a new window] |
Figure 6. Western blots of p56Ick in Jurkat cells. (A) Untreated
(lanes 1–3), PMA- (24 h, 160 nM; lanes 4–6), or 4-PMA- (24 h, 160
nM; lanes 7–9) treated cells (5 x 106 lymphocytes)
were left unstimulated (lanes 1, 4, and 7) or were exposed (2 min) to
the anti-Ti C305 (lanes 2, 5, and 8) or to the anti-CD3 Leu 4 (lanes 3,
6, and 9) mAb. Cell lysates were sized by electrophoresis (10%
polyacrylamide) and electrotransfered to a membrane. Western blots were
performed with an anti-p56Ick mAb and proteins were
revealed by chemiluminescence using a peroxidase-conjugate anti-mouse
Ig pAb. The relative molecular sizes (kDa) of the protein bands are
indicated on the left. (B) Effect of the phosphotyrosine phosphatase
inhibitor peroxovanadium bpV(pic) on p56Ick. Untreated
(lanes 1–3), PMA- (24 h, 160 nM; lanes 4–6) and bpV(pic)- (15 min, 10
µM; lanes 7–9) treated Jurkat (5 x 106) cells were
left unstimulated (lanes 1, 4, and 7) or were exposed (2 min) to the
anti-Ti C305 (lanes 2, 5, and 8) or the anti-CD3 Leu 4 (lanes 3, 6, and
9) mAb. Cell lysate processing and detection were as above. The
relative molecular sizes (kDa) of the protein bands are indicated on
the left. The figure is representative of five similar experiments.
|
60 kDa). We investigated whether the shift in electrophoretic
mobility of p56Ick observed here was due to
hyperphosphorylation. Jurkat cells were treated with bpV(pic), a
peroxovanadium derivative that is a specific inhibitor of
phosphotyrosine phosphatases [55
]. Western blotting
revealed that bpV(pic) dramatically increased the proportion of the
p60–62 band (Fig. 6B
, lanes 7–9) with respect to untreated cells
(Fig. 6B , lanes 1–3), in a manner similar to the effect of the PMA
treatment (Fig. 6A
, lanes 4–6). The ratio of p60–62 to p56 in
pbV(pic)-treated cells was 5.71 ± 0.18 (n = 3).
The electrophoretic mobilities of p59fyn and ZAP-70 were
not modified in PMA-treated Jurkat cells (not shown). We investigated whether the decreased level of protein tyrosine phosphorylation observed in PMA-treated Jurkat cells (Fig. 5) could be related in part to a decreased activity of hyperphosphorylated p56Ick that would result in defective signal transduction in stimulated Jurkat cells. The activity of p56Ick was determined in immunoprecipitates from untreated and PMA-treated Jurkat lymphocytes that were left unstimulated or exposed to mAb C305 or Leu 4. Quantitation of incorporated label (Fig. 7 ) showed that mAb C305 increased the level of incorporated label approximately 1.6-fold in p56Ick and enolase (with respect to unstimulated cells, ratio set to 1.0), whereas activating the cells through the CD3 complex resulted in an approximate 2.4-fold increase in both substrates. In marked contrast, p56Ick activity was not up-regulated in the case of Jurkat cells that had been treated with PMA and exposed to mAb C305 or Leu 4.
 View larger version (24K): [in a new window] |
Figure 7. Quantification of the activity of p56Ick. The activity of
p56Ick was determined in immunoprecipitates using
[-32P]ATP and enolase as substrates. Phosphorylated
proteins were sized by SDS-PAGE (12% polyacrylamide), the gel dried
under vacuum, and then exposed to a Kodak RP XRP-1 film. Bands
corresponding to enolase (Mr 30 kDa) and
p56Ick were cut out and counted. Results are expressed as
the ratio of incorporated label in Jurkat cells exposed to cross-linked
anti-Ti ß-subunit mAb C305 (open bars) or to cross-linked anti-CD3
mAb Leu 4 (hatched bars) with respect to unstimulated (secondary pAb
only) cells (value set to 1.0, filled bars). *Significant
differences (Student’s t test for paired data;
P < 0.01) with respect to unstimulated cells. The
figure is representative of two separate experiments done in
duplicate.
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, ßI,
ßII), whereas isoforms of the novel family were significantly ()
or partially () decreased or up-regulated (). As expected
[50
], the atypical PKC isoform was not down-regulated
but its expression was up-regulated (Fig. 1)
. Up-regulation of PKC
isozymes as a result of cell treatment with PMA has been reported. For
instance, PKC is up-regulated in the T84 human epithelial cell line
exposed to a pharmacological concentration of PMA [57
],
an observation that has been attributed to a phenomenon of cross-talk
between the different isoforms of PKC [58
].
Chronic treatment of Jurkat cells with PMA did not affect the
Ca2+ response to ligation of Ti (Figs. 2)
, which showed the
same characteristic as untreated cells (Fig. 2
, Table 1
). It is
interesting that cells that responded initially to mAb C305 failed to
respond to a subsequent challenge with anti-CD3 mAb and reciprocally
(not shown), suggesting that the Ti- and CD3-dependent Ca2+
signal targeted the same InsP3-sensitive Ca2+ store. It is
interesting that PMA-treated cells failed to generate a
Ca2+ response to mAb Leu 4 (Fig. 2)
and did not produce IP3
(Fig. 3)
. The lack of Ca2+ response was independent of the
mAb used (Leu 4 or OKT 3) or their physical state (monomeric or
cross-linked) and was not due to a reduced level of expression of Ti or
CD3 (Fig. 4) . Data shown in Figures 2
and 3
suggested an absence of
up-regulation of PLC-1 activity that can be assessed by increased
tyrosine phosphorylation [18
]. Here, Western blotting
experiments failed to show significant differences (three separate
experiments) in the levels of tyrosine-phosphorylated PLC-1 in
PMA-treated and untreated cells (not shown). These observations are in
agreement with the report of Schröder et al. [59
]
who have presented indirect evidence for the involvement of a PLC
isoform different than PLC-1, which would act downstream of
PMA-sensitive PKC in activated T cells.
We investigated whether the treatment with PMA influenced the patterns of tyrosine-phosphorylated proteins in Jurkat cells exposed to anti-Ti or anti-CD3 mAb. In this connection, it has been reported that down-regulation of PKC in Jurkat cells does not prevent tyrosine phosphorylation of the components of CD3 [60 ]. Our data showed an overall decrease in the levels of tyrosine phosphorylation of most of the proteins in PMA-treated lymphocytes (Fig. 5 , Table 2 ) independently of mAb (anti-Ti or anti-CD3) stimulation. The reduced levels of tyrosine phosphorylation were not related to cytotoxicity, differences in cell growth, or unequal protein loads on the gels. These results were consistent with the interpretation that conventional (PMA-sensitive) PKC were involved at an early step in the cascade of events leading to the TcR/CD3-dependent activation of Jurkat lymphocytes.
Strauss and Weiss [13
] have generated a mutant of Jurkat
T cells (J.CaM1) that is devoid of p56Ick activity due to a
defect in alternative splicing of the p56Ick gene. This
mutant possesses some characteristics that are relevant to observations
reported here. For instance, the J.CaM1 mutant displayed a biphasic
Ca2+ response to an anti-CD3 mAb but failed to respond to
mAb C305 [13
], in contrast with our findings in the case
of PMA-treated Jurkat cells (Fig. 2)
. In addition, the mutant was
severely defective in the induction of tyrosine-phosphorylated proteins
in response to stimulation through Ti or the CD3 complex
[13
] in agreement with our data (Fig. 5)
. Close
examination of the tyrosine-phosphorylated protein patterns of Figure 5
showed that a protein band of Mr 60–62 kDa was
present to a greater extent in PMA-treated cells than in controls. This
band was not revealed by an anti-p59fyn mAb but by an
anti-p56Ick mAb that also recognized a faint band of
Mr 56 kDa (Fig. 6A)
. This result contrasted with
the observation that the same anti-p56Ick mAb recognized a
major phosphotyrosine-positive band of Mr 56 kDa
and a faint band of Mr 60 kDa in untreated
and in 4-PMA-treated cells (Fig. 6A)
. The reduced electrophoretic
mobility of p56Ick suggested a state of
hyperphosphorylation [54
] that was confirmed by treating
the cells with the phosphotyrosine phosphatase inhibitor pbV(pic)
[55
]. In this case, p56Ick shifted to a
major phosphotyrosine-positive band of Mr 60–62
kDa, accompanied by a faint band of Mr 56 kDa
(Fig. 6B)
. We tested the hypothesis that the hyperphosphorylated state
of p60–62Ick interfered with its up-regulation, thus
inhibiting the p56Ick-dependent arm of the signal
transduction pathways in a manner similar to that observed in the case
of the J.CaM1 mutant [13
]. Whereas stimulating untreated
Jurkat lymphocytes with mAb C305 or Leu 4 up-regulated
p56Ick activity, these TcR/CD3-directed mAb failed to
up-regulate the activity of the enzyme in PMA-treated cells (Fig. 7)
.
The role of PKC in T cell activation is slowly emerging. For example,
the PKC isoform that is predominantly expressed in hematopoietic and
skeletal cells plays a critical role in the regulation of
transcriptional activation of early-activation genes [reviewed in 61]
and activation-induced apoptosis in T cells [62
].
Furthermore, PKC is the only PKC isoform to localize at the contact
site between the TcR/CD3 complex of antigen-responding T cells and the
MHC of antigen-presenting cells [63
, 64
].
Other PKC isoforms are involved early in signal transduction as well.
For instance, Miranti et al. [38
] have used transfection
experiments in Cos7 cells to obtain evidence that classical and novel
PKC isoforms act upstream of the Shc adaptor protein and Schröder
et al. [59
] have recently reported that PKC is involved
in a modulation of the MAP kinase pathway associated with the
up-regulation of p56Ick in activated T cells, in agreement
with data obtained with the J.CaM1 p56Ick-deficient mutant
[40
]. Data presented here are in agreement with these
reports of an early involvement of PKC in T cell signaling but do not
exclude the possibility of further involvement of PKC downstream of
p21ras or p56Ick, as reported by several
laboratories [34
, 39
, 40
]. Our
results suggest that members of the classical and novel families of PKC
isozymes play a critical role in the tight coupling of Ti to the CD3
signal-transducing complex in Jurkat cells and the up-regulation of
p56Ick activity. However, the possibility remains that
these PKC isoforms could also be involved in the regulation of protein
tyrosine phosphatase activity associated with the regulation of
signaling in Jurkat T cells.
Received August 28, 1998; revised March 2, 2000; accepted March 4, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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