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Department of Microbiology, Michigan State University, East Lansing, MI 48824
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Abstract |
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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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T cell activation proceeds through at least three parallel and
interacting pathways (7). The process of T cell activation
is initiated by ligation of the TCR and CD4 (or CD8) by an MHC peptide
Ag complex displayed on the surface of an APC. The first steps leading
to activation involve a wave of tyrosine phosphorylation of the CD3
component of the TCR by the tyrosine kinase Lck (7). Other
tyrosine kinases (such as ZAP-70) and adaptor molecules (such as LAT
(8) and SLP-76 (9)) are rapidly recruited to
the TCR, and each becomes further phosphorylated (7, 10).
Subsequently, the MAPK pathway is activated via Grb2, SOS, and Ras. The
JNK pathway is activated via small GTPases (such as Rac/CDC42) and
requires costimulation through CD28 (11). A third major
pathway in T cell activation proceeds through activation of PLC
and
PKC and the release of Ca2+ from the endoplasmic
reticulum stores (reviewed in Ref. 12). TCR engagement
results in the activation of phosphatidylinositol PLC1, which
catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate into
inositol 1,4,5-trisphosphate and diacylglycerol. Inositol
1,4,5-trisphosphate stimulates the release of calcium from
intracellular stores, and cytoplasmic Ca2+
triggers the opening of plasma membrane calcium channel (CRAC), causing
an influx of extracellular calcium (12). It is the
extracellular Ca2+ entering through the CRAC
channel that is largely responsible for the lengthy, sustained
cytoplasmic Ca2+ levels observed after TCR
stimulation (12, 13). At the end of the pathway,
cytoplasmic calcium binds to the calcium-dependent regulatory protein,
calmodulin, and the complex activates the phosphatase calcineurin,
which activates NF-AT by dephosphorylation (12). Efficient
T cell responses to Ags also require a cosignal provided by CD28
receptor on T cells and B7 ligand on APC. CD28-B7 interaction, in
conjunction with TCR stimulation, increases the duration of the
response and augments the production of lymphokines, whereas TCR
stimulation in the absence of CD28 ligation leads to anergy
(14).
The critical role of CD45 in the TCR signaling process is in the
initial tyrosine phosphorylation events involving the tyrosine kinase,
Lck. CD45 dephosphorylates the C-terminal, inhibitory site at position
Tyr505 and thus keeps Lck in an active state
(10, 15, 16). CD45-negative cells have lowered Lck
activity in the membrane-associated pool of the PTK (17).
This paradigm predicts that the loss of CD45 results in the loss of all
signaling activity because of the lack of sufficient Lck in an
activated state. Further support for the idea that Lck is a primary
substrate for CD45 has been provided by the observation that
F1 hybrids between CD45 knockout mice and mice
expressing an activated form of Lck regain the ability to be stimulated
by Ag (18). Lymphocytes isolated from CD45 knockout mice
cannot be stimulated through the TCR (19). Alternate views
have been proposed that CD45 may also exert a negative regulatory
effect on Lck (20). In addition to Lck, CD45 is likely to
act on other substrates, such as TCR
or ZAP-70 (21, 22). In T cells CD45 is the most abundant membrane protein,
comprising about 10% of the T cell membrane protein and >90% of the
membrane-associated PTP activity (23).
The unique nature and high phylogenetic conservation of the 19-aa acidic region in the CD45 D2 domain led us to hypothesize that this insert serves as a regulatory module in lymphocyte activation. We have addressed the question of the role of the CD45 D2 in downstream signaling in T cells by performing mutational analysis of certain highly conserved sites in the 19-aa, acidic insert in the D2. The effect of mutation was determined by reconstitution of CD45- cells followed by functional analysis of various signaling pathways. A potential role for the D2 acidic insert has been identified in the regulation of the Ca2+/NF-AT pathway in resting and activated T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jurkat cells (clone E6-1) (a human acute T cell leukemia line) and CD45-deficient Jurkat cells (clone J45.01) were obtained from Dr. Gary Koretzky (University of Iowa). H45.01, a CD45-deficient variant of HPB.ALL (24), was obtained from Dr. Arthur Weiss (University of California, San Francisco, CA). The cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml streptomycin/penicillin (Life Technologies, Gaithersburg, MD), and 50 mM 2-ME (Sigma, St. Louis, MO). Cyclosporin A (CsA) was purchased from Alexis Biochemicals (San Diego, CA). HRP-conjugated goat anti-rabbit and goat anti-mouse secondary Abs were purchased from Bio-Rad (Hercules, CA). Chemiluminescent Western blotting detection reagents were purchased form Amersham (Arlington Heights, IL).
Monoclonal anti-CD3 clone 235 (IgM type) and anti-CD28 clone NE51 (IgG type) Abs were provided by Dr. Shu Man Fu (University of Virginia, Charlottesville, VA). Anti-Lck Ab was obtained from Dr. Bart Sefton (The Salk Institute, La Jolla, CA). Phospho-p44/42 MAPK Ab was purchased from New England Biolabs (Beverley, MA); MAPK Ab was purchased from Sigma. Anti-phosphotyrosine Ab (4G10) was purchased from Upstate Biotechnology (Lake Placid, NY). CD45 Ab for Western blotting was purchased from Transduction Laboratories (Lexington, KY), and CD45 Ab for immunoprecipitation was purified from 9.4 hybridoma (American Type Culture Collection, Manassas, VA). Fluorescent CD45 Ab, PE anti-human CD45, was purchased from PharMingen (San Diego, CA). Anti-ZAP 70 Ab and anti-PLC Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
DNA constructs and site-directed mutagenesis
The expression vector hLCA-NEO 3, which contained the intact wild-type CD45 cDNA under the control of SFFV promoter, was provided by Dr. Arthur Weiss. The construct expresses full-length CD45 with the low m.w. form (RO isoform) extracellular domain. NF-AT-luciferase vector, containing the IL-2 minimal promoter and three copies of NF-AT-1 binding sites, was provided by Dr. G. Crabtree (Stanford University, Stanford, CA). PRSV-ßgal reporter vector was purchased from Clontech (Palo Alto, CA).
Mutations of the four serine residues in the acidic insert of the CD45 D2 domain were performed using QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) as described by the manufacturer. Desired mutations (underlined) were incorporated into a pair of oligonucleotide primers, each complementary to opposite strands of the parental DNA template. The primers used for Ser to Ala (815, 818, 819, and 823 in the CD45 RO form) mutagenesis were: 5'-GTGAGCATGATGCAGATGAAGCCGCTGATGATGACGCTGATTCAGAGG-3' and 5'-CCTCTGAATCAGCGTCATCATCAGCGGCTTCATCTGCATCATGCTCAC-3'. The mutagenesis of Ser to Glu (815, 818, 819, and 823) was performed in two steps; each step substitutes two Ser with Glu. The primers were 5'-GTGAGCATGATGAGGATGAAGCCGAGGATGATGACGCTG-3' and 5'-CAGCGTCATCATCCTCGGCTTCATCCTCATCATCCTCAC-3' for Ser to Glu (815 and 819) replacement, and 5'-GATGAGGATGAAGAGGAGGATGATGACGAGGATTCAGAGGAACC-3' and 5'-GGTTCCTCTGAATCCTCGTCATCATCCTCCTCTTCATCCTCATC-3' for Ser to Glu (818 and 823) replacement. Each pair of primers was extended by Pfu DNA polymerase during a short temperature cycling (95°C for 30 s, 55°C for 1 min, and 65°C for 13.5 min (2 min/kb of plasmid length), 18 cycles), and the parental DNA template was then digested by DpnI endonuclease. Mutants were selected after the synthesized DNA was transformed into Escherichia coli XL1-Blue and later verified by sequencing.
Deletion of the 19-aa acidic region (position 808–826) was performed using the GeneEditor In Vitro Site-Directed Mutagenesis System (Promega, Madison, WI) as described by the manufacturer. Briefly, dsDNA template was denatured and annealed with the phosphorylated mutagenic oligonucleotides and the modified drug selection oligonucleotides. The mutagenic oligonucleotides with the desired deletion of 57 nucleotides were hybridized to the template DNA at a higher molar ratio compared with the drug selection oligonucleotides. The mutant strand DNA was synthesized and ligated in vitro and transformed into the repair minus strain of E. coli BMH 71–8 mutS. Transformants in the presence of GeneEditor antibiotic selection mix contained enriched mutant DNA and were used to purify plasmid DNA. The isolated DNA was transformed into E. coli JM109 to segregate mutant and wild-type plasmids. Transformants were further analyzed by sequencing. The mutagenic oligonucleotide used for the acidic region deletion was: 5'-GCCACTTAAA CATGAGCTGGAAATGGAACCAAGCAAATACATAATGCATC-3'.
Transient transfection and luciferase assay
Transient transfection of Jurkat T cells was performed using DMRIE-C reagent (Life Technologies) as described by the manufacturer. Typically, 6 µl of DMRIE reagent was mixed with 3 µg of NF-AT-luciferase reporter DNA, 4 µg of hLCA.neo CD45 DNA, and 1 µg of pßgal reporter (Clontech, Palo Alto, CA) DNA for 45 min at room temperature to form lipid-DNA complexes. Each transfection reaction contained 2 x 106 cells in 200 µl of serum-free medium, which was incubated with the lipid-DNA complex for 5 h at 37°C followed by addition of growth medium containing 15% serum. Transfected cells were treated with anti-TCR Abs 2 days post-transfection and were collected 8 h after stimulation. ß-Galactosidase activity was measured using a Turner ED 20e luminometer after incubating the lysate with the chemiluminescent substrate Galacton-Star (Clontech) for 1 h at room temperature, and the luciferase activity of the cell lysate was measured by luciferase reporter gene assay kit (Roche, Indianapolis, IN).
Stable transfection and clone selection
CD45-negative HPB cells (1 x 107) were washed twice with cold PBS and resuspended in 500 µl of RPMI 1640 medium. Mutant and wild-type CD45 cDNA (20 µg) were linearized with XhoI digestion and mixed with the cell suspension. Electroporation was performed using the Bio-Rad Gene Pulser at 250 V and 960 µF, followed by incubation in RPMI 1640 medium with 10% FCS. After 48 h, transfected cells were placed into RPMI 1640 medium containing 2 mg/ml geneticin (Life Technologies) and seeded into 96-well plates by limited dilution. Drug-resistant single colonies were screened by Western blotting with CD45-specific Ab (Transduction Laboratories). Positive clones were further confirmed by FACS analysis using PE-anti-human CD45 (PharMingen). Clones that expressed comparable levels of CD45 surface protein were chosen for further functional analysis.
FACS analysis
For CD45 staining, stable transfectant cells (1 x 106) were collected and washed twice with PBS (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, and 1 mM KH2PO4, pH 7.4) supplemented with 2% FBS, and resuspended in 50 µl of PBS/2% FBS. PE-anti-human CD45 Ab (20 µl) was added to the cell suspension and incubated at 4°C in the dark for 30–45 min. Labeled cells were washed twice with PBS/2% FBS and resuspended in 50 µl of filtered PBS/2% FBS. For fixation of the stained cells, 20 µl of 1% formaldehyde was added, and the samples were stored at 4°C. Staining of H45.01 cells, which are CD45 negative, was used as a negative control. Each sample was resuspended in 300 µl of PBS/2% FBS before FACS analysis. For CD3 staining, primary anti-CD3 mAb (clone 235) was diluted in PBS/2% FBS (1/500) and added to 1 x 106 cells. After incubation at 4°C in the dark for 30 min, the cells were washed twice with PBS/2% FBS and incubated with diluted secondary Ab, FITC-goat anti-mouse Ab (Sigma), for another 30 min at 4°C in the dark. Samples were then washed, fixed, and subjected to FACS analysis as described above. Cells stained with secondary Ab alone were used as negative controls. FACS was performed with a Becton Dickinson Vantage FACS (San Jose, CA) at the Michigan State University Flow Cytometry Facility.
PTP assay
Stable transfectant cells (5 x 107) were washed twice with 20 mM Tris (pH 8.0) and 137 mM NaCl and lysed for 30 min at 4°C in Nonidet P-40 lysis buffer containing 1% Nonidet P-40 (Pierce, Rockford, IL), 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 5 mM EDTA, 2 mM PMSF, 0.23 U/ml aprotinin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin A, and 10 µg/ml DNase I. After removal of nuclei by centrifugation, the lysate supernatant was incubated with anti-CD45 (clone 9.4, American Type Culture Collection) for 30 min followed by addition and incubation with GammaBind Plus Sepharose (Pharmacia Biotech, Piscataway, NJ) for another 30 min at 4°C. The immunoprecipitates were washed once with 20 mM Tris (pH 8.0) and 137 mM NaCl, once with LiCl, once with 20 mM Tris (pH 8.0), and once with 1x PTP buffer (25 mM HEPES (pH 7.3), 5 mM EDTA, and 10 mM DTT) and were resuspended in 40 µl of 1x PTP. The PTP assay (Promega) was conducted at 30°C, and each assay contained 5 µl of tyrosine phosphopeptide substrate END(pY)INASL. After incubation for 30 min, the supernatant was added to 96-well plates, and the reaction was stopped by a mixture of malachite green and molybdate. The amount of released free phosphate was measured by the absorbance of the dye-phosphate complex at 600 nm wavelength using a microtiter plate reader (Molecular Devices, Eugene, OR), and the data were analyzed using SoftMaxPro (version 2.1.0) software. The Sepharose G-bound CD45 was boiled in SDS sample buffer, loaded in 10% SDS-polyacrylamide gel, and transferred to nitrocellulose membrane after electrophoresis. Western blotting was performed to confirm equal amounts of CD45 in the immunoprecipitates.
TCR stimulation and detection of MAPK activation
Cells were washed twice with PBS, resuspended into RPMI 1640 medium at 1 x 108/ml, and incubated at 37°C for 5 min. Anti-CD3 mAb (clone 235, 1/500) and anti-CD28 (clone NE51, 1/1000) Ab were added to the cell suspension and incubated for 5 and 30 min, respectively. Stimulated cells were washed with PBS once and lysed in 1% Nonidet P-40 lysis buffer containing PTP inhibitors (2 mM sodium orthovanadate and 4 mM sodium molybdate) at 4°C for 30 min. The lysates were centrifuged at 12,000 rpm at 4°C to remove nuclei. Forty micrograms of each lysate was boiled with SDS sample buffer and loaded onto 10% SDS polyacrylamide gels. Lysate proteins were transferred onto nitrocellulose membrane following electrophoresis and blotted with anti-phospho-MAPK Ab. The same membrane was treated with membrane-stripping buffer (2% SDS, 0.7% 2-ME, and 62.5 mM Tris (pH 6.7)), and specific proteins were detected with anti-MAPK and anti-CD45 Abs.
In vitro kinase assay
Stable transfectant cells (5 x 106
cells) were stimulated with Abs and lysed in 1% Nonidet P-40 lysis
buffer as described above. Each lysate was incubated with
anti-ZAP-70 or anti-Lck Abs followed by the addition of protein
A agarose (Life Technologies). Immunoprecipitated ZAP-70 and Lck PTKs
were washed once with PBS, twice with 0.5 M LiCl (phosphorylation,
7.4), once with 20 mM Tris (pH 7.4), and once with 1x kinase buffer
(20 mM Tris (pH 7.4), 10 mM MnCl2, and 0.07%
2-ME) and were resuspended in 50 µl of 1x kinase buffer. The kinase
assay was performed by adding 10 µM of cold ATP and 5 µCi of
[-32P]ATP (3000 Ci/mmol; NEN Life Science
Products, Boston, MA) and was incubated at 30°C for 20 min. The
reaction was stopped by adding 1 ml of washing buffer containing 20 mM
Tris (pH 7.5), 150 mM NaCl, and 20 mM EDTA. The pellet was boiled in
SDS sample buffer and subjected to electrophoresis on 10%
SDS-polyacrylamide gel followed by transfer onto nitrocellulose
membrane. Autophosphorylation of ZAP-70 and Lck PTKs was detected by
PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA), and the
same membrane was incubated with anti-ZAP-70 or anti-Lck Ab to
assure the presence of equal amount of proteins in each sample.
Calcium flux analysis
Cells (1 x 107) were washed with cell loading medium (RPMI 1640, 2% FCS, and 25 mM HEPES, pH7.4) and incubated with 1.5 µM indo-1/AM (Molecular Probes) at 37°C for 1 h in the dark. The loaded cells were washed twice with DMEM (Life Technologies) plus 2% FCS and resuspended in 10 ml of cell loading medium. Aliquots of 1 ml of loaded cells were analyzed relative to time by flow cytometry using an EPICS Elite flow cytometer (Coulter, Hialeah, FL). The ratio of fluorescence at 420 nm to that at 510 nm was used to indicate changes in calcium flux. Some samples were treated with 1 µM ionomycin or 8 mM EGTA, and stimulated with anti-CD3 and/or anti-CD28 Abs (1 µg each/106 cells/ml). The flow cytometry data were analyzed using WinMDI software written by Joe Trotter (The Salk Institute, Flow Cytometry Laboratory).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown previously that Ser phosphorylation of the D2 domain
acidic insert by CK2 was blocked by mutation of Ser at positions 815,
818, 819, and 823. To study the role of the acidic insert Ser residues
in CD45-mediated lymphocyte signal transduction, mutations were
incorporated into expression vector hLCA.neo as shown in Fig. 1
. These mutants were designated: S/A,
containing the simultaneous replacement of four Ser at positions 815,
818, 819, and 823 to Ala; S/E, containing the simultaneous replacement
of the four Ser to Glu; and 
19, deletion of aa 808–826. The
C667S mutant in the D1 domain alters the
essential cysteine residue and abolishes all PTP activity of CD45.
Deletion of the entire D2 domain, D2, was also prepared, but failed
to express at the protein level, although mRNA was detected by Northern
blot (data not shown). Two other control mutants used were
Ser789Ala and Ser789Glu
(not shown), which is in the substrate interaction loop at the opening
of the putative active site (25, 26).
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Mutant CD45 cDNA was transiently expressed into CD45-deficient
Jurkat T cells (J45.01) together with NF-AT-luciferase or
AP-1-luciferase reporter constructs. pßgal reporter DNA was also
cotransfected to normalize for transfection efficiency. Two days
post-transfection, cells were incubated with anti-CD3 and
anti-CD28 Abs for 8 h, and luciferase activity was determined
using a luminometer (Fig. 2). Stimulation
of J45.01 cells with anti-CD3/CD28 Abs did not result in NF-AT
luciferase activation, confirming that efficient CD45 expression was
required for TCR-mediated T cell activation (Fig. 2, vector only).
Introduction of wild-type CD45 into J45.01 cells restored the capacity
of the cells to activate NF-AT-luciferase reporter upon TCR engagement
(Fig. 2, wt), whereas introduction of the PTP inactive form of CD45 did
not rescue signaling (Fig. 2, C667S). When CD45
with S/A mutations in the D2 domain acidic region was transiently
expressed, NF-AT luciferase was activated by the anti-CD3/CD28
stimulation to a similar extent as the wild type transfectant (Fig. 2, S/A). However, an elevated basal level of NF-AT activity was observed
in S/A transfectants. A smaller, but reproducible, basal elevation of
NF-AT activity was also observed for S/E and 19 transfectants,
without alteration in the overall magnitude of response to
anti-CD3/CD28 stimulation (Fig. 2, S/E and 19). The S/A mutant
exhibited an average 9- to 10-fold increase in basal activity, while
intermediate values were observed for the S/E mutant and 19 mutant
(5- and 3-fold increases, respectively; Fig. 2B). The
success of transfection for each mutant was judged to be at the same
efficiency, because all the mutant constructs exhibited comparable
overall stimulation with anti-CD3/CD28 (Fig. 2A), and
comparable ß-galactosidase activity was detected in each transfectant
(data not shown). Although the cells were clearly functionally
reconstituted, FACS analysis using PE-anti-CD45 Ab staining of the
transiently transfected populations indicated CD45 expression in only
about 5–8% of cells, compared with empty vector transfection (data
not shown). Parallel experiments performed using an AP-1-luciferase
demonstrated only a small activation after stimulation regardless of
the CD45 expression (data not shown).
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A). The basal NF-AT
activity in Jurkat cells was not elevated by the transfection of S/A
mutant CD45 compared with that observed upon transfection of J45.01
cells (Fig. 3B). This result indicates that the effect of
S/A mutant was overcome by the presence of wild-type CD45 in Jurkat
cells. NF-AT is activated by the serine/threonine phosphatase,
calcineurin (PP2B), which dephosphorylates cytoplasmic NF-AT and allows
its translocation into the nucleus. CsA (100 ng/ml), an inhibitor of
calcineurin, completely blocked the activation of NF-AT by TCR
engagement, and it also abolished the elevated basal NF-AT activity in
CD45 S/A transfectants (Fig. 3C). Thus, the elevation of
NF-AT in CD45 S/A transfected resting T cells depended on the function
of the Ca2+ pathway and calcineurin.
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D). At the same time, the basal elevation of NF-AT
activity in CD45 S/A transfectant decreased to background levels,
suggesting that the effect of CD45 S/A expression was due to a loss of
regulation of calcium influx. Stable transfectants expressing mutant CD45
The experiments described above suggested that the alterations caused by mutation of CD45 were primarily in the Ca2+/NF-AT pathway. However, this was difficult to study further in a Jurkat transient transfection system because the overall expression of CD45 was quite low. We therefore endeavored to develop a system in which stable cell lines expressing mutated CD45 could be evaluated for multiple TCR signaling pathways and for Ca2+ flux under various experimental conditions.
CD45-negative H45.01 cells were transfected with mutant CD45 constructs
by electroporation, and single, stable transfectants were selected by
limiting dilution and G418 resistance. Selected clones were screened by
immunoblotting with anti-CD45, and CD45 expression was confirmed by
flow cytometric analysis of PE-anti-CD45-labeled cells (Fig. 5A). For each CD45 mutant construct, at least three
individual clones expressing comparable levels of CD45 protein were
chosen for functional analysis. CD45-expressing H45.01 clones were also
stained with anti-CD3 (235 mAb) and FITC-goat anti-mouse Ab and
subjected to flow cytometric analysis. Each stable transfectant used
was shown to express essentially the same level of CD3 (Fig. 4B). As further confirmation
of CD45 expression, the CD45 PTP activity of stably transfected cells
was compared (Fig. 5). Wild-type and
mutant CD45 were immunoprecipitated from individual transfectant clones
(5 x 107 cells) and subjected to tyrosine
phosphatase assay using tyrosine phosphopeptide END(pY)INASL as
substrate. Immunoprecipitates obtained from
CD45- H45.01 cells were used as negative
controls. The acidic insert mutants were shown to have comparable PTP
activity as wild-type transfectants (Fig. 5), while the
C667S mutant lacked PTP activity. The data
presented here are representative of different individual clones.
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One of the earliest signaling events upon TCR stimulation is the
activation of TCR-associated PTKs. The activated PTKs phosphorylate
diverse downstream substrates, which leads to activation of the MAPK
pathway and the Ca2+/calcineurin/NF-AT pathway.
To determine the effect of the CD45 mutation we compared the
autophosphorylation and kinase activities of Lck and ZAP-70 after TCR
stimulation. All the acidic insert mutations (S/A, S/E, and 19)
exhibited similar activation patterns for both kinases, which peaked at
5 min after stimulation and returned to basal level by 30 min (data not
shown). In addition, the overall tyrosine phosphorylation patterns of
the CD45 mutant clones after stimulation were not detectably different
from that of wild-type CD45 (data not shown). We next examined the
activation of p44/42 MAPK in the mutant transfectants (Fig. 6). TCR-mediated activation of MAPK was
detected by a phospho-MAPK-specific Ab that recognizes
catalytically activated Erk1 and Erk2. Minutes after TCR
cross-linking, phospho-MAPK was detected in wild-type CD45
transfectants, while CD45- H45.01 cells failed
to induce the phosphorylated form of MAPK (Fig. 6A). All
three mutants of the D2 acidic region appeared to activate p44/42 MAPK
to approximately the same extent as wild-type CD45 after stimulation
for 5 min, followed by a decrease after 30 min of stimulation. We
concluded, therefore, that mutations in the D2 acidic region did not
detectably affect the MAPK pathway.
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CD45- H45.01 cells did not exhibit any
detectable Ca2+ flux upon stimulation (Fig. 7C). The intracellular calcium concentration of the
wild-type CD45 transfectant increased within a few minutes after TCR
cross-linking, peaked at about 5–6 min, and then gradually decreased
to the baseline (Fig. 7A). When the CD45 S/A mutant
transfectants were stimulated by anti-TCR, the calcium flux
observed was of the same magnitude as that seen for cells expressing
the wild type CD45 (Fig. 7B). However, the calcium flux in
the S/A mutant appeared more sustained than that observed for the
wild-type transfectant, and after longer incubation (up to 2 h) it
was observed that the CD45 S/A mutant did not recover to the baseline
(Fig. 7B plus additional clones shown in Fig. 7E). Ca2+ flux in cells expressing
wild-type CD45 returned to baseline during this incubation (Fig. 7A and additional clones shown in Fig. 7D). When
EGTA was added to the medium containing the CD45 S/A clone, the
sustained calcium signal returned to baseline immediately (Fig. 7E), indicating that the abnormally sustained
Ca2+ level required extracellular
Ca2+. This result suggests that the elevated
calcium level in CD45 S/A mutant cells results from the loss of
regulation of a plasma membrane calcium channel. Interestingly,
analysis of the CD45 S/E and 19 mutants showed that these clones
exhibited a calcium flux that was intermediate and close to the
wild-type pattern (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CD45 acidic insert mutant proteins supported TCR-mediated activation. The only mutant that did not support activation was the C667S mutant that lacked PTP activity. For the stable mutants, the activations of Lck, Zap-70, and MAPK were similar regardless of whether the cells expressed wild-type CD45 or the mutated CD45 S/A. For these signaling molecules, the expression of CD45 S/A did not affect the magnitude or the duration of activation. In addition, the overall patterns of tyrosine-phosphorylated proteins after stimulation were similar regardless of the CD45 form expressed. Examination of the Ca2+ pathway, however, revealed that the CD45 S/A mutant significantly altered the down-regulation of extracellular Ca2+ influx through membrane channels. Cells expressing CD45 S/A exhibited a sustained Ca2+ flux after activation that lasted long beyond the point when the Ca2+ levels of wild-type CD45-expressing cells returned to basal levels. The sustained Ca2+ level in CD45 S/A-expressing cells immediately returned to normal when EGTA was added to the medium. Because EGTA chelates external Ca2+ and does not enter cells, we conclude that the extended flux was due to a loss of ability to down-regulate Ca2+ influx and not to the release of internal stores of Ca2+, which are usually depleted quickly after TCR activation. CD45 could act directly on CRAC channels to prevent their inactivation, or there could be other intermediates involved (such as Lck). Previous evidence supports the idea that inactivation of Ca2+ channels is a regulated event. For example, the inactivation of L-type channels is regulated by Ca2+/calmodulin and depends on specific short sequences in the channel cytoplasmic tail (27). Because little is known about the physical nature of CRAC channels in lymphocytes, the interaction with CD45 described in this report may provide novel approaches to the study of Ca2+ flux in T cells.
Corroboration of our results with the H45.01 stable clones was provided by the observation of a similar deregulation of the Ca2+/NF-AT pathway by an independent, transient transfection system. In this system we transiently transfected a CD45- Jurkat line, J45.01, with the acidic insert mutants, and the effect on downstream pathways was evaluated. The Ca2+/NF-AT pathway was deregulated in a way that led to aberrantly high basal levels of active NF-AT in resting cells. The elevated basal level was completely abrogated by treatment with CsA, showing that the calmodulin/calcineurin pathway was involved in generation of the observed NF-AT activation. In addition, treatment of the cells with EGTA, which chelates external Ca2+, completely suppressed the high basal activity, suggesting that an influx of external Ca2+ is necessary to maintain the aberrant NF-AT levels. Increased levels of Ca2+ were not detectable in the transient system due to the low number of transfected cells.
The results obtained using the two model systems described in this report support each other, but the same measurements were not possible with both transfection systems. In addition, the cell lines used may also differ somewhat with regard to signaling pathways. For example, NF-AT activity measurements in transfected H45.01 cells might not have been successful due to the low expression of NF-AT in these cells (28). Other reports have suggested that the HPB cell line may exhibit low activation of PLC and PKC upon TCR stimulation (13, 29). Despite these differences, the results we obtained were quite complementary, and both transfection systems supported the conclusion that CD45 plays a role in the regulation of Ca2+/NF-AT signaling pathways.
This conclusion is also supported by the observations of others concerning the role of CD45 in the activation of T cells. For example, Leitenberg and Bottomly, who examined the effect of anti-CD45 Abs on Ca2+ flux during TCR activation, concluded that CD45 played a role in the regulation of influx of extracellular Ca2+ (30). In addition, experiments in which the D2 domain of CD45 was replaced with the homologous D2 of LAR PTP suggested that the CD45 D2 domain was required for Ca2+/NF-AT-dependent IL-2 secretion (31). The observation that the D2 domain of LAR (which does not contain the 19-aa insert region) could not substitute the D2 domain of CD45 suggests that the insert was essential for CD45 function.
The D2 domain of CD45 is also necessary for the full expression of D1 activity. Mutagenesis studies have revealed that the intact D2 domain was required for the enzymatic activity of CD45 expressed in rabbit reticulocyte in vitro transcription/translation system (5). Our studies have also shown that the intact D2 domain was essential for CD45 expression in H45.01 cells used in the current study (unpublished data). Most studies of the expression of the cytoplasmic domain of CD45 have shown that the D2 domain was essential for the expression of D1 PTP activity, and deletion of the D2 19-aa insert reduced activity of the D1 (5). More recently, recombinant CD45 D1 domain alone was expressed in a bacterial system and was shown to have enzymatic activity, but the presence of the D2 domain was found to increase the thermostability of D1 domain (32). Taken together these studies and the current report show that the D2 domain is important in the expression and function of CD45.
Further work will be needed to determine the exact mechanism by which the intact acidic insert is required for maintaining Ca2+ homeostasis in a basal state. Ser to Ala mutation exhibited the largest effect in our studies, suggesting the hydroxyl groups of the Ser residues in the wild-type protein allowed CD45 to regulate the levels of Ca2+. Such regulation could occur by interaction of CD45 with the CRAC channel or by altering substrate selectivity of CD45. The recently reported crystal structure of LAR (33) suggests that the acidic insert could extend to the region of the D1, where it could alter the accessibility of substrates to the active site. Introduction of CD45 S/A in J45.1 cells could have resulted in higher Ca2+/NF-AT levels by not down-regulating the Ca2+ levels after fluctuations during normal activity (such as the cell cycle or stimulated growth after addition of serum). A similar mechanism could function to produce sustained Ca2+ levels after TCR ligation in HPB cells. Because the physical nature of the Ca2+ channels is unknown, it will be important to direct future experiments toward elucidating the mechanism of interaction of CD45 with Ca2+ regulation in the cell.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Walter J. Esselman, 344 Giltner Hall, Department of Microbiology, Michigan State University, East Lansing, MI 48824-1101. E-mail address:
3 Abbreviations used in this paper: PTP, protein tyrosine phosphatase; PTK, protein tyrosine kinase; PVDF, polyvinylidene difluoride; CsA, cyclosporin A; CK2, casein kinase 2; CRAC channels, calcium release-activated calcium channels; ER, endoplasmic reticulum; Lck, p56lck protein; MAPK, mitogen-activated protein kinase; PLC, phospholipase C; PKC, protein kinase C.
Received for publication September 22, 1999. Accepted for publication December 20, 1999.
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chain. Proc. Natl. Acad. Sci. USA 91:10928. in vivo. J. Biol. Chem. 273:33856.This article has been cited by other articles:
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  P. Contini, M. Ghio, A. Merlo, A. Poggi, F. Indiveri, and F. Puppo Apoptosis of Antigen-Specific T Lymphocytes upon the Engagement of CD8 by Soluble HLA Class I Molecules Is Fas Ligand/Fas Mediated: Evidence for the Involvement of p56lck, Calcium Calmodulin Kinase II, and Calcium-Independent Protein Kinase C Signaling Pathways and for NF-{kappa}B and NF-AT Nuclear Translocation J. Immunol., December 1, 2005; 175(11): 7244 - 7254. [Abstract] [Full Text] [PDF]
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 Y. Wang and P. Johnson Expression of CD45 Lacking the Catalytic Protein Tyrosine Phosphatase Domain Modulates Lck Phosphorylation and T Cell Activation J. Biol. Chem., April 8, 2005; 280(14): 14318 - 14324. [Abstract] [Full Text] [PDF]
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G. A. Robichaud, B. Barbeau, J.-F. Fortin, D. M. Rothstein, and M. J. Tremblay Nuclear Factor of Activated T Cells Is a Driving Force for Preferential Productive HIV-1 Infection of CD45RO-expressing CD4+ T Cells J. Biol. Chem., June 21, 2002; 277(26): 23733 - 23741. [Abstract] [Full Text] [PDF]
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B. Barbeau, G. A. Robichaud, J.-F. Fortin, and M. J. Tremblay Negative Regulation of the NFAT1 Factor by CD45: Implication in HIV-1 Long Terminal Repeat Activation J. Immunol., September 1, 2001; 167(5): 2700 - 2713. [Abstract] [Full Text] [PDF]
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S. F. Greer, Y.-n. Wang, C. Raman, and L. B. Justement CD45 Function Is Regulated by an Acidic 19-Amino Acid Insert in Domain II That Serves as a Binding and Phosphoacceptor Site for Casein Kinase 2 J. Immunol., June 15, 2001; 166(12): 7208 - 7218. [Abstract] [Full Text] [PDF]
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TCR,
arrow), and subjected to flow cytometric analysis. The ratio of
fluorescence at 420 nm to that at 510 nm was used to indicate changes
in calcium flux. D, Two clones of H45.01 cells
expressing wild-type CD45 were stimulated with anti-CD3/CD28, and
intracellular calcium levels were measured for about 10–15 min and
then 2 h later (x-axis scale break).
E, Three independent clones of H45.01 cells expressing
CD45 S/A were stimulated, and intracellular calcium levels were
measured for 15 min and then 2 h later. Additions of
anti-CD3/CD28 Abs (