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(Received for publication, March 16, 1995; and in revised form, August 25, 1995) From the
The CD45 family of transmembrane protein-tyrosine phosphatases
plays a crucial role in the regulation of lymphocyte activation by
coupling activation signals from antigen receptors to the signal
transduction apparatus. Multiple CD45 isoforms, generated through
regulated alternative mRNA splicing, differ only in the length and
glycosylation of their extracellular domains. Differential distribution
of these isoforms defines subsets of T cells having distinct functions
and activation requirements. While the requirement for the
intracellular protein-tyrosine phosphatase domains has been documented,
the physiological role of the extracellular domains remains elusive.
Here we report the generation of CD45-antisense transfected Jurkat T
cell clones that lack CD45 or have been reconstituted to uniquely
express either the smallest, CD45(0), or the largest, CD45(ABC),
isoform. These cells exhibited marked isoform-dependent differences in
IL-2 production and tyrosine phosphorylation of cellular proteins,
including Vav after anti-CD3 stimulation. These results demonstrate
that the distinct CD45 extracellular domains differentially regulate T
cell receptor-mediated signaling pathways. Furthermore, these findings
suggest that alterations in CD45 isoform expression by individual T
cells during thymic ontogeny and after antigen exposure in the
periphery directly affects the signaling pathways utilized.
Activation of resting T lymphocytes through the T cell receptor
(TCR) ( While the requirement for the intracellular
PTPase domains has been
documented(10, 11, 12, 13) , the
function of the CD45 extracellular domain in lymphocyte signal
transduction remains a major unresolved issue. In humans, five CD45
isoforms, ranging in size from 180-220 kDa, are generated by the
regulated alternative mRNA splicing of three exons, encoded by a single
gene(14, 15, 16) . The alternatively spliced
exons, commonly referred to as A, B, and C, are located near the 5` end
of the gene and give rise to isoforms that differ only in their
extracellular regions. Individual lymphocytes simultaneously express
more than one CD45 isoform(17, 18) . However, the
expression of certain isoforms is highly regulated, resulting in their
differential expression on lymphocytes of different lineage (e.g. T versus B cells), as well as on distinct functional
subsets of T cells(19, 20, 21, 22) .
Furthermore, individual T cells alter their isoform expression in a
highly regulated manner during thymic selection and upon antigen
exposure in the
periphery(18, 23, 24, 25, 26) .
The tight regulation of CD45 isoform expression by lymphocytes having
distinct functions argues that these differences are likely to be of
biologic importance. However, attempts to study the role of individual
CD45 isoforms in signaling have been severely hampered by great
difficulty in re-expressing different single intact CD45 isoforms into
the same cellular background. Recent studies have clearly
demonstrated that TCR-mediated signaling can be reconstituted in
CD45
Rabbit polyclonal Ab to Vav was developed
by immunization of rabbits with a synthetic peptide containing residues
575-594 of (murine) Vav. Immunoprecipitation of a 95-kDa band
with the antiserum was specifically blocked by addition of the
immunizing peptide to the lysate mixture (data not shown).
Figure 2:
IL-2
secretion by Jurkat versus CD45
Like peripheral T cells, the Jurkat human T cell leukemia
line normally expresses CD45 at high levels, and individual cells
express multiple isoforms
simultaneously(17, 18, 19) . To examine the
role of CD45 and its individual isoforms, free from the potential
confounding influences of unknown mutations, we directly targeted
endogenous CD45 expression by stable transfection of a plasmid
construct (AS-CD45) expressing an antisense RNA directed at a 270-base
pair region of genomic CD45 just upstream from the coding region. (Fig. 1A). Of the several independent CD45
Figure 1:
A, schematic representation of DNA
inserts encoding antisense CD45 RNA (AS-CD45), CD45(0), and CD45(ABC)
isoforms. The arrow under each insert indicates the direction
of transcription once inserted into the respective expression vectors.
Sense and antisense constructs have minimal overlap. B,
representative immunofluorescence analysis of cell surface expression
of various markers on Jurkat, CD45
J-AS-1
was then stably transfected with CD45 cDNA constructs modified to
minimize overlap with AS-CD45 antisense RNA and encoding either the
smallest isoform, denoted CD45(0), lacking alternative exons, or the
largest isoform, denoted CD45(ABC), which includes all three
alternative exons. These isoforms best exemplify differential
distribution on T cell subsets having distinct functions and activation
preferences(19, 21, 23) . Each of the
CD45 Current
evidence indicates that CD45 regulates the activity of proximal
components of the signaling apparatus such as the Src family PTKs, Lck
and Fyn, and, presumably, their
substrates(3, 4, 5, 6) . First,
TCR/CD3-induced IL-2 secretion, which depends on the coordinated
activation of multiple transcription factors(34) , was examined
as an integrated measure of such signaling events. The dose-response
curve to anti-CD3 (Fig. 2A) reveals that, in contrast
to Jurkat, the CD45 Similar
responses by J-AS-1 and each of its single isoform-reconstituted
derivatives after stimulation by Ab-mediated cross-linking of CD3 and
CD28 (Fig. 2B), or with PMA plus ionomycin (Fig. 2C), documents similar inherent capacity of each
cell line to secrete IL-2 when the proximal signaling machinery, or the
requirement for CD45(30) , are bypassed, respectively. Thus,
after stimulation with anti-CD3 (at 0.05 to 1 µg/ml), IL-2
secretion by J[0] transfectants is not significantly
different from wild-type cells, despite 6-7-fold lower CD45
expression. Nonetheless, it is possible that decreased IL-2 secretion
by J[ABC] cells compared to J[0] cells is due to
their somewhat lower levels of CD45 expression. To rule out this
possibility, we sorted J[ABC] clones to obtain CD45
expression equal to that of J[0] cells and then compared IL-2
secretion by these cell populations after anti-CD3 stimulation (see Fig. 3). As before, clones expressing CD45(0) secrete wild-type
levels of IL-2. As shown, increased CD45 expression by J[ABC]
transfectants did not augment IL-2 secretion. Both sorted and unsorted
J[ABC] populations averaged just 24% of the wild-type levels
of IL-2.
Figure 3:
A,
representative immunofluorescence analysis of CD45 expression on
J[ABC] transfectants both before (solid line) and
after fluorescence activated cell sorting (dotted line) to
obtain J[ABC] cells expressing CD45 at levels equal to those
expressed by J[0] transfectants (upper panel). Log
fluorescence of isotype-matched negative controls for each clone was in
the first decade (not shown). B, IL-2 secretion by Jurkat,
J[0] transfectants, and J[ABC] transfectants before
and after cell sorting for increased CD45 expression. Cell populations
depicted in A (plus wild-type Jurkat) were stimulated with
anti-CD3 at doses of either 0.5 µg/ml or 1 µg/ml, and 24-h
supernatants were assessed for IL-2 secretion, as described under
``Materials and Methods.'' IL-2 secretion was normalized to
the response of Jurkat cells. Anti-CD45 treatment itself had no effect
on IL-2 secretion by J[ABC] clones (not shown). Data depicted
are the average of four experiments ±
S.E.
Given differential anti-CD3-induced IL-2 secretion by these
cell lines, more proximal signaling events were next examined.
Comparison of Lck and Fyn activities by immune complex kinase assays
failed to reveal isoform-dependent differences (data not shown). T cell
activation is associated with alterations in the tyrosine
phosphorylation of a number of cellular proteins. Therefore, we
compared the tyrosine phosphorylation of cellular proteins in each cell
line before, and at various time points after, anti-CD3 stimulation (Fig. 4). Under basal conditions, J-AS-1 consistently revealed
hyperphosphorylation of a limited set of bands at
Figure 4:
Comparison of tyrosine phosphorylation of
cellular proteins in Jurkat and J-AS-1 (A) and representative
single-isoform transfectants J[0]-1 and J[ABC]-1 (B) before and after anti-CD3 stimulation for the times
indicated. Whole cell lysates were immunoblotted with anti-Tyr(P). Brackets and arrows to the right indicate
bands exhibiting the most consistent differences between cells in each
panel. Approximate molecular mass of bands indicated (from top to bottom): A, 95, 70-75, 60, 52, and
38-40 kDa; B, 95, 58-60, and 32
kDa.
After anti-CD3 stimulation of Jurkat,
there was rapid phosphorylation (peaking at 30 s to 1 min) and
subsequent dephosphorylation of a number of bands. Although many of the
same bands were ultimately phosphorylated (within 5-10 min) after
stimulation of J-AS-1 cells, the kinetics were significantly slowed.
Furthermore, once phosphorylated, these bands did not undergo
dephosphorylation, consistent with decreased action of the CD45 PTPase
and perhaps of other cellular PTPases whose activities depend on
regulated tyrosine phosphorylation (35, 36). Re-expression of either
the CD45(0) or the CD45(ABC) isoforms generally restored basal and
activation-related tyrosine phosphorylation, although the kinetics were
somewhat prolonged compared to wild-type Jurkat (Fig. 4B). This may reflect the lower overall levels of
CD45 expression in these cells. More importantly, direct comparison
reveals clear isoform-dependent differences in the relative
phosphorylation of several bands. For example, J[ABC] cells
consistently exhibited relative hyperphosphorylation of a band at
This prompted a
comparison in our cells of the tyrosine phosphorylation of p95 Basal Vav tyrosine
phosphorylation was minimal but detectable in each of our cell lines (Fig. 5A). Anti-CD3 stimulation consistently induced
significantly greater tyrosine phosphorylation of Vav within 1 min, in
Jurkat and particularly in all three J[ABC] transfectants
compared to either of the two J[0] transfectants or the
CD45
Figure 5:
A,
tyrosine phosphorylation of Vav in Jurkat, J-AS-1, and single isoform
expressing transfectants expressing either CD45(ABC) or CD45(0)
isoforms before (0 min) or after (1 min) stimulation with anti-CD3. Vav
was immunoprecipitated from each cell line and immunoblotted with
anti-Tyr(P). C (first lane) indicates control
immunoprecipitation from lysates of stimulated Jurkat cells using
rabbit pre-immune serum. B, reprobing the same membrane with
anti-Vav antisera to demonstrate similar amounts of Vav
protein/lane.
Our results are the first to demonstrate that
signaling pathways utilized by the TCR are differentially regulated by
the extracellular domain of distinct CD45 isoforms. Stimulation of the
TCR leads to the phosphorylation of a number of cellular proteins
including Vav. Although the signaling pathways involving Vav have not
yet been clarified, Vav contains an array of signaling and DNA-binding
motifs, including SH2 and SH3 domains, a Dbl domain, and a
helix-loop-helix, which all appear to be involved in the generation of
downstream signals (33, 42, 43, 44, 45) .
Activation-related tyrosine phosphorylation directs SH2-mediated
interactions between Vav and several other signaling molecules. Thus,
Vav has been shown to associate with Shc, Grb2, ZAP-70,
phosphatidylinositol 3-kinase (p85), CD19, VAP-1, and several other
uncharacterized bands through SH2 and/or SH3-mediated interactions
after activation of B or T
lymphocytes(37, 46, 47, 48) . How
different CD45 isoforms might regulate this pathway remains
speculative. Particular CD45 isoforms might directly dephosphorylate
Vav or could differentially regulate the activity of the PTK(s)
responsible for Vav phosphorylation. However, preferential
dephosphorylation of Vav by a particular isoform is difficult to
envision given that Vav phosphorylation is decreased in both
CD45 As mentioned above, chimeric PTPase
molecules lacking the CD45 transmembrane or extracellular domains are
able to restore nearly normal patterns of tyrosine phosphorylation and
calcium flux(11, 12, 13) . In general
agreement, we showed that either the CD45(0) or CD45(ABC) isoforms are
capable of reconstituting activation-related tyrosine phosphorylation.
However, isoform-specific differences in IL-2 production and the
tyrosine phosphorylation of cellular proteins indicate that the
extracellular domain can superimpose regulatory influences on a
``constitutive'' cellular PTPase requirement. Thus, even
though many of the signaling pathways are conserved, a subset (that
includes Vav) appears subject to differential regulation by the various
CD45 extracellular domains. Differences in the utilization of these
pathways can lead to rather substantial isoform-dependent differences
in IL-2 secretion as demonstrated in our model and in the mouse
thymocyte model of Novak et al.(49) . Exactly how
the CD45 extracellular domains may regulate the PTPase domains is
unknown. One long-standing hypothesis supported by our findings is that
the distinct extracellular domains of the various CD45 isoforms
interact with different molecules on the surface of the same or other
cells, thereby directing the cytoplasmic phosphatase domains toward
distinct substrates. In this regard, the co-capping studies of Dianzani et al.(50) support the notion that differential
interactions between CD45 isoforms and other molecules on the surface
of the same cell can occur. In conclusion, our results indicate that
the regulated expression of distinct CD45 isoforms on different
developmental and functional subsets of T cells may impose preferential
utilization of particular TCR-mediated signaling pathways. Alterations
in CD45 isoform expression by individual T cells in response to thymic
selection or peripheral antigen exposure, may consequently allow that
cell to respond to TCR ligation using a different subset of signals. We
speculate that the delivery of these different signals to the cell
nucleus might have a significant influence on cell differentiation, the
expression of functional repertoire, or in allowing T cells to
``fine-tune'' their responsiveness. A more complete
understanding of these differences is likely to have important
implications for signal transduction and for the interpretation of the
highly regulated expression of CD45 isoforms in lymphocytes.
Volume 270,
Number 42,
Issue of October 20, 1995 pp. 24949-24954
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)requires expression of the CD45 family of
transmembrane protein-tyrosine phosphatases (PTPases) (1, 2) . CD45 has been shown to regulate the basal
activity of the Fyn and Lck protein-tyrosine kinases (PTKs) by
dephosphorylation of their respective regulatory carboxyl-terminal
tyrosine
residues(3, 4, 5, 6, 7) .
However, it is not clear that these are CD45's sole functions.
For example, new evidence suggests that CD45 can also dephosphorylate
certain PTK substrates, such as the TCR chain (8) and the
32-kDa CD45-associated phosphoprotein, LPAP(9) . Thus, the
precise functions of the CD45 phosphatase in signal transduction are
incompletely understood.
mutants by transfection of chimeric molecules
containing the conserved PTPase domains, but lacking the CD45
transmembrane or extracellular regions (11, 12, 13) . However, these results do not
exclude a potentially important role for the CD45 extracellular domain,
since the various extracellular domains could superimpose distinct
regulatory constraints upon the cytoplasmic domain. Our present
findings strongly support this hypothesis. Utilizing a unique model
system, we now demonstrate that the expression of different individual
CD45 isoforms is associated with differences in IL-2 production, as
well as differences in the activation-related phosphorylation of
cellular proteins including Vav. These findings demonstrate the
preferential utilization of different signaling pathways by distinct
CD45 isoforms.
DNA Constructs
A 270-base pair segment (from the
P1 transcription initiation site to the initiation codon) was amplified
by polymerase chain reaction from genomic CD45 DNA (clone LCA.512, from
Dr. H. Saito, Dana-Farber Cancer Institute, Boston, MA(16) ).
Polymerase chain reaction primers incorporating unique restriction
sites allowed ligation into the RcSR
plasmid vector (27) in an antisense orientation, generating the AS-CD45
plasmid vector. To generate CD45 cDNA constructs in the sense
orientation, the pSPSR LCA.1 and LCA.6 constructs(28) ,
encoding the smallest, CD45R(0), and largest, CD45R(ABC), isoforms,
respectively (from Dr. M. Streuli, Dana-Farber Cancer Institute), were
modified by removing a 5` SacI-SphI segment, to
reduce overlap between AS-CD45 and these CD45 cDNAs to 40 base pairs.Cell Lines and Transfections
The Jurkat human
leukemic CD4 T cell line was maintained in RPMI 1640
media containing 10% fetal calf serum, 4 mML-glutamine, and 50 µg/ml gentamycin at 37 °C in
humidified atmosphere with 5% CO
. Cells were transfected by
electroporation, and G418-resistant colonies were screened for loss of
CD45 by immunofluorescence. Two of the
CD4CD45
clones (J-AS-1 and J-AS-2)
were selected for further study. J-AS-1 was co-transfected at a 10:1
ratio with cDNA constructs for either the CD45R(0) or CD45R(ABC)
isoform plus the pPGKhyg plasmid (29) encoding Hygromycin B
resistance. Resistant colonies (G418 and Hygromycin) were screened by
immunofluorescence for CD45 expression as well as for expression of
appropriate cell surface markers described below. Clones were sorted
(fluorescence activated cell sorting) as necessary to obtain similar
CD45 and CD4 expression, as described below.
Antibodies, Immunofluorescence Phenotyping, and Cell
Sorting
Immunofluorescence analysis was performed as described
previously(26) . Mouse anti-human mAbs reactive with CD2, CD3,
CD4, CD28, CD45, and CD45RA were obtained as ascites (generously
provided by Dr. Chikao Morimoto, Dana-Farber Cancer Institute) or as
phycoerythrin conjugates (from Coulter Immunology, Hialeah, FL),
anti-CD45RO (obtained with the kind permission of Dr. Peter Beverly,
University College Hospital, London), and goat anti-mouse IgG-FITC
(from Southern Biotechnology, Birmingham, AL). Cell phenotype was
routinely monitored for these markers using a BD FACSTAR IV (10,000
cells/sample). Cell sorting was performed using a BD FACSTAR IV, after
staining by direct or indirect immunofluorescence, as
described(26) . Fluorochrome conjugates were dialyzed to remove
sodium azide prior to use.IL-2 Secretion
10
cells/well in
triplicate flat bottom 96-well tissue culture plates were stimulated
with anti-CD3 (purified OKT3) at the concentrations indicated,
essentially as described(27) . Cross-linking was performed with
goat anti-mouse (GAM) (at a 1:1 ratio with anti-CD3). For anti-CD3
anti-CD28 cross-linking, anti-CD3 (1 µg/ml) and anti-CD28
(1:400 dilution of ascites) were cross-linked with GAM (2 µg/ml).
PMA (1 ng/ml) was added to all wells as described(30) . 24-h
supernatants were assayed for IL-2 concentration using CTLL-2 cells.
IL-2 units were determined in each assay by comparison to a standard
curve using human rIL-2 (generously supplied by Chiron Corp.,
Emeryville, CA). The data were normalized to the response of Jurkat
cells at maximal anti-CD3 (1 µg/ml) in each experiment. 100%
response averaged 40-70 units/ml (see Fig. 2legend).
antisense-transfected Jurkat cell lines (J-AS), CD45(0)
transfectants (J[0]) (open symbols), and CD45(ABC)
transfectants (J[ABC]) (closed symbols), after
stimulation with various doses of anti-CD3 (cross-linked with GAM) (A), anti-CD28 (1:400) and anti-CD3 (1 µg/ml)
(cross-linked with GAM) (B), and ionomycin (1 µM)
plus PMA (1 or 5 ng/ml) (C). Each point represents the mean
values for two J-AS clones, two J[0] clones, and three
J[ABC] clones each examined in three independent experiments.
Data for each experiment were normalized to the response of Jurkat to
the maximal stimulus (100% response averaged 50 units/ml in A,
40 units/ml in B, and 70 units/ml in C, where 1 ng of
rIL-2 = 50 units). Vertical lines indicate
S.E.
Cellular Activation
For whole cell lysates, 3
10
cell aliquots were prewarmed to 37 °C and
then treated for the indicated time periods with prewarmed goat
anti-mouse (GAM) alone (unstimulated control), or anti-CD3 (30
µg/ml) plus GAM (7.5 µg/ml) (stimulated), as
described(31) . For analysis of Vav tyrosine phosphorylation,
15 10 cells were stimulated with anti-CD3 for 1 min
at 37 °C, as described above. Unstimulated controls were treated
with anti-CD3 after addition of lysis buffer. At the appropriate times,
an excess of ice-cold stop solution (phosphate-buffered saline
containing 5 mM EDTA and phosphatase inhibitors) was added,
followed immediately by brief centrifugation in a Microfuge, removal of
the supernatant, and resuspension of the pellets in ice cold lysis
buffer (described below).Cell Lysis, Western Blotting, and
Immunoprecipitation
Cells were lysed in 1% Nonidet P-40 lysis
buffer containing 25 mM Tris-HCl (pH 8.0) with 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 10 mM iodoacetamide, 1
mM sodium vanadate, 10 mM NaF, and 10 mM sodium pyrophosphate, for 20 min at 4 °C, followed by
centrifugation at 14,000 rpm for 15 min, as
described(31, 32) . For experiments using whole cell
lysates, postnuclear supernatants were boiled in Laemmli sample buffer
and loaded onto SDS-PAGE gels. For analysis of CD45 isoform expression,
(1-2 10 cell eq for Jurkat and 5
10 for transfectants) were run on 6% SDS-PAGE, transferred
to nitrocellulose membranes, and immunoblotted with anti-CD45 (GAP 8.3,
ATCC). For analysis of tyrosine phosphorylation of cellular proteins, 3
10 cell eq/lane were lysed before or after
stimulation as above, and postnuclear supernatants separated on 10%
SDS-PAGE (nonreducing conditions). Following transfer to
nitrocellulose, membranes were blocked with 5% milk in
phosphate-buffered saline and probed with anti-phosphotyrosine
(anti-Tyr(P)) (mAb 4G10 generously provided by Dr. Brian Drucker,
Oregon Health Sciences Center, Portland, OR). For immunoprecipitation
of Vav, 15 10 cells/lane were activated and lysed
as described above, and postnuclear supernatants were precleared as
described(31, 32) , twice. Protein concentration was
determined using the micro-BCA protein assay kit (Pierce), and
equivalent amounts of protein in precleared lysates were incubated with
anti-Vav antisera, followed by immunoprecipitation with Protein
A-Sepharose (Pharmacia Biotech Inc.). Immunoprecipitates were washed
five times with lysis buffer, boiled in sample buffer, and separated on
8% SDS-PAGE under reducing conditions. After transfer to
nitrocellulose, blots were blocked as above and then probed with
anti-Tyr(P) (4G10), as described (31, 33) . After
stripping, the same membrane was reprobed with anti-Vav (kindly
provided by Dr. A. Altman, La Jolla Research Institute, La Jolla, CA,
or from UBI, Lake Placid, NY). All immunoblots were developed using
horseradish peroxidase-conjugated secondary reagents and developed
using ECL (Amersham).
colonies selected and subcloned, two, denoted J-AS-1 and J-AS-2,
were selected on the basis of CD4 expression comparable to that of
parental Jurkat. (Fig. 1B). Jurkat expresses high
levels of total CD45 and lower levels of both the smallest CD45 isoform
(CD45RO) and the largest (two) isoforms which contain exon A (CD45RA).
J-AS-1 and -2 lack detectable CD45 expression either on the surface or
in the cytoplasm (Fig. 1, B and C).
(J-AS-1), and
single CD45 isoform transfectants expressing either the CD45(0) isoform
(J[0]-1) or the CD45(ABC) isoform (J[ABC]-1).
Isotype-matched negative controls are depicted as dotted
lines. The x and y axes represent log
fluorescence and cell number, respectively. C, anti-CD45
immunoblotting of whole cell lysates from: Raji (human B cell line),
Jurkat, CD45
(J-AS-1), CD45(0) transfectants
(J[0]-1 and -2), and CD45(ABC) transfectants
(J[ABC]-1 and -2). 300-19 (mouse pre-B cell) is shown
as a negative control. Arrows on left indicate previously
established human isoforms at 220, 205, 190, and 180 kDa(17) .
Lower M
bands represent immature forms, not yet
glycosylated at O-linked sites(17) . Arrows at
right indicate nonspecific bands present in control 300-19
cells.
clones arising expressed solely the transfected
CD45 isoform by both immunofluorescence and by immunoblotting. (Fig. 1, B and C). Three independent CD45(ABC)
transfected isolates (J[ABC]-1, -2, and -3) and two CD45(0)
transfected isolates (J[0]-1 and -2) were selected for
further study, based on CD45 expression and wild-type levels of CD3.
The clones were then sorted to obtain stable populations expressing
similar levels of CD4 and CD45. When matched for their surface
expression, J[0] and J[ABC] clones expressed
identical levels of CD45 by immunoblotting, indicating no inherent
differences in the relative distribution of intracellular and
extracellular CD45 (data not shown). Although total CD45 expression was
lower in the transfectants, the expression of individual CD45(0) and
CD45(ABC) isoforms by J[0] and J[ABC] cells,
respectively, was similar to their expression in wild-type cells. The
expression of CD3, CD2, and CD28 was nearly identical in each of the
cell lines (Fig. 1B and data not shown).
(J-AS) cell lines secreted
minimal IL-2 in response to all doses of anti-CD3 tested. Furthermore,
no enhancement was seen after co-stimulation by cross-linking anti-CD3
and anti-CD4 (data not shown). Reconstitution with the CD45(0) isoform
resulted in wild-type levels of IL-2 secretion after stimulation with
anti-CD3 (1.0 µg/ml). In contrast, the CD45(ABC) transfected cell
lines produced significantly less IL-2 than either Jurkat or the
CD45(0) transfectants at both 0.05 and 1.0 µg/ml anti-CD3,
secreting at maximum, 30% of wild-type levels. Increasing the anti-CD3
dose to 5 µg/ml had no additional effect on IL-2 secretion by any
of the cell lines (data not shown). However, at lower doses of anti-CD3
(0.005 µg/ml), transfectants expressing either individual isoform
secrete much less IL-2 than Jurkat, possibly owing to the lower levels
of overall CD45 expression. Stimulation with anti-CD2 gave overall
results similar to those observed above (not shown).
70-76 kDa
and decreased tyrosine phosphorylation of several other bands
(
105,
95, and
50-52 kDa) when compared to Jurkat (Fig. 4A).
95 kDa when compared to J[0] cells.
(Vav) which is rapidly and transiently phosphorylated on tyrosine
after ligation of the TCR(33, 37) , CD28(38) ,
or upon the binding of IL-2 to its receptor(39) . While the
exact function of this proto-oncogene product in signal transduction is
unclear, gene ablation studies document the important role of Vav in
the activation and proliferation of mature lymphocytes as well as in
the normal developmental expansion of lymphocyte precursors in the
marrow and thymus(40, 41) .
J-AS-1 cells. Reprobing the same membrane with
anti-Vav antisera confirmed similar loading of Vav protein in each lane (Fig. 5B). These differences are not secondary to
altered kinetics, since the same pattern is observed 4 min after
anti-CD3 stimulation, at which time phosphorylation of Vav in Jurkat
and single-isoform transfectants is decreasing (data not shown and (33) ).
cells and those expressing CD45(0), yet
increased in wild-type cells (which express both CD45(0) and CD45(ABC)
isoforms) and in cells expressing CD45(ABC). Activation-related
phosphorylation of Vav clearly does not directly correlate with
absolute levels of CD45 expression. Our results are more consistent
with augmented activity of the PTK responsible for Vav phosphorylation
by cells expressing the CD45(ABC) isoform. However, the PTK(s)
responsible for the in vivo phosphorylation of Vav are
presently unknown. Although Lck is capable of phosphorylating Vav in vitro, IL-2 mediated phosphorylation of Vav occurs in the
absence of Lck(39) . CD28 ligation results in Itk
phosphorylation followed temporally by that of Vav, suggesting a
possible link(38) . Recently, it was reported that ZAP-70 can
physically associate with the Vav-SH2 domain after T cell activation,
although it is unknown whether Vav serves as a substrate for this
PTK(47) . Further analysis of these PTKs and Vav-associated
molecules in our single isoform transfectants may help delineate those
pathways relevant to CD45.
)
We are grateful to Dr. Tomas Mustelin and Dr. Alfred
Bothwell for their critical reading of this manuscript, Drs. Yoshihisa
Nojima, Chris Rudd, and Toshiaki Tanaka for reagents and helpful
advice, and to Drs. Amnon Altman, Brian Drucker, Chikao Morimoto, Haruo
Saito, and Michel Streuli for generously providing various mAbs and
CD45 genomic segments and cDNAs.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 D. M. Rothstein, M. F. A. Livak, K. Kishimoto, C. Ariyan, H.-Y. Qian, S. Fecteau, M. Sho, S. Deng, X. X. Zheng, M. H. Sayegh, et al. Targeting Signal 1 Through CD45RB Synergizes with CD40 Ligand Blockade and Promotes Long Term Engraftment and Tolerance in Stringent Transplant Models J. Immunol., January 1, 2001; 166(1): 322 - 329. [Abstract] [Full Text] [PDF]
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 A. A. ROSSINI, D. L. GREINER, and J. P. MORDES Induction of Immunologic Tolerance for Transplantation Physiol Rev, January 1, 1999; 79(1): 99 - 141. [Abstract] [Full Text] [PDF]
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N. Kashio, W. Matsumoto, S. Parker, and D. M. Rothstein The Second Domain of the CD45 Protein Tyrosine Phosphatase Is Critical for Interleukin-2 Secretion and Substrate Recruitment of TCR-zeta in Vivo J. Biol. Chem., December 11, 1998; 273(50): 33856 - 33863. [Abstract] [Full Text] [PDF]
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 G. P. Basadonna, L. Auersvald, C. Q. Khuong, X. X. Zheng, N. Kashio, D. Zekzer, M. Minozzo, H.-Y. Qian, L. Visser, A. Diepstra, et al. Antibody-mediated targeting of CD45 isoforms: A novel immunotherapeutic strategy PNAS, March 31, 1998; 95(7): 3821 - 3826. [Abstract] [Full Text] [PDF]
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H. Onodera, D. G. Motto, G. A. Koretzky, and D. M. Rothstein Differential Regulation of Activation-induced Tyrosine Phosphorylation and Recruitment of SLP-76 to Vav by Distinct Isoforms of the CD45 Protein-tyrosine Phosphatase J. Biol. Chem., September 6, 1996; 271(36): 22225 - 22230. [Abstract] [Full Text] [PDF]
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S. Dornan, Z. Sebestyen, J. Gamble, P. Nagy, A. Bodnar, L. Alldridge, S. Doe, N. Holmes, L. K. Goff, P. Beverley, et al. Differential Association of CD45 Isoforms with CD4 and CD8 Regulates the Actions of Specific Pools of p56lck Tyrosine Kinase in T Cell Antigen Receptor Signal Transduction J. Biol. Chem., January 11, 2002; 277(3): 1912 - 1918. [Abstract] [Full Text] [PDF]
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