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Department of Pediatric Oncology, Dana-Farber Cancer Institute, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterial and MethodsResultsDiscussionReferences |
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can
be induced by PTP inhibitors, but not by anti-CD3. Detailed
characterization of this cell-free system showed that the pattern and
the order of induced tyrosine phosphorylation is similar to that
induced in intact cells. Upon removal of the PTP inhibitor, the
tyrosine-phosphorylated proteins, including Lck, Fyn, Syk, Zap70, and
CD3, are rapidly dephosphorylated. Preliminary characterizations
indicate that a PTP distinct from CD45, SHP1, and SHP2 is present in T
cell membranes and the inhibition of this yet unidentified PTP is most
likely responsible for the Lck-dependent tyrosine phosphorylation
triggered by PTP inhibitors. |
Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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is tyrosine phosphorylated,
most likely by activated Lck. This is followed by the association and
activation of Zap70/Syk family PTKs with phosphorylated CD3
(9, 10, 11). The activation of TCR/CD3-associated PTKs appears
to be responsible for the phosphorylation and activation of
phospholipase C 
1 and the subsequent induction of the
phospholipid/calcium second-message pathway (2, 12). The
activation of these PTKs also results in the formation of complexes of
adapter proteins such as Shc/Grb2/Sos, p36/Grb2/Sos, Grb2/Cbl, and
Crk/C3G, which link the TCR to the downstream
Ras/mitogen-activated protein kinase pathway (3, 13, 14).
Tyrosine phosphorylation is controlled by the coordinated action of
PTKs and protein tyrosine phosphatases (PTPs) (15, 16, 17).
The role of PTPs in the regulation of T cell activation is not as well
elucidated as the role of the PTKs. Accumulating data indicate that
PTPs have both positive and negative effects on T cell signaling
(18, 19, 20, 21, 22, 23, 24, 25, 26, 27). CD45, the receptor-like PTP, exerts primarily a
positive effect through the dephosphorylation of negative regulatory
tyrosyl residues of Src family PTKs (18, 19, 20).
CD45-deficient T cell clones and cell lines are largely nonresponsive
to CD3 stimulation (21, 22). Recently, two SH2-containing
PTPs, SHP1 (also known as HCP, SHPTP1, PTP1C) and SHP2 (previously
called Syp, SHPTP2) were proposed as negative regulators of T cell
activation (23). T cells from SHP1-deficient
me/me mice are hypersensitive to TCR stimulation, reflecting
a possible negative regulation of TCR activation by SHP1 (24, 25). In Jurkat T cells, upon T cell stimulation, SHP1 was found
associated with Zap70. This interaction resulted in a decrease in the
kinase activity of Zap70 (26). Transient expression of
dominant negative SHP1 appeared to decrease the activation threshold of
Jurkat T cell toward CD3 or PMA stimulation. SHP2 was recently
demonstrated to specifically associate with CTLA-4, a homologue of the
T cell costimulator molecule CD28 (27). In T cells from
CTLA-4-/- mice, Lck, Fyn, and Zap70 were found to be
constitutively active, and Shc, an adapter molecule for the
Ras/mitogen-activated protein kinase pathway, was found associated with
tyrosine phosphorylated CD3. These findings suggest that the
recruitment of SHP2 to the T cell membrane through CTLA-4 negatively
regulates T cell activation.
H2O2 has been described as a PTP inhibitor
(28). Like other oxidants, H2O2
may oxidize the thiolate anion of a cysteine residue in the
PTP-reactive center, blocking the formation of a phosphoryl-cysteine
intermediate, which is a critical step in dephosphorylation
(29). In T lymphocytes, exposure to
H2O2 induces a rapid tyrosine phosphorylation
of a variety of proteins, including Src family and Syk family PTKs
(30, 31, 32). H2O2 has also been
reported to induce the tyrosine phosphorylation of mitogen-activated
protein kinases and the activation of AP-1 and nuclear factor 
B
(NF-B) (33, 34). Thus, there are many similarities
between CD3-mediated tyrosine phosphorylation and PTP inhibitor-induced
T cell tyrosine phosphorylation. It is not clear, however, whether
H2O2 mimics CD3 stimulation by triggering an
ordered signaling cascade, or if H2O2 functions
by inducing or maintaining random tyrosine phosphorylation as a
consequence of the inhibition of PTPs.
To study the tyrosine phosphorylation triggered by inhibition of
membrane PTPs, we developed a cell-free system by subcellular
fractionation of T cells. We found that upon
ATP/H2O2 induction, target proteins in the T
cell membrane fraction became tyrosine phosphorylated, whereas proteins
in a cytosolic fraction did not. The inhibition of the
membrane-associated PTPs triggered a cascade of tyrosine
phosphorylation, which resembled that induced by TCR/CD3 stimulation in
intact T cells. The tyrosine phosphorylation of the proteins in the T
cell membrane fraction, including CD3-associated PTKs and CD3, was
rapidly reversed upon removal of H2O2. Lck is
required for the H2O2 induction of tyrosine
phosphorylation. Preliminary characterization indicates that a PTP
distinct from CD45, SHP1, and SHP2 is present in T cell membranes, and
may play a critical role in the negative regulation of TCR
signaling.
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Jurkat T cell line J77, a variant of clone E6-1 (American Type
Culture Collection (ATCC), Rockville, MD), was cultured in RPMI 1640
medium supplemented with 10% FCS at 37°C in a 5%
CO2-humidified atmosphere. JCAM (J.CaM1.6) and
JCD45-(J45.01) Jurkat T cells were obtained from the ATCC.
Total thymocytes were obtained from 12- to 14-day-old normal and
me/me mice (C3HeB/FeJLe-a/a-me strain; the
Jackson Laboratory, Bar Harbor, ME). Anti-phosphotyrosine
(anti-ptyr) (RC20), anti-SHP1, anti-SHP2, and
anti-Zap70 mAbs were purchased from Transduction Laboratories
(Lexington, KY). Anti-CD3 monoclonal, anti-Fyn polyclonal,
anti-Syk polyclonal, and anti-Zap70 polyclonal Abs were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Lck
polyclonal Ab was purchased from Upstate Biotechnology (Lake Placid,
NY). Anti-CD3 Ab (OKT3) was prepared from a hybridoma obtained from
the ATCC.
Immunoprecipitation and Western blot analysis
Jurkat T cells (2 x 107) were washed and resuspended in 1 ml of PBS. For CD3 stimulation, cells were incubated with OKT3 (2 µg/ml) for 5 min on ice, cross-linked by rabbit anti-mouse IgG (5 µg/ml) on ice for a further 5 min, then incubated at 37°C for 3 min. For H2O2 stimulation, cells were incubated with 5 mM H2O2 at 37°C for 3 min. After washing with PBS, cells were lysed in 1 ml of Nonidet P-40 (NP-40) lysis buffer (1% NP-40, 150 mM NaCl, 20 mM Tris, pH 7.4, 0.5% sodium deoxycholate, 50 mM NaF, 1 mM PMSF, 1 µg/ml leupeptin, and 2 µg/ml antipain) at 4°C for 30 min. The NP-40 lysate was centrifuged at 12,000 x g for 15 min at 4°C. Immunoprecipitation was carried out at 4°C overnight or at room temperature for 4 h with protein A-Sepharose beads. The beads were washed twice with 0.1% Triton X-100/TBS and once with TBS. The protein was eluted from the beads by boiling for 5 min in 50 µl of Laemmli reducing SDS sample buffer. Proteins from about 107 cells were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was blocked with 3% BSA/TBST and incubated with Abs in TBST for 2 h at room temperature. Following four 15-min washes with TBST, the membranes were incubated with second Ab for 30 min, washed three times for 5 min with TBST, and developed by enhanced luminol reagent (Amersham, Arlington Heights, IL).
Subcellular fractionation of Jurkat T cells
After washing in PBS, T cells (2 x 108) were incubated in 2 ml of hypotonic buffer (42 mM KCl, 10 mM HEPES (pH 7.4) and 5 mM MgCl2) for 15 min at 4°C. The cells were passed through a 30-gauge needle 10 times. The extract was centrifuged at 250 x g for 10 min to remove the nuclei and intact cells. The postnuclear supernatant (PNS) was centrifuged at 150,000 x g for 30 min at 4°C to separate the cytoplasm from the membrane fraction.
Induction of T cell membranes by PTP inhibitors and dephosphorylation assay
Membranes from 108 T cells were resuspended in 0.5 ml hypotonic buffer containing 1 mM ATP, then stimulated with 10 mM H2O2 or 1 mM vanadate at 37°C for 3 min. The reaction mixtures were centrifuged at 12,000 x g for 3 min at 4°C. The membrane pellets were washed once with hypotonic buffer at 4°C. For the dephosphorylation assay, pelleted membranes were resuspended in 0.5 ml hypotonic buffer, incubated at 37°C for varying times. After centrifugation, membranes were solublized in NP-40 lysis buffer.
In vitro kinase assay
Immunoprecipitates were incubated together with 0.1 µg of
enolase and 1 µCi of [-32P]ATP in 50 µl of kinase
reaction buffer (20 mM HEPES, pH 7.4, 10 mM MnCl2) at room
temperature for 30 min. The reaction was stopped by adding 25 µl of
2 x SDS sample buffer and boiling for 5 min. Proteins were then
resolved by SDS-PAGE, transferred to polyvinylidene difluoride
membrane, and autoradiographed.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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To determine the dose dependence of
H2O2-induced tyrosine phosphorylation, Jurkat T
cells were incubated with H2O2 at
concentrations ranging from 0.1 mM to 10 mM for 2 min or for 5 min.
Tyrosine-phosphorylated proteins were immunoprecipitated by an
anti-ptyr (4G10). After SDS-PAGE and transfer, proteins were
immunoblotted with another anti-ptyr (RC20).
H2O2 at 0.2 mM induced detectable tyrosine
phosphorylation while 5 to 10 mM induced maximal tyrosine
phosphorylation. The time of incubation had little effect on tyrosine
phosphorylation (Fig. 1
A).
H2O2-induced tyrosine phosphorylation is a
rapid event that can be detected within 1 min (data not shown). The
major tyrosine-phosphorylated proteins observed have the apparent
molecular weights of 18 kDa, 38 kDa, 56 kDa, 70 kDa, and 100 kDa. To
compare the patterns of tyrosine phosphorylation induced by
H2O2 to that induced by CD3 stimulation, Jurkat
T cells were stimulated either by 10 mM H2O2 or
by anti-CD3 cross-linking followed by immunoprecipitation with
anti-ptyr or Abs specific for Lck, Fyn, Syk, Zap70, and CD3. The
tyrosine phosphorylation induced by H2O2 and by
anti-CD3 appeared quite similar (Fig. 1B). Both modes of
stimulation increased the tyrosine phosphorylation of Lck, Fyn, Syk,
Zap70, and CD3. Coimmunoprecipitation of Zap70 PTK with CD3, as
well as the coimmunoprecipitation of ZAP70 with Lck, was also observed,
indicating that both types of stimulation resulted in the association
of Zap70 with CD3 and the association of Zap70 with Lck.
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The above results demonstrated that incubation with
H2O2 induces tyrosine phosphorylation of PTKs
and TCR subunits involved in the early steps of TCR signaling, which
suggested that H2O2 may be targeted at
molecules on T cell membranes. To assess this possibility, Jurkat T
cells were homogenized in a hypotonic buffer, then fractionated by
differential centrifugation. The PNS, a fraction obtained by 250
x g centrifugation to remove the nuclei from homogenates,
was further separated into crude membrane and cytosolic fractions by
150,000 x g centrifugation. After
H2O2 stimulation, these fractions were
subsequently solubilized by NP-40 lysis buffer and immunoprecipitated
with an anti-ptyr Ab. The results show that
H2O2 induces strong protein tyrosine
phosphorylation in the PNS, very weak tyrosine phosphorylation in the
membrane fraction, and no tyrosine phosphorylation in the cytosolic
fraction (Fig. 2A). When the
cytosolic fraction was mixed with the membrane fraction,
H2O2-induced tyrosine phosphorylation was
reconstituted. Considering that some essential elements for tyrosine
phosphorylation such as ATP and divalent ions, could be missing in the
membrane fraction, it was supplemented with 1 mM ATP and 5 mM
Mg2+. In the presence of 1 mM ATP,
H2O2 induced strong tyrosine phosphorylation in
the crude membrane fraction (Fig. 2B). In the PNS and
cytosolic fractions, H2O2-induced tyrosine
phosphorylation was not affected by adding exogenous ATP. In contrast,
CD3 stimulation does not induce tyrosine phosphorylation in analogous
experiments using subcellular fractions.
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). Vanadate (0.1 mM) was as potent as 2
mM H2O2 in the induction of tyrosine
phosphorylation. Pervanadate was extremely potent, with 0.1 µM
inducing maximal tyrosine phosphorylation. The pattern of tyrosine
phosphorylation induced by H2O2, vanadate, and
pervanadate appears quite similar.
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subunit (Fig. 4A). In the membrane fraction,
tyrosine phosphorylation of Lck, Fyn, and CD3 was induced by
H2O2. In the presence of cytosolic components,
as shown in the PNS fraction, the tyrosine phosphorylation of Zap70 and
the coimmunoprecipitation of Zap70 with CD3 and Lck were observed.
This indicated that Zap70 can be recruited into the TCR/CD3 complex in
cell-free conditions in response to H2O2
stimulation. When the cytoplasmic fraction was stimulated by
H2O2, tyrosine phosphorylation of Zap70 was not
observed (Fig. 4B). These data indicate that
H2O2 does not induce Zap70 tyrosine
phosphorylation directly but, instead, through the activation of PTKs
in the T cell membrane which, in turn, phosphorylate Zap70. Thus, the
tyrosine phosphorylation induced by PTP inhibition is a
membrane-related event.
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To study the inhibition of PTPs in the T cell membrane, we
designed a dephosphorylation assay by using the fractionated T cell
membranes. T cell membranes were stimulated by
H2O2/ATP for 3 min, then
H2O2 was removed from the reaction mixture by
adding catalase and washing. Following additional incubation, the level
of tyrosine phosphorylation was determined. We found that, after
removal of H2O2, substantial dephosphorylation
was observed within 15 min while phosphorylation was maintained for
over 60 min in the presence of H2O2 (Fig. 5A). These reactions were
performed in the presence of 1 mM ATP and reflect an equilibrium
between phosphorylation and dephosphorylation. By contrast, when ATP
was removed after stimulation, a rapid dephosphorylation was observed
(Fig. 5B). In the absence of H2O2,
dephosphorylation was also completed within 5 min. In the presence of
H2O2, the dephosphorylation required about 30
min, demonstrating that T cell membranes contain PTP activity and that
H2O2 inhibition of PTPs is reversible and
incomplete. The membrane dephosphorylation assay was also performed
following vanadate stimulation, and similar results were obtained (Fig. 5C). To further characterize this observation, the
dephosphorylation of Lck, Syk, Zap70, and CD3 was studied in a
similar way by specific immunoprecipitation. The results showed that
all TCR-associated PTKs studied and CD3 were rapidly
dephosphorylated in isolated T cell membrane upon removal of PTP
inhibitors (Fig. 5D).
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A). When blotted with
anti-Lck, a similar amount of Lck protein was observed, confirming
that there is no apparent loss of Lck protein in these assays (Fig. 6B). To determine whether Lck is inactivated by
dephosphorylation, Lck activity was determined in an in vitro kinase
assay following its dephosphorylation (Fig. 6C). Lck is
almost completely inactivated 15 min after removal of
H2O2, while most of the Lck activity is
maintained in the presence of H2O2. These
results indicate that the inhibition of membrane-associated PTPs
triggers the tyrosine phosphorylation in T cells.
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CD45 is a known transmembrane phosphatase that regulates Lck
activity (18, 19, 20, 21, 22). In order to study the role of CD45 in
PTP inhibitor-induced tyrosine phosphorylation, we used a
CD45- Jurkat T cell line. As in Jurkat T cells,
H2O2- and vanadate-induced tyrosine
phosphorylation was observed in CD45- T cells and their
membrane preparation, indicating that CD45 is not involved in
H2O2-induced tyrosine phosphorylation (Fig. 7, A and B).
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, A and
B). This indicates that, in the absence of Lck, inhibition
of PTPs is not sufficient to trigger tyrosine phosphorylation. This was
confirmed by showing that tyrosine phosphorylation in a JCAM cell line
transfected with Lck was H2O2 inducible (data
not shown). These data suggested that the PTPs inhibited in T cell
membranes could be involved in the negative regulation of Lck
signaling.
SHP1 and SHP2, two SH2-containing PTPs, have been reported to be
involved in the dephosphorylation of T cell membrane-associated
proteins such as Lck, Zap70, and CD3 (23, 24, 25, 26, 27). As
cytosolic PTPs, SHP1 and SHP2 may associate with phosphorylated
membrane proteins, and therefore be present in T cell membrane. To
study the role of SHP1 and SHP2 in the regulation of tyrosine
phosphorylation, we examined the subcellular distribution of SHP1 and
SHP2. The membrane and cytosolic fractions were prepared from T cells,
as well as fibroblasts, as a control. Western blot analysis with
anti-SHP1 and anti-SHP2 showed that both SHP1 and SHP2 were
expressed in T cells and fibroblasts predominantly as cytosolic
proteins with only a small amount of SHP1 detectable in the T cell
membrane preparation (Fig. 8A). The presence of SHP2 in
the T cell membrane fraction was not detected in our experiment. This
suggested that SHP1 may be the membrane-associated PTP which, when
inhibited, triggers tyrosine phosphorylation. Neither SHP1 nor SHP2 was
detected in 3T3 cell membrane preparations. Therefore, the PTP activity
observed in our membrane preparations is unlikely to be due to
cytosolic contaminants.
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, B and C).
Without stimulation, the basal tyrosine phosphorylation in
me/me mice is at a low level, and is only slightly above
that in normal littermate mice. The exception is an 18-kDa protein
determined to be CD3, which is constitutively tyrosine
phosphorylated in me/me mice. In normal mice, tyrosine
phosphorylation of CD3 was induced by H2O2
and vanadate stimulation. This indicates that SHP1 does play a role in
the regulation of protein tyrosine phosphorylation of CD3. However,
it is clear that additional PTPs are present in me/me T cell
membranes, whose activity affects the tyrosine phosphorylation observed
in isolated T cell membranes. |
Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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). These results suggest that the PTP
inhibitors H2O2 and vanadate specifically
affect the early steps of TCR signaling. To further study this
question, we established a cell-free system by subcellular
fractionation of Jurkat T cells. In cell-free conditions, tyrosine
phosphorylation of proteins in the T cell membrane fraction can be
induced by PTP inhibitors, H2O2, or vanadate,
but not by TCR/CD3 stimulation (Figs. 2 and 3). In cell-free
conditions, we found that membrane-associated Lck, Fyn, Zap70, and
CD3 were phosphorylated by ATP/H2O2 (Fig. 4). Furthermore, the regulation of tyrosine phosphorylation in isolated
membrane was studied by a dephosphorylation assay (Fig. 5). Lck and
other TCR-associated PTKs and CD3 were rapidly dephosphorylated in T
cell membranes upon removal of PTP inhibitors (Figs. 5 and 6). The
dephosphorylation of Lck also resulted in the inactivation of Lck
kinase activity (Fig. 6). Therefore, we established a cell-free system
in which tyrosine phosphorylation may be regulated by the addition or
removal of PTP inhibitors. To our knowledge, this is the first report that regulation of tyrosine phosphorylation/dephosphorylation may be studied in cell-free conditions. Characterization of this new system indicates that isolated T cell membranes keep the dynamic balance between tyrosine phosphorylation and dephosphorylation and demonstrates an ordered pattern of tyrosine phosphorylation. We expect this system to prove useful to explore early signal transduction cascades. By reconstitution of membranes and cytosol from different cell types or different tissues, it may be possible to determine the specific cytosolic substrates of membrane-associated kinases and the interactions between cytosolic factors and membrane proteins. In cell-free conditions, these factors may be conveniently added into the assay mixture or specifically removed by Abs. Also, these cell-free systems may be useful to test drugs or reagents that are not permeable to the cell membranes.
The presence of Lck in T cell membranes is required for
H2O2- and vanadate-induced tyrosine
phosphorylation. Lck is activated when T cells are treated with
H2O2 (Fig. 7). It has been hypothesized that
H2O2 and other oxidants may activate Src family
kinases by changing the intracellular redox state. Redox state is
coupled to the oxidation of cysteine residues in proteins by a complex
thiol/disulfide exchange mechanism that may result in the
conformational change and activation of the Src family kinases
(35, 36, 37). However, H2O2 and all
other in vivo-effective oxidants and alkylating agents fail to activate
Src family kinases in vitro (36, 37). Only HgCl can
activate Src family kinases in vitro, presumably by bridging two
adjacent sulfydryl groups to form an R-S-Hg-S-R bond (36, 37). Currently, there is no evidence that such an intramolecular
or intermolecular disulfide bridge could be formed through the
H2O2 induction. Our results showed that
tyrosine phosphorylation of Lck is induced by vanadate or
H2O2 in a cell-free system, but is not induced
when isolated Lck is treated with H2O2 or
vanadate (data not shown). It supports the hypothesis that the
activation of Src family kinases by H2O2 and
vanadate is indirect, and a common mechanism, the inhibition of PTPs,
is the most likely explanation for H2O2, and
other oxidants such as phenylarsine oxide- and diamideinduced tyrosine
phosphorylation in T cells (38).
The identity of the PTPs inhibited by H2O2 and
vanadate that associate with T cell membranes and regulate Lck and
other TCR-associated PTKs is currently under investigation. In JCAM T
cells, which are deficient in Lck, tyrosine phosphorylation was not
induced by H2O2 (Fig. 7) (8).
Therefore, the membrane-associated PTPs targeted by PTP inhibitors are
most likely to be involved in negative regulation of Lck. This is
consistent with previous reports that H2O2 may
activate Lck (30, 31, 32). CD45, a transmembrane PTP, has been
reported to regulate Lck (20, 21). However, there must be
PTPs other than CD45 in T cell membranes, as our results show that
CD45-deficient T cells respond to H2O2 with a
signal similar to J77 T cells (Fig. 7). A role for SHP1 and SHP2 in the
negative regulation of T cell activation has been reported
(23). T cells deficient in SHP1 are hypersensitive to TCR
stimulation (26, 27). SHP1 can dephosphorylate and
inactivate bacterially produced Lck (39). Lck, Zap70, and
CD3 were dephosphorylated by SHP1 when cotransfected in a
Spodoptera frugiperda cell line (40). SHP1, a
primarily cytosolic PTP, could be present in T cell membranes via an
interaction with CD3
and CD5 (25). SHP2 may also be
present in the T cell membrane in association with tyrosine
phosphorylated CTLA-4 (27). We have studied the
subcellular distributions of SHP1 and SHP2 and found a small amount of
SHP1, but not SHP2, in T cell membrane preparations. To further study
the potential role of SHP1 in regulating tyrosine phosphorylation, the
membranes from thymocytes of SHP1 deficient me/me mice were
stimulated by H2O2 (Fig. 8). Strong tyrosine
phosphorylation was induced by H2O2 in both
me/me mice and normal littermate control mice, although
CD3 was found to be constitutively tyrosine phosphorylated in
me/me mice but not in normal control mice, consistent with
previous reports (24, 25). These data indicate that, in
addition to SHP1 and CD45, a novel PTP may be present in T cell
membranes, the inhibition of which is more likely related to the
overall tyrosine phosphorylation observed upon
H2O2 stimulation. The further characterization
and identification of this membrane-associated PTP is of great interest
and will be facilitated by the ability to study the regulation of
tyrosine phosphorylation in this cell-free system.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yong-Jiu Jin, Department of Pediatric Oncology, Room M654, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115.
3 Abbreviations used in this paper: PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; TBS, Tris-buffered saline; TBST, TBS0.01% Tween-20; PNS, postnuclear supernatant; NF-B, nuclear factor B; anti-ptyr, anti-phosphotyrosine Ab; NP-40, Nonidet P-40.
Received for publication January 5, 1998. Accepted for publication April 15, 1998.
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References |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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S. W. Feigelson, V. Grabovsky, E. Winter, L. L. Chen, R. B. Pepinsky, T. Yednock, D. Yablonski, R. Lobb, and R. Alon The Src Kinase p56lck Up-regulates VLA-4 Integrin Affinity. IMPLICATIONS FOR RAPID SPONTANEOUS AND CHEMOKINE-TRIGGERED T CELL ADHESION TO VCAM-1 AND FIBRONECTIN J. Biol. Chem., April 20, 2001; 276(17): 13891 - 13901. [Abstract] [Full Text] [PDF]
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