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*
Department of Microbiology and Immunology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan;
Institute for Protein Research, Osaka University, Suita, Japan; and
University of Chicago School of Medicine, Chicago, IL 60637
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Ab
demonstrated that Lyn associated with the resting BCR was
constitutively phosphorylated and activated in CD45-negative cells. In
the parental cells, both regulatory sites were phosphorylated on BCR
ligation. Taken collectively, these results suggest that CD45 keeps
both BCR-associated and total cytoplasmic pools of Lyn in an inactive
state, and a mechanism by which Lyn is activated by relative reduction
of CD45 effect may be operative on BCR ligation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Extensive analyses have demonstrated that one of the crucial enhancers of BCR signaling is the receptor-type protein tyrosine phosphatase, CD45 (8, 9, 10, 11, 12). CD45 has been implicated in both T and B cell activation based on experiments using CD45-deficient cells (13, 14, 15, 16, 17) and cells from CD45 gene-targeted mice (18, 19). It has been shown that CD45 in T cells dephosphorylates Lck and Fyn at the COOH-terminal negative regulatory sites, Tyr505 (20, 21, 22, 23, 24) and Tyr531 (25, 26, 27), respectively. From these findings, a view has emerged that the binding of the phosphorylated COOH-terminal tyrosine residue of a Src-family PTK to its own SH2 domain causes the PTK to assume an inactive conformation. CD45 activates the kinase by dephosphorylating the COOH-terminal tyrosine, thereby releasing the inhibitory intramolecular conformation. However, there are also discrepant results in which Lck and Fyn are hyperphosphorylated and activated in CD45-deficient YAC-1 T cell clones. In these experiments, the sites of phosphorylation include both the negative COOH-terminal tyrosine (Tyr505) and the positive autophosphorylation site (Tyr394) of Lck (28, 29).
We have previously demonstrated that in CD45-deficient clones generated from immature WEHI-231 cells, Lyn is selectively hyperphosphorylated and activated in the absence of BCR ligation. Furthermore, receptor stimulation did not significantly enhance phosphorylation and activation of Lyn (30). BCR-induced Ca2+ mobilization, growth arrest, and apoptosis were also negatively regulated by CD45 in WEHI-231 cells (16). In contrast, CD45 exerted positive, crucial effects on BCR-induced growth arrest and, to a lesser extent, BCR-induced tyrosine phosphorylation in mature BAL-17 B cells (17). Recent studies on two different B cell lines revealed somewhat different mode of action of CD45. In CD45 gene-targeted clones from the chicken DT40 B cell line, Lyn was shown to be hyperphosphorylated on BCR ligation at the COOH-terminal negative regulatory residue as well as the positive regulatory tyrosine. However, the kinase activity of Lyn was reduced as compared with the activity of wild-type cells (31). In CD45-negative, BCR-transfected J558Lµm3 plasmacytoma cells, only the COOH-terminal tyrosine of Lyn was constitutively hyperphosphorylated, and the enzymatic activity of Lyn was significantly lower than its counterpart isolated from CD45-positive cells (32). The reasons for these differences have not yet been well defined.
This study was initiated to elucidate the mechanisms whereby CD45
regulates Lyn tyrosine kinase in immature B cells. Studies on Lyn in
immunoprecipitates from 32P-labeled WEHI-231
CD45-deficient cells with anti-Ig Ab demonstrated that Lyn,
total cellular as well as BCR-associated, was constitutively tyrosine
phosphorylated and activated. Cyanogen bromide (CNBr) cleavage mapping
of Lyn clearly demonstrated that both the COOH-terminal negative
regulatory residue (Tyr508) and the positive
regulatory residue (Tyr397) were phosphorylated
before BCR stimulation in CD45-deficient cells. In the parental cells,
by contrast, phosphorylation of not only Tyr397 but also
Tyr508 was induced by BCR ligation. Thus, in WEHI-231
cells, the main function of CD45 is to dephosphorylate two major
regulatory residues, Tyr508 and Tyr397, and
inactivate Lyn kinase in both the receptor-associated and total
cellular pools before BCR ligation. These results also suggest a
mechanism by which BCR ligation reduces the negative effect of CD45,
thereby inducing the phosphorylation and activation of Lyn kinase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The WEHI-231 cell line and its CD45-deficient clone, 10-5, were described in previous reports (16, 30). These cells were maintained in RPMI 1640 supplemented with 10% FBS, 50 µM 2-ME, 100 µg/ml streptomycin, and 100 U/ml penicillin.
Abs and reagents
Goat (Fab')2 fragments of anti-mouse
IgM Ab and intact anti-mouse IgM Ab were purchased from Cappel,
Organon Teknika (Durham, NC). Polyclonal Ab against Lyn was purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine
(PY) mAb (4G10) was purchased from United Biotechnology (Lake Placid,
NY). Anti-Ig Ab was raised by immunizing rabbits with GST fusion
proteins containing the cytoplasmic tails of the protein. Abs were then
purified using the immunogen. Alkaline phosphatase (AP)-conjugated goat
anti-mouse IgG and mouse anti-rabbit IgG were obtained from
Bio-Rad (Richmond, CA), and Jackson Immunoresearch Laboratories (West
Grove, PA), respectively.
Enolase was purchased from Sigma (St. Louis, MO).
Cell stimulation, immunoprecipitation, and Western blot analysis
Cells were harvested from log phase cultures, resuspended in fresh prewarmed RPMI 1640 containing 10% FBS supplemented with 20 mM HEPES, and incubated for 3 h at 37°C. The cells were then stimulated with 25 µg/ml F(ab')2 fragments of anti-IgM Ab for 1 min, and the reactions were terminated with ice-cold PBS containing 2 mM Na3VO4 and 2 mM EDTA (PBS-VE). The cells were centrifuged and solubilized in TNE lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM Na3VO4, 2 mM EDTA) or digitonin lysis buffer (1% digitonin, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM Na3VO4, 2 mM EDTA) supplemented with Protease Inhibitor Mixture (Boehringer Mannheim GmbH, Mannheim, Germany). The lysates were centrifuged at 10,000 x g at 4°C for 30 min, and the supernatants were subjected to further analysis.
Immunoprecipitation and Western blot analyses were performed as
previously described (30). Each sample was
immunoprecipitated with protein G-Sepharose (Pharmacia Biotech,
Uppsala, Sweden) coupled with Ab against Lyn or Ig. Anti-Lyn
immunoprecipitates were boiled with SDS sample buffer under reducing
conditions and subjected to 10% SDS-PAGE. Separated proteins were
blotted onto nitrocellulose membranes, and the membranes were incubated
overnight with anti-PY mAb and anti-Lyn Ab, followed by
AP-conjugated goat anti-mouse IgG and mouse anti-rabbit IgG,
respectively. The blots were visualized by developing them with an AP
Conjugate Substrate kit (Bio-Rad, Hercules, CA). Anti-Ig
immunoprecipitates were washed with 0.5% digitonin lysis buffer, and
dissolved in 1 µl of 1% Nonidet P-40, 0.1%SDS lysis buffer. The
supernatants were immunoprecipitated with anti-Lyn Ab- or normal
rabbit IgG-coated protein G beads. The precipitates were washed with
TNE and then subjected to SDS-PAGE or to in vitro kinase assays. The
intensity of each band was measured with a Bio-Rad densitometer.
In vitro kinase assay
In vitro kinase assay was performed as previously described
(30). Cells were solubilized in TNE or digitonin lysis
buffer, and the supernatants were immunoprecipitated with anti-Lyn
Ab or anti-Ig and anti-Lyn Abs. The immunoprecipitates were
washed with lysis buffer and then with kinase buffer (20 mM HEPES, pH
7.5, 150 mM NaCl, 10 mM magnesium acetate, 20 mM
MnCl2). For in vitro kinase assays, 0.35 MBq
[
-33P]ATP (37–110 TBq/mmol, Amersham,
Arlington Heights, IL) in kinase buffer containing 10 mM cold ATP was
added to the anti-Lyn immunoprecipitates. For anti-Ig and
anti-Lyn immunoprecipitates from digitonin lysates, reactions were
performed in the same buffer without cold ATP together with exogenous
substrate enolase. To assess phosphorylation of Tyr508 of
Lyn, 1 µl of recombinant COOH-terminal Src kinase (Csk)
(33) was added to the anti-Lyn immunoprecipitates
during the in vitro kinase assay. The reactions were terminated by
adding SDS sample buffer, and the samples were subjected to 10%
SDS-PAGE analysis. The resulting gels were treated with 1 N KOH at
60°C for 90 min to hydrolyze phosphoserine and phosphothreonine,
dried, and analyzed with a BAS 2000 Bio-Imaging Analyzer (Fuji Photo
Film, Tokyo, Japan).
Metabolic labeling and CNBr digestion
Cells were washed with phosphate-free RPMI 1640 and cultured at 4 x 106 cells/ml for 3 h at 37°C in phosphate-free medium supplemented with 10% dialyzed FBS. The cells were harvested and labeled with 370 MBq of [32P]orthophosphate in 2 ml of medium for 1 h at room temperature. After addition of 8 ml of phosphate-free medium to the culture, cells were incubated for 3 h at 37°C. Labeling was stopped by adding ice-cold PBS-VE, and the labeled cells were lysed and immunoprecipitated with anti-Lyn Ab as described above. Bound proteins were eluted in SDS sample buffer, applied to 10% SDS-PAGE, and transferred onto a nitrocellulose membrane. The Lyn bands (p53 and p56), which were identified by autoradiography and subsequent immunoblotting, were excised and eluted with 250 µl of 150 µg/ml CNBr in 70% formic acid at room temperature for 2 h. The eluted proteins were dried in a vacuum concentrator (Tomy Seiko, Tokyo, Japan) to remove formic acid, and the final pellets were dissolved in SDS sample buffer and applied to SDS-PAGE in 15–25% gradient gel. The separated proteins were transferred to a polyvinylidine difluoride (PVDF) membrane and subjected to BAS 2000 analysis.
Phosphoamino acid analysis
Phosphoamino acid analysis was performed as described earlier (34). 32P-labeled proteins were excised from a PVDF membrane and hydrolyzed with 6 N HCl at 110°C for 2 h. The samples were dried, dissolved in 5 µl of distilled water, and spotted onto TLC plates. Electrophoresis was conducted in pH 1.9 buffer for 20 min at 1.5 kV. After drying, the plates were subjected to second dimension electrophoresis in pH 3.5 buffer for 16 min at 1.3 kV. Separated phosphoamino acids were analyzed with a BAS 2000.
In vitro dephosphorylation assay
WEHI-231 cells stimulated with or without anti-IgM Ab were lysed with TNE, and Lyn was immunoprecipitated and subjected to in vitro kinase assays to phosphorylate the autophosphorylation site. CD45 was immunoprecipitated from WEHI-231 cells by using anti-CD45 mAb. The immunoprecipitates were washed with TNE, and CD45 was eluted with 50 µl 0.17 M glycine-HCl buffer, pH 2.0. The eluted supernatants were neutralized immediately by adding 0.45 ml 1 M Tris-HCl buffer, pH 9.0, and dialyzed to PBS. A phosphatase assay was performed by incubating autophosphorylated Lyn with 0.5 ml purified CD45 for 60 min at 37°C. After incubation, the precipitates were washed with TNE and subjected to SDS-PAGE. The results were visualized by autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the immature B cell line WEHI-231, absence of CD45 led to
constitutive hyperphosphorylation and activation of Lyn, but not other
PTKs including Lck, Blk, Btk, and Syk (30) (T.K. and H.Y.,
unpublished observations). Representative results from anti-PY blot
analyses and in vitro kinase assays of Lyn in WEHI-231 and its
CD45-deficient clone (10-5) are shown in Fig. 1
A. Anti-IgM stimulation of
the parent cells induced a 7.7-fold increase in tyrosine
phosphorylation, but in the CD45-deficient clone, tyrosine
phosphorylation was constitutively elevated 3.6-fold and was not
enhanced further by BCR ligation. The kinase activity of Lyn was
induced only after BCR ligation in the parent, whereas Lyn was
constitutively activated (3.8-fold) in CD45-deficient cells, and the
activity was slightly increased (to 5.5-fold) on BCR ligation.
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B). These results suggest that CD45 selectively
dephosphorylates and inactivates Lyn in WEHI-231 cells. Negative
regulation of a Src-family kinase by CD45 is in apparent contrast to
the prevailing view that CD45 dephosphorylates the COOH-terminal
negative regulatory tyrosine residue, thereby activating the kinase
activity (8, 11). Both negative and positive regulatory tyrosine residues are dephosphorylated by CD45
To address the mechanisms by which CD45 regulates Lyn activity,
the dephosphorylation sites were determined by the CNBr cleavage
method. CD45-positive and -negative WEHI-231 cells were metabolically
labeled with [32P]orthophosphate, and Lyn
immunoprecipitations were treated with CNBr. The resulting fragments
were resolved by SDS-PAGE. CNBr cleavage of Lyn is expected to yield a
fragment of 4.1 kDa containing the COOH-terminal negative regulatory
tyrosine residue (Tyr508) and a fragment of 8.2 kDa
containing the autophosphorylation site (Tyr397), among
others (Fig. 2A). In the
parental cells, BCR ligation induced phosphorylation of 8.2- and
4.1-kDa fragments by 2.7- and 3.5-fold, respectively (Fig. 2B). In CD45-deficient cells, however, phosphorylation of
the 8.2-kDa fragment was 
2-fold greater than control even in the
absence of anti-IgM stimulation, and the 4.1-kDa fragment was even
more strongly phosphorylated (4.1-fold). The phosphorylation levels of
the respective fragments were not significantly enhanced by BCR
stimulation in the CD45-deficient clone, 10-5. The 8.2-kDa fragment
corresponded to a band containing the positive regulatory tyrosine
residue Tyr397, as assessed by its size and phosphorylation
in an in vitro kinase assay (Fig. 2C). The 4.1-kDa fragments from both
WEHI-231 and 10-5 were phosphorylated by Csk (Fig. 2D),
suggesting that it contained the negative regulatory residue
Tyr508. These results suggest that BCR ligation induces
phosphorylation of Lyn not only at the autophosphorylation site but
also at the negative regulatory tyrosine residue. The net result of
phosphorylation at both sites is an activation of the kinase.
Furthermore, in the absence of CD45, both the positive and negative
regulatory tyrosine residues of Lyn were constitutively phosphorylated;
nevertheless, the kinase activity was increased.
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C, Lyn prepared by this
method was phosphorylated predominantly at Tyr397 but not
at Tyr508. When incubated with autophosphorylated Lyn for
60 min, CD45 strongly dephosphorylated Lyn (Fig. 3, lane 2 vs lane
4). Thus, CD45 dephosphorylates not only the negative but also the
positive regulatory tyrosine residues both in vitro and in
vivo.
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Previous reports suggested that CD45 exerts its regulatory effects
differently on the total pool and the TCR- or CD4-associated pools of
Src-family PTKs (27, 35). It is therefore possible that
the observed regulation of CD45 in WEHI-231 applied only to the total
pool of Lyn and not to the more physiologically relevant subpool of Lyn
associated with the BCR. To exclude this possibility, we examined the
phosphorylation state and the kinase activity of Lyn in the BCR
complex. WEHI-231 and its CD45-deficient clone 10-5 were labeled with
[32P]orthophosphate. After incubating with or
without 25 µg/ml anti-IgM Ab for 1 min, cells were lysed with
digitonin and immunoprecipitated with anti-Ig Ab. The
immunoprecipitates were dissolved in 1% Nonidet P-40,
immunoprecipitated with anti-Lyn Ab or control IgG, and subjected
to SDS-PAGE analysis. The results revealed that on BCR ligation,
p53Lyn and p56Lyn were
phosphorylated in the parental cells (Fig. 4A, lane 2) but
that in the CD45-deficient cells, Ig-associated Lyn was
hyperphosphorylated even in the absence of BCR stimulation (Fig. 4A, lane 3). Control IgG did not precipitate Lyn
(Fig. 4A, lanes 5 and 6). Similar results were
obtained with anti-Ig
immunoprecipitation (data not shown).
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Ab.
Compared with the parental cells, Lyn activity, both
autophosphorylation and phosphorylation of enolase was significantly
increased even before BCR ligation in the CD45-deficient clone 10-5
(Fig. 5A, lanes 1, 2 vs
lanes 3, 4). These results suggest that both
receptor-associated and not associated Lyn pools were constitutively
hyperphosphorylated and activated in the absence of CD45. Thus, CD45
dephosphorylates both the positive and negative regulatory tyrosine
residues of Lyn, and the net effect is inactivation of the kinase.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Phosphorylation was observed in the absence of BCR
stimulation and occurred on both the negative regulatory
Tyr508 and the positive regulatory Tyr397 (Fig. 2). This negative regulation was observed not only in the total
cellular Lyn pool but also in the more physiologically important pool
of kinases associated with the BCR (Figs. 4 and 5). Thus, CD45
dephosphorylates the two major regulatory tyrosine residues and
inactivates Lyn kinase in WEHI-231 cells.
Accumulating evidence suggests that Src-family PTKs are regulated in
part by tyrosine phosphorylation of the inhibitory site in the
COOH-terminal tail and the stimulatory site in the kinase domain
(36). The different phosphorylation states stabilize a
repressed or an activated conformation respectively. Csk phosphorylates
the COOH-terminal tyrosine, keeping Src-family PTKs in a repressed
conformation (33). Therefore, one way to activate
Src-family PTKs is by dephosphorylating the COOH-terminal tyrosine.
Mutations in the kinase and SH2 and SH3 domains led to activation of
Src, suggesting that conformational changes in these regions are also
important factors for activating Src-family PTKs (36). Our
results showing that a Src-family PTK can be active even if its
negative regulatory COOH-terminal tyrosine is phosphorylated appear to
contradict the conventional model of regulation. However, there are
several explanations for our findings. One is that the phosphorylation
state of the positive regulatory tyrosine is dominant over that of the
negative regulatory tyrosine as has been suggested previously
(29). An alternative possibility is that in Src-family
PTKs that have phosphorylated COOH-terminal tyrosines, activation can
occur by a phosphorylation-independent mechanism. It is also possible
that the observed phosphorylation at both sites could be caused by the
presence of two populations of Lyn, each phosphorylated at different
residues. In this case, the enhanced kinase activity is a reflection of
the dominant presence of Lyn phosphorylated at the stimulatory
Tyr397. This possibility has not been examined in this or
any other studies. Given that phosphorylation at the inhibitory
Tyr508 was 2.2-fold higher than that at the stimulatory
Tyr397 in activated Lyn (Fig. 2B), we think this
possibility is unlikely.
It has been argued that the main function of CD45 is to activate Src-family PTKs. A consensus model is that Csk phosphorylates the COOH-terminal negative regulatory tyrosine residue of Src-family PTKs, keeping PTKs in an inactive conformation, and that on Ag receptor ligation CD45 dephosphorylates the negative regulatory tyrosine, activating their enzymatic activity. This was based on the initial descriptions of CD45-negative T cell lines in which the Src-family PTKs were found to be hyperphosphorylated at their COOH-terminal tyrosines and inactive (20, 21, 22, 23, 24, 25, 26, 27, 37). However, subsequent studies revealed that the function of CD45 might be more complex. In CD45-negative human HPB-ALL T cells, anti-CD4-induced activation and tyrosine phosphorylation of Lck, particularly the CD4-associated pool, was higher than those of CD45-positive cells (35). Studies on three T cell lines, YAC-1, SAKRTLS, and HPB-ALL, demonstrated that Lck and Fyn are constitutively hyperphosphorylated and paradoxically activated in the CD45-negative clones and that in vitro exposure of CD45 to Lck leads to decreases, rather than increases, in the kinase activity (28). CNBr cleavage mapping showed that Lck in the CD45-negative clones is hyperphosphorylated at both the negative regulatory tyrosine residue (Tyr505) and the autophosphorylation site (Tyr394) (28). Phosphoamino acid analysis confirmed that the increased phosphorylation in CD45-negative YAC-1 cells is restricted to tyrosine residues, Tyr505 and, to a lesser extent, Tyr394 (29). Further mutational analysis revealed that mutation of Tyr505 to phenylalanine results in increased kinase activity whereas mutation of Tyr394 to phenylalanine decreases enzymatic activity. The double mutation, Tyr505-Phe and Tyr394-Phe, led to inactivation of the kinase (29). These results suggest that without CD45, phosphorylation at Tyr394 induces activation of Lck despite hyperphosphorylation at Tyr505 and that the phosphorylation state of Tyr394 may have a dominant role in the regulation of Lck.
In B cells, there have been discrepant reports as well. Lyn from CD45-negative, BCR-reconstituted J558Lµm3 plasmacytoma cells was shown to be hyperphosphorylated at the COOH-terminal tyrosine residue. The kinase was neither activated nor recruited to the BCR complex (32). A study on the chicken DT40 B cell line showed that CD45 dephosphorylates both the positive and the negative regulatory tyrosine residues of Lyn but the activity of Lyn is enhanced in the presence of CD45 and that BCR-induced tyrosine phosphorylation and Ca2+ mobilization are severely impaired in the absence of CD45 (31). Although the sites dephosphorylated by CD45 are different, both studies underscore the importance of dephosphorylation of the COOH-terminal tyrosine of Lyn in certain processes of BCR-mediated activation. In contrast, our previous reports (16, 30), together with the present study, propose a different mode of CD45 action, in which CD45 dephosphorylates both Tyr508 and Tyr397 of Lyn and keeps the kinase in an inactive state, exerting negative modulatory effects on BCR-induced Ca2+ mobilization, growth arrest, and apoptosis. It is also important that BCR ligation induces phosphorylation of not only Tyr397 but also Tyr508, yet activates the kinase. All these results suggest that there must be a mechanism whereby BCR ligation negates the effect of CD45, thus inducing phosphorylation of two regulatory sites and activation of the kinase.
What accounts for all the phenotypic differences is not clearly elucidated at present. Recent crystal structural and functional analyses on the inactive form of Src-family PTKs, c-Src and Hck (38, 39, 40), may give us an important hint as to the role of CD45 in the regulation of this family of PTKs. These studies demonstrated that although the SH2 domain binds to the phosphorylated COOH-terminal tail, this interaction does not block the catalytic site. Rather, the kinase is probably inactive because the linker sequence between the catalytic and SH2 domains binds the SH3 domain (38, 39, 41). Furthermore, the PTK that is phosphorylated at the COOH-terminal tyrosine was shown to be activated by disturbing these intramolecular interactions with exogenous SH2 and SH3 ligands (40). Thus, regulation of Src-family PTKs seems to be very complex such that the state of phosphorylation at the two major regulatory tyrosine residues is but one determiner of activation. Various factors, including unknown SH2 and SH3 ligands, contribute to the net enzymatic activity. It may also be possible that tyrosine phosphorylation at the two sites is a consequence of activation or inactivation induced by conformational alterations that dictate the accessibility of Src-family PTK phosphorylation sites to other PTKs and protein tyrosine phosphatases (36). In light of these structure-function relationships, our findings are not necessarily idiosyncratic but reflect a common regulatory activity of CD45. This is supported by observations in other CD45-negative B cell clones in which BCR-induced tyrosine phosphorylation of total cellular proteins is slightly reduced, but phosphorylation of a few species of proteins is almost completely defective (17). Differences observed in the PTK activity in a variety of CD45-deficient cells may be explained partly by the different availability of molecules within the cell capable of affecting the conformation of the Src-family PTKs (42).
In summary, CD45 has an inhibitory effect on Lyn by dephosphorylating the autophosphorylation site as well as the COOH-terminal regulatory tyrosine in immature WEHI-231 cells. This regulation exerted on not only total cellular Lyn but also the BCR-associated pool of Lyn. Furthermore, the fact that both regulatory tyrosines are phosphorylated and activated by BCR ligation suggests that BCR signaling machinery may have a mechanism by which the negative effect of CD45 is somehow inhibited.
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Acknowledgments |
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Footnotes |
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2 Current address: Howard Hughes Medical Institute, Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
3 Current address: Department of Microbiology and Immunology, Tokyo Women’s Medical University, School of Medicine, Tokyo 162-8666, Japan.
4 Address correspondence and reprint requests to Dr. Hidetaka Yakura, TMIN, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan. E-mail address:
5 Abbreviations used in this paper: BCR, B cell Ag receptor; PTK, protein tyrosine kinase; PY, anti-phosphotyrosine; AP, alkaline phosphatase; PVDF, polyvinylidine difluoride.
Received for publication August 17, 1998. Accepted for publication May 24, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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