(Received for publication, August 30, 1995)
From the
It has been well established that protein-tyrosine phosphatase CD45 is critically involved in the regulation of initial tyrosine phosphorylation and effector functions of T and B cells. However, the signaling pathway governed by CD45 is not completely understood. In B cells, it has not been unequivocally resolved as to which protein-tyrosine kinases (PTKs) associated with B cell antigen receptor are regulated by CD45 in intact cells. As a first step toward the elucidation of CD45-initiated signaling events, we have tried to identify physiological substrates for CD45 by analyzing PTK activity in CD45-deficient clones recently generated from the immature B cell line WEHI-231. The results clearly demonstrated that among PTKs examined (Lyn, Lck, and Syk), only Lyn kinase is dysregulated in the absence of CD45 such that without B cell antigen receptor ligation, Lyn is hyperphosphorylated and activated in CD45-negative clones. Thus, Lyn seems to be a selective in vivo substrate for CD45 in immature B cells.
Ligation of B cell antigen receptor (BCR) ()by
anti-IgM antibody or multivalent antigens rapidly induces tyrosine
phosphorylation of a number of cellular
proteins(1, 2, 3) . This process is initiated
by the activation of protein-tyrosine kinases (PTKs) associated with
BCR; Src family PTKs (Lyn, Blk, Lck, and Fyn) (4, 5, 6) and a non-Src family PTK
(Syk)(7) . Signals are then propagated by phosphorylating
downstream substrates. Among the substrates characterized,
phospholipase C
(8, 9) catalyzes the breakdown of
phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate
and diacylglycerol, which results in the release of Ca
from cytoplasmic stores and the activation of protein kinase C,
respectively. BCR ligation also induces activation of
phosphatidylinositol 3-kinase (10, 11) and
p21
(12, 13) among others. All
these signaling events ultimately lead to activation, cell death, or
anergy, depending on the maturational stages of B cells or the nature
of the initial stimuli (14, 15) .
It has been well
established that protein tyrosine phosphorylation is tightly balanced
by protein-tyrosine phosphatases in a reversible
fashion(16, 17) . One of the key protein-tyrosine
phosphatases is a prototypic receptor-type
CD45(18, 19) . The requirement for CD45 in T and B
cell activation has been substantiated by studies in which
CD45-deficient cells were
used(20, 21, 22, 23, 24) .
In T cells, CD45 dephosphorylates CD4- or CD8-associated Lck at a
C-terminal negative regulatory site (Tyr-505) both in vivo(25) and in vitro(26, 27, 28) and TCR-associated Fyn at a
negative regulatory site
(Tyr-531)(29, 30, 31) . Furthermore, CD45 has
been shown to be physically associated with Lck (32) and
Fyn(33) . It is thus likely that Lck and Fyn constitute
physiological substrates for CD45 in T cells. In contrast, although
there have been reports showing that CD45 dephosphorylates
BCR-associated Ig- (CD79a) and Ig-
(CD79b) in vitro(22) and is physically associated with Ig-
, Ig-
,
and Lyn, but not Blk or Fyn in spleen B cells(34) ,
physiologically relevant substrates for CD45 in B cells have not been
firmly established.
We have previously shown that in CD45-deficient clones generated from the immature B cell line WEHI-231, tyrosine phosphorylation of total cell lysates is constitutively higher than in the parental cells(23) . In this study, we tried to elucidate how CD45 regulates the activity of PTKs associated with BCR complex in in vivo conditions, using the combination of these cells. The results presented herein demonstrated that Lyn is hyperphosphorylated and activated in CD45-negative clones without BCR stimulation, whereas the activities of Syk and Lck are not significantly changed between CD45-positive and -negative cells. Thus, CD45 selectively dephosphorylates Lyn kinase and yet inhibits its kinase activity. The precise regulatory mechanism of Lyn by CD45 remains to be defined.
This study was initiated to examine the regulation of PTKs
associated with BCR complex by CD45 and to determine which PTKs
constitute in vivo substrates for CD45 in immature B cells. To
this end, we used CD45-deficient clones generated from the immature B
cell line WEHI-231 in which tyrosine phosphorylation was dysregulated (23) . The expression of signal-transducing molecules was
examined in the parent WEHI-231 cells, CD45-negative clones (10-5 and
39-2), and a revertant clone (39-2rvt) by Western blot analysis (Fig. 1). The parent and CD45 expression mutants equally
expressed Src family PTKs, Lyn and Lck, and non-Src family PTKs, Syk
and Csk (data not shown). Phospholipase C(8, 9) ,
phosphatidylinositol 3-kinase(10, 11) , and
hematopoietic cell protein-tyrosine phosphatase (protein-tyrosine
phosphatase 1C), which is also implicated in the regulation of
BCR-mediated signaling(35, 36) , were all expressed
comparably among different groups of cells (data not shown). However,
Fyn was not detected in all groups (Fig. 1). The absence of Fyn
in WEHI-231 cells was reported previously(37) , and it has been
suggested that this may be one of the characteristics of immature B
cells(38) .
Figure 1: Expression of Src family and Syk PTKs in CD45-negative and -positive WEHI-231 cells. Total cell lysates were separated on 10% SDS-PAGE gels, transferred to a nitrocellulose membrane, and probed with Abs against CD45, Lyn, Lck, Fyn, and Syk.
Because loss of CD45 led to an increase in
constitutive tyrosine phosphorylation(23) , candidate substrate
PTKs for CD45 are expected to be more activated in CD45-negative clones
without BCR stimulation than in the parent. First, enzymatic activity
of Lyn was examined. CD45-negative and -positive WEHI-231 cells were
unstimulated or stimulated with anti-IgM Ab for 1 min and
immunoprecipitated with anti-Lyn Ab. In vitro kinase assays
were then performed on these immunoprecipitates. As shown in Fig. 2, Lyn kinase activity was induced 10-fold upon BCR
stimulation in CD45-positive cells. In contrast, in the absence of BCR
stimulation, both autophosphorylation and phosphorylation of the
exogenous substrate enolase were much greater in the CD45-negative
clones than in the parent. Densitometric analysis revealed that
autophosphorylation and phosphorylation of enolase were increased by
6- and
9-fold, respectively. Anti-IgM stimulation induced an
increase in Lyn kinase activity only marginally in the CD45-negative
clones (1-3-fold). These results were consistently observed in
five separate experiments.
Figure 2: Increase in Lyn kinase activity in CD45-negative WEHI-231 clones. CD45-positive (parent and revertant) and CD45-negative (39-2 and 10-5) cells were either unstimulated(-) or stimulated with 25 µg/ml anti-IgM Ab for 1 min (+), lysed, and incubated with protein G-Sepharose coupled with anti-Lyn Ab. In vitro kinase assays were performed for 3 min without (Experiment 1) or with exogenous substrate enolase (Experiment 2).
To examine how the tyrosine
phosphorylation state of Lyn is altered in cells without CD45,
immunoblot analysis with anti-phosphotyrosine Ab was performed on Lyn
in CD45-negative and -positive cells. As shown in Fig. 3,
anti-IgM stimulation induced a 2-fold increase in tyrosine
phosphorylation of Lyn from the parent, whereas Lyn in the
CD45-negative clones was phosphorylated
2-fold greater than in the
parent even without BCR stimulation. As in the kinase activity,
anti-IgM Ab did not increase significantly the level of Lyn
phosphorylation in the CD45-negative clones. Taken together with the
results of in vitro kinase assays, it is suggested that CD45
decreases the degree of tyrosine phosphorylation of Lyn and inhibits
its kinase activity.
Figure 3: Tyrosine phosphorylation of Lyn in parent and CD45-negative clones. Cells were either stimulated with 25 µg/ml anti-IgM Ab for 1 min (+) or unstimulated(-), solubilized, and immunoprecipitated with protein G-Sepharose coated with anti-Lyn Ab. The immunoprecipitates were subjected to SDS-PAGE and blotting to a membrane. The blots were probed sequentially with anti-phosphotyrosine 4G10 Ab and then anti-Lyn Ab.
The activity of Src family PTKs is regulated by tyrosine phosphorylation of them. In T cells, it has been clearly demonstrated that CD45 dephosphorylates the C-terminal negative regulatory residue of Lck (25, 26, 27, 28) or Fyn(29, 30, 31) , activating their enzymatic activity. Thus, negative regulation of Lyn activity by CD45 in WEHI-231 cells is in apparent contrast to the mode of CD45 action previously defined. One of the interpretations for this phenomenon is that CD45 dephosphorylates the positive regulatory site or autophosphorylation site of Lyn, thereby decreasing the enzymatic activity. Given the recent report that loss of CD45 in T cell lines results in hyperphosphorylation of the C-terminal tyrosine of Lck (Tyr-505) and paradoxically increases Lck kinase activity(39) , it is also possible that CD45 dephosphorylates the C-terminal negative regulatory residue of Lyn and inhibits its kinase activity. We are currently differentiating these alternatives by investigating which tyrosine residue(s) is hyperphosphorylated in the CD45-negative clones.
To address the selectivity of CD45 substrates, the enzymatic activity of other PTKs, Lck and Syk, was examined in the CD45-negative clones. Fig. 4shows the results of in vitro kinase analyses on Lck. The constitutive kinase activity was low and not significantly different among cells with or without CD45. BCR stimulation induced similar levels of Lck activity both in the CD45-negative and -positive cells, suggesting that Lck kinase activity is regulated independently of CD45. Next, tyrosine phosphorylation and kinase activity of Syk were analyzed. As shown in Fig. 5A, Syk was tyrosine-phosphorylated at 1 min after BCR stimulation in the parental cells, and the degree of phosphorylation in the CD45-negative clones was similar to the parent. In vitro kinase assays demonstrated that constitutive and BCR-induced autophosphorylation and phosphorylation of an exogenous substrate (myelin basic protein) in the CD45-deficient clones are comparable with those in the parent (Fig. 5B). These results suggest that regulation of Syk is also independent of CD45. Given the absence of a negative regulatory tyrosine residue analogous to the Src family PTKs in Syk, it seems reasonable that CD45 is not directly involved in Syk regulation. The selective regulatory action of CD45 on Lyn kinase is consistent with the report that CD45 is physically associated with Lyn, but not Blk or Fyn, in splenic B cells(34) .
Figure 4: Lck kinase activity in CD45-negative and -positive WEHI-231 cells. Cells were unstimulated(-) or stimulated with 25 µg/ml anti-IgM Ab for 1 min (+), lysed, and immunoprecipitated with anti-Lck-coupled protein G-Sepharose. In vitro kinase assays were performed for 15 min with enolase. The results are representative of three separate experiments.
Figure 5: Tyrosine phosphorylation and kinase activity of Syk in parent and CD45-negative clones. A, cells, unstimulated(-) or stimulated with 25 µg/ml anti-IgM Ab for 1 min (+), were immunoprecipitated with anti-Syk Ab. The immunoprecipitates were separated by SDS-PAGE and blotted to a membrane. The blots were probed sequentially with anti-phosphotyrosine 4G10 Ab and then anti-Syk Ab. B, cells were unstimulated or stimulated with anti-IgM Ab for 1-15 min, and Syk protein was immunoprecipitated. In vitro kinase analyses were performed for 15 min in the presence of myelin basic protein (MBP) as an exogenous substrate.
It has been demonstrated that Syk is constitutively associated with BCR (40, 41) and TCR complex(42) . However, regulation of Syk activation and the relationship between Syk and Src family PTKs in the cascades of antigen receptor signaling have not been fully elucidated. For example, there is a report showing that TCR-induced Syk activation is not dependent on Lck, but Lck can enhance phosphorylation of cellular proteins. Thus, Src family PTKs may be downstream of Syk and serve as amplifiers of signals generated from Syk(42) . Further, coexpression of Syk and Lck in COS cells resulted in the activation of Lck but not Syk, suggesting that activating signals are delivered from Syk to Lck(43) . The other report that BCR-induced tyrosine phosphorylation and activation of Syk kinase are greatly reduced in lyn-negative chicken B cell line (44) points to a possibility of Syk activation by Lyn. In contrast, the results presented herein clearly showed that activated Lyn in CD45-negative clones does not induce phosphorylation and activation of Syk kinase, signifying an alternative possibility that Syk and Lyn may be functioning independently.
What causes these discrepancies is not known at the present time. However, different cell types used in these studies may be one of the explanations. We have recently generated CD45-deficient clones from the mature B cell line BAL-17(24) . In these clones, BCR-induced tyrosine phosphorylation was almost identical to the parent except for a few species of protein, but growth inhibition induced by BCR stimulation was completely abolished(24) . These biochemical and functional alterations by the absence of CD45 regulation are clearly different from the situations in immature WEHI-231 cells(23) . It is thus important to understand how CD45 regulates Src family and Syk PTKs in mature B cells and whether CD45 governs distinct downstream signaling pathways at different maturational stages of B cells. These problems are under investigation.