By
From the * Department of Oncology, Institute of Medical Science, University of Tokyo, Tokyo 108, Japan; Department of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University,
Fukuoka 812-62, Japan; § Department of Biochemistry, Fukui Medical School, Fukui 910-11, Japan;
Research Institute for Bioscience, Science University of Tokyo, Noda 278, Japan; and the ¶ Department of Biochemistry, Kobe University, School of Medicine, Kobe 650, Japan
The 75-kD HS1 protein is highly tyrosine-phosphorylated during B cell antigen receptor (BCR)-mediated signaling. Owing to low expression of HS1, WEHI-231-derived M1 cells, unlike the parental cells, are insensitive to BCR-mediated apoptosis. Here, we show that BCR-associated tyrosine kinases Lyn and Syk synergistically phosphorylate HS1, and that Tyr378 and Tyr-397 of HS1 are the critical residues for its BCR-induced phosphorylation. In addition, unlike wild-type HS1, a mutant HS1 carrying the mutations Phe-378 and Phe-397 was unable to render M1 cells sensitive to apoptosis. Wild-type HS1, but not the mutant, localized to the nucleus under the synergy of Lyn and Syk. Thus, tyrosine phosphorylation of HS1 is required for BCR-induced apoptosis and nuclear translocation of HS1 may be a prerequisite for B cell apoptosis.
Stimulation of the antigen receptor on B lymphocytes
(BCR) induces intracellular biochemical events that include rapid tyrosine phosphorylation of cellular proteins.
Accumulating data reveal that cytoplasmic kinases such as
the Syk kinase and Src-like kinases are associated with the
BCR (1, 2) and play important roles in the signal transduction cascade through the BCR (3). We previously demonstrated that tyrosine phosphorylation of various cellular
proteins was greatly enhanced in COS7 fibroblasts transfected with both Lyn and Syk expression plasmids as compared with those transfected with either the Lyn or Syk
plasmid alone (6). Thus, these kinases may cooperate in
phosphorylating substrates crucial for BCR-mediated B cell
activation.
The 75-kD HS1 protein is highly tyrosine phosphorylated upon BCR cross-linking (7). Studies with HS1 Cell Culture and Antibodies.
Retrovirus producer Plasmid Construction and Preparation of Recombinant Viruses.
The cDNA fragments encoding human Lyn and porcine Syk
were inserted into the expression vector pME-18S (13), to generate pME-Lyn and pME-Syk, respectively. The human HS1
cDNA and its mutant cDNA, encoding a product that lacks 23 amino acids from Tyr-378 to Val-400, were inserted into pME18S to generate pME-HS1 and pME-HS1- DNA Transfection and Viral Infection.
CV-1 cells were transfected with various combinations of pME-Lyn, pME-Syk, pMEHS1, and pME-HS1- Immunoprecipitation and Immunoblotting.
The proteins in the
cell lysates were subjected to immunoprecipitation with Abs to
the human HS1 protein as described (7). The proteins in the lysates or the immunoprecipitates were resolved by 8.5% SDS-PAGE
and transferred to a polyvinylidene difluoride membrane (Bio
Rad, Richmond, CA). Then, the blot was probed with mAb to
phosphotyrosine, PY-20, or anti-human HS1 mAb.
Analysis for BCR-induced Tyrosine Phosphorylation and Apoptosis.
WEHI-231 cells or M1 cells were incubated with or without 20 µg/ml of anti-IgM Ab at 37°C for 1 min. Then, the cells
were lysed and the proteins in the lysates were subjected to immunoprecipitation and immunoblotting. To examine the degree
of the apoptotic death, cells were incubated with or without 4 µg/ml
of anti-IgM for 48 h and the DNA content of the cells was measured as described (9).
Analysis of HS1 Subcellular Localization.
COS7 cells were transfected with various combinations of pME-Lyn, pME-Syk, pMEHS1, and pME-HS1-FF. The cells seeded onto cover slides were
fixed and permeabilized as described (15). Then, the permeabilized cells were incubated for 1 h with anti-human HS1 mAb in
PBS containing 1% BSA at room temperature. The cells on cover
slides were incubated with FITC-labeled goat anti-mouse IgG
and observed using fluorescence microscopy (Zeiss). To examine the amount of nuclear HS1 in the BCR-stimulated WEHI-231
cells, cells were incubated with 20 µg/ml of anti-IgM Ab for the
indicated time. Then, nuclei were separated from the cell homogenates by centrifugation and solubilized as described (7).
The HS1 protein is highly tyrosine-phosphorylated upon
BCR cross-linking (7), probably by Lyn and/or Syk. Because tyrosine phosphorylation of various cellular proteins
was greatly enhanced in fibroblasts transfected with both
Lyn and Syk expression plasmids as compared with those
transfected with either the Lyn or Syk plasmid alone (6),
we examined whether Lyn and Syk can synergistically phosphorylate HS1 in the cells. The results showed that HS1
was highly tyrosine-phosphorylated only when both Lyn
and Syk were coexpressed (Fig. 1). This is consistent with
our previous observation that another Src family member,
Fyn, cooperates with ZAP-70, an analogue of Syk, in phosphorylating HS1 in T cells (12). It has been shown that Lyn
activates Syk when they are coexpressed in fibroblasts (6)
and that BCR cross-linking induces little activation of Syk
in the splenic B cells (Nishizumi, H., unpublished data) and
mast cells (16) of lyn
Of the 17 tyrosine residues in human HS1, Tyr-378 and
Tyr-397 are preceded by acidic residues (ENDY378 and
EGDY397), which are characteristic of many tyrosine phosphorylation sites (17, 18). In fact, the EGDY397EEV sequence of HS1 is the best known substrate for Syk kinase (19). Because tyrosine-phosphorylated HS1 interacts with the SH2 domains of Src family kinases (7), and because a phosphorylated pYED/E sequence shows the highest affinity for
these domains (20), we predicted that the relevant phosphorylation sites on HS1 would be followed by two acidic
residues. Of all the tyrosine residues in HS1, only Tyr-378
and Tyr-397 match the consensus (Y378ED and Y397EE) (Fig.
2 A). These amino acids are also conserved in mouse HS1
(21). Thus, Tyr-378 and Tyr-397 of human HS1 are likely phosphorylated by the BCR-associated kinases. Indeed, when
coexpressed with Lyn and Syk in CV-1 cells, a deletion mutant HS1-
To verify BCR-mediated phosphorylation on Tyr-378
and Tyr-397 of HS1, we generated three HS1 mutants,
HS1-FY, HS1-YF, and HS1-FF, in which Tyr-378, Tyr397, and both have been substituted by phenylalanine,
respectively. These mutants and wild-type HS1 were expressed in WEHI-231 cells by retroviral infection. By probing the anti-human HS1 immunoprecipitates with the antiphosphotyrosine antibody, we showed that HS1-FF was
not detectably tyrosine-phosphorylated upon BCR crosslinking, whereas wild-type HS1 was highly and rapidly tyrosine-phosphorylated (Fig. 2 C). Both HS1-FY and HS1-YF
were phosphorylated at a very low level. Thus, Tyr-378 and
Tyr-397 are important for tyrosine phosphorylation of HS1
upon BCR stimulation.
WEHI-231-derived M1 cells are resistant to mIgMinduced apoptosis, unlike their parental cells. This resistibility is due to very low expression of HS1 in the cells (9).
M1 cells are rendered sensitive to BCR-mediated apoptosis
by the exogenous expression of wild-type HS1 (9). Furthermore, peritoneal B cells from HS1
Despite the presence of a putative nuclear localization
signal, HS1 localizes mainly in the cytoplasm of resting B cells
(7). Consistently, HS1 expressed in COS7 cells was present
in the cytoplasm. However, HS1 coexpressed together
with Lyn and Syk was mostly in the nucleus. In contrast,
HS1-FF remained in the cytoplasm in the presence of Lyn
and Syk (Fig. 4 A). Thus, tyrosine phosphorylation of HS1
appears to be required for its own translocation from the cytoplasm to the nucleus. These data suggest that BCR
cross-linking causes a significant fraction of tyrosine-phosphorylated HS1 to localize to the nucleus. Consistently, subcellular localization experiments showed that the amount
of HS1 in the nucleus is increased after BCR cross-linking
(Fig. 4 B). Similarly, the amount of wild-type human HS1
but not HS1-FF mutant expressed in M1 cells was increased in the nuclei upon BCR stimulation (Nishizumi,
H., unpublished data). Tyrosine phosphorylation on HS1
may trigger its conformational alteration that allows its nuclear translocation and thereby signaling to its downstream
targets.
Because de novo protein synthesis is required for BCRmediated apoptosis of WEHI-231 cells (22), the transcriptional and/or translational regulation of an as yet unidentified gene(s) is involved in the process. HS1 possesses motifs
characteristic of transcription factors (11). Therefore, it may
regulate gene expression as a transcription factor following
translocation into the nucleus, as is proposed for the STAT
family of proteins. Alternatively, HS1 may interact with the
other transcription factors or may transport a protein(s)
critical for apoptosis into the nucleus. Molecules that may
interact with the other motifs, such as the SH3 domain, of
HS1 have yet to be determined.
Basing on the present data we propose a model that,
upon BCR cross-linking, Syk becomes fully activated by
Lyn and phosphorylates HS1 on Tyr-378 and Tyr-397. Because the Lyn SH2 domain binds tyrosine-phosphorylated HS1 (7), phosphorylated Y378ED and Y397EE could be the
binding sites. This interaction would then allow Lyn to
phosphorylate HS1 on other tyrosine residues. The model is consistent with the sequential phosphorylation model in
which the primary kinase phosphorylates a residue that is
directly recognized by the secondary phosphate-directed
kinase. The secondary kinase then phosphorylates another
residue nearby (23). A similar mechanism is proposed for
tyrosine phosphorylation of p130CAS by Abl (24). Accordingly, not only wild-type HS1 but also HS1-YF and HS1FY would be phosphorylated upon BCR stimulation on
Tyr-378 and Tyr-397, respectively, and thereby would interact with Lyn, allowing their further phosphorylation.
However, HS1-YF and HS1-FY were much less tyrosine
phoshorylated than wild-type HS1 (Fig. 2 C), as though no
secondary kinases were available. It should be noted that
Syk can interact with unphosphorylated HS1 and that the
interaction terminates once the HS1 protein becomes tyrosine-phosphorylated at appropriate sites (Fukuda, T., unpublished data). This allows us to speculate that Lyn interacts with HS1 only when the two residues, Tyr-378 and
Tyr-397, become phosphorylated and HS1 is dissociated
from Syk. It is likely that HS1-YF and HS1-FY are still
associated with Syk even after BCR stimulation, which
prevents Lyn from interacting with HS1. Once the two tyrosine residues are both phosphorylated, processive phosphorylation of HS1 by Lyn and the other Src family kinases
would take place, producing hyperphosphorylated form of
HS1. Finally, it is this hyperphosphorylated form of HS1
that translocates to the nucleus and activates B cell apoptosis.
/
mice (8) and with a mutant WEHI-231 cell line that expresses very low level of HS1 (9) suggest that HS1 plays
roles in not only B cell proliferation but also apoptosis
upon BCR cross-linking. In this study, we addressed molecular mechanisms of HS1 phosphorylation and significance of HS1 phosphorylation in BCR-mediated apoptosis.
CRE cells,
CV-1 monkey kidney fibroblasts, and COS7 cells, a derivative of
CV-1 cells expressing SV40 large T antigen, were maintained in
Dulbecco's modified Eagle medium containing 10% calf serum.
WEHI-231 cells and its variant M1 cells (10) were maintained in
RPMI-1640 as described (9). A mouse monoclonal antibody (mAb)
specific to human HS1 was raised against 15 amino acids from
Val-306 to Ser-320 of human HS1 (11). Specificity of the mAb
was confirmed by immunoblotting the lysates of parental and human
HS1-transfected WEHI-231 cells. Rabbit anti-human HS1 sera
were as described (12). Affinity-purified goat Ab to mouse IgM
F(ab
)2 was from Southern Biotechnology (Birmingham, AL),
FITC-labeled goat Ab to mouse IgG was from PharMingen (San Diego, CA), and an anti-phosphotyrosine mAb PY-20 was from
ICN (Irvine, CA).
YY, respectively.
The cDNA fragments encoding human HS1 mutants, HS1-FY,
HS1-YF, and HS1-FF, which carry Phe-378, Phe-397, and Phe378/Phe-397, were also inserted into pME-18S to generate
pME-HS1-FY, pME-HS1-YF, and pME-HS1-FF, respectively. The site-directed mutagenesis was performed as described (14). The retroviral vectors were constructed by cloning the cDNAs of human HS1 and its mutants into the pM5-neo plasmid as described (9).
YY by the standard calcium-phosphate method. 2 d after transfection, cells were washed with serum-free medium and lysed with TNE (1% NP-40, 50 mM Tris, pH 8, 20 mM EDTA, 0.2 mM sodium orthovanadate with aprotinin at
10 µg/ml) buffer (7). To obtain high titer retroviruses carrying
the sequences for the HS1 and mutant proteins,
CRE helper
cells (9) were transfected with the vector plasmids. The WEHI231 cells and M1 cells were infected by the recombinant viruses
and infectants expressing the highest amount of exogenous HS1
or its mutants were cloned as described (9).
/
mice. Accordingly, lyn
/
splenocytes failed to induce tyrosine phosphorylation of HS1 upon BCR cross-linking (4). In contrast, Lyn is not detectably activated by Syk (6). Therefore, our results suggest that
Syk activated by Lyn phosphorylates HS1 directly in BCRmediated signaling.
Fig. 1.
Cooperation between Lyn and Syk in HS1 phosphorylation. CV-1 cells (1.5 × 106) were transfected with the
expression plasmids (5 µg), pMELyn (+), pME-Syk (+), pMEHS1 (+), or with H2O in place
of DNA solution (), and lysed
with TNE buffer. The lysates
were subjected to immunoprecipitation with anti-serum to
HS1. The immunoprecipitates were immunoblotted with PY20 (top). Aliquots (1/50) of the
lysates were immunoblotted with anti-HS1 Ab (bottom). Positions of HS1 and standard protein markers (kD) are indicated.
[View Larger Version of this Image (70K GIF file)]
YY, lacking 23 amino acids from Tyr-378 to
Val-400, was tyrosine phosphorylated at a greatly reduced
level compared with wild-type HS1 (Fig. 2 B).
Fig. 2.
Requirement of Tyr-378 and Tyr-397 in tyrosine phosphorylation of HS1 upon BCR stimulation. (A) Schematic structure of the human HS1 protein. The 23 amino acids sequence of HS1, Tyr-378 to
Val-400, deleted to generate HS1-YY, is shown on the top. Possible
target sites of the SH2 domain of the Src-like kinase are underlined. Two
tyrosine residues, Tyr-378 and Tyr-397, in the sequence are indicated in
bold. The positions of the two tyrosine residues (YY), three helix-turn-
helix repeats (HTH repeats) and the SH3 domain (SH3) are indicated in
the schematic drawing of HS1. The amino acid numbers (from Met-1 to
Glu-486) are shown on the bottom. (B) Tyrosine phosphorylation of HS1
and HS1-
YY in CV-1 cells. CV-1 cells (1.5 × 106) were transfected
with the expression plasmids (5 µg), pME-Lyn, pME-Syk, and pMEHS1-
YY (
YY) or pME-HS1 (HS1), then lysed with TNE buffer. The
lysates were equally divided into two samples and subjected to immunoprecipitation with antiserum to HS1. The immunoprecipitates were analyzed by anti-HS1 (HS1) or by PY-20 immunoblotting. The levels of Lyn
and/or Syk-mediated tyrosine phosphorylation of the other cellular proteins were virtually the same between the cells expressing wild-type HS1
and those expressing HS1-
YY (data not shown). Positions of HS1,
HS1-
YY, and a 96-kD standard protein marker are indicated. (C) Tyrosine phosphorylation of HS1 mutants in WEHI-231 cells. WEHI-231derived cells (1 × 107), which express human HS1 (lanes 1 and 2), HS1FF (lanes 3 and 4), HS1-FY (lanes 5 and 6), or HS1-YF (lanes 7 and 8),
were incubated with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) anti-IgM Ab. The cell lysates were subjected to immunoprecipitation with the
human HS1-specific Ab and the immunoprecipitates probed by anti-phosphotyrosine (PY blot) or anti-human HS1 (hHS1 blot) immunoblotting. BCRmediated tyrosine phosphorylation of the other proteins was not affected by expressing exogenous HS1 or its mutants (data not shown). Positions of HS1
and standard protein markers are indicated.
[View Larger Versions of these Images (19 + 39 + 35K GIF file)]
/
mice do not undergo apoptosis upon BCR cross-linking (8). These data
indicate that HS1 is a critical molecule for BCR-mediated apoptosis. However, unlike wild-type HS1, the exogenously introduced HS1-FF protein failed to restore the sensitivity of M1 cells to BCR-mediated apoptosis (Fig. 3 A).
This was not due to differences in the expression levels of
these proteins. In addition, the HS1-FF protein in M1
cells, as in WEHI-231 cells, was not tyrosine-phosphorylated by BCR cross-linking (Fig. 3 B). Therefore, tyrosine
phosphorylation of HS1 is essential for BCR-mediated
apoptosis.
Fig. 3.
HS1 tyrosine phosphorylation is required for
BCR-mediated apoptosis. (A)
Restoration of BCR-mediated
apoptosis by exogenous HS1 but
not by HS1-FF in M1 cells.
WEHI-231 cells (1 and 2), M1 cells (3 and 4), M1-derived cells
expressing wild-type HS1 (5 and 6), and M1-derived cells expressing the HS1-FF protein (7 and 8) (1-2 × 105) were incubated for 48 h with (2, 4, 6, and
8) or without (1, 3, 5, and 7)
anti-IgM Ab, stained with propidium iodide, and analyzed by
flow cytometry. The DNA content of the cells (non-gated) was expressed as the histogram of
propidium iodide fluorescence
intensity. The percentage of apoptotic cells is denoted in each histogram (above horizontal bars).
The results are representative of
three independent experiments.
Cells containing subdiploid DNA
represent apoptotic cells. (B) Expression and tyrosine phosphorylation of wild-type HS1 and HS1-FF in M1 cells. M1-derived cells (1 × 107), which express
the human HS1 (lanes 1 and 2) or HS1-FF (lanes 3 and 4), were incubated with (lanes 2 and 4) or without (lanes 1 and 3) anti-IgM Ab. The cells were
then lysed in TNE buffer and the lysate were analyzed by immunoprecipitation with the anti-HS1 mAb and by anti-phosphotyrosine (PY blot) or antihuman HS1 (hHS1 blot) immunoblotting of the immunoprecipitates. Positions of HS1 and standard protein markers are indicated.
[View Larger Versions of these Images (39 + 25K GIF file)]
Fig. 4.
Subcellular localization of HS1. (A) Nuclear localization of HS1 coexpressed with Lyn
and Syk. COS7 cells (1.5 × 106)
were transfected with the expression plasmids (5 µg), pME-Lyn, pME-Syk, and either pME-HS1
(b), or pME-HS1-FF (c). As a control, cells were transfected with
pME-HS1 alone (a). Subcellular
localization of the HS1 protein was
investigated by indirect immunofluorescence microscopy using antiHS1 mAb and FITC-labeled second Ab. No significant signal was
detectable in the absence of primary
Ab and in untransfected COS7 cells.
(B) Increment of nuclear HS1 in
BCR-stimulated WEHI-231 cells.
WEHI-231 cells were incubated
with anti-IgM Ab for the time indicated above. Then the cells were
subjected to subcellular fractionation. Samples of nuclear fraction
from 2 × 105 cells per lane were
tested for HS1 by immunoblotting.
[View Larger Versions of these Images (34 + 18K GIF file)]
Address correspondence to Tadashi Yamamoto, Department of Oncology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan. The current address for Y. Yamanashi is the Department of Biology, MIT, 77 Massachusetts Ave., Cambridge, MA 02139.
Received for publication 5 August 1996 and in revised form 29 January 1997.
We thank A. Tanaka, B. Chen, C. Roman, S. Cherry, and R. Gandi for critical reading of this manuscript. Y. Yamanashi is grateful to people at the Yamamoto and Baltimore laboratories for generous help and encouragement.
This work was supported by grants from the Ministry of Education, Sciences, Sports, and Culture of Japan and a grant from the Human Frontier Scientific Program.
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