(Received for publication, March 27, 1997, and in revised form, May 7, 1997)
From the Wistar Institute, Philadelphia, Pennsylvania 19104 and the
Department of Molecular Genetics, Institute for Hepatic
Research, Kansai Medical University, Moriguchi 570, Japan
The tyrosine kinases Syk and Lyn are activated in
B lymphocytes following antibody induced cross-linking of the B cell
receptor for antigen (BCR). It has been suggested that activation of
Syk is dependent on Lyn. We tested this hypothesis by comparing the phosphorylation and activation of several downstream effector molecules
in parental DT40, DT40Syk and DT40Lyn
B cells. The phosphorylation and activation of p90Rsk was ablated in
Syk-deficient B cells but unaffected in Lyn-deficient B cells while the
phosphorylation/activation of Ras GTPase activating protein (Ras GAP)
and mitogen activated protein (MAP) kinase required both Syk and Lyn.
Thus, these data indicate that Syk can be activated in the absence of
Lyn after BCR cross-linking and results in the activation of p90Rsk via
a MAP kinase-independent pathway in DT40Lyn
cells. We
also demonstrated that BCR mediates the activation of p70S6k. However,
activation of p70S6k in DT40Syk
and DT40Lyn
cells was comparable with that observed in parental cells. Thus, either
Syk or Lyn may be sufficient for activation of p70S6k, or activation of
p70S6k occurs independently of both Syk and Lyn. The kinase activity of
Syk was required for the phosphorylation/activation of each of these
downstream effector molecules but only the phosphorylation of Ras GAP
was affected in cells expressing a mutant of Syk in which tyrosines 525 and 526 were substituted to phenlyalanines.
The cytoplasmic protein tyrosine kinase
(PTK)1 Syk is expressed in a wide variety
of hematopoietic cells including B and T lymphocytes, thymocytes,
myeloid lineage cells, mast cells, and platelets (1-5). Syk has been
implicated in the signal transduction pathways of several cell surface
receptors such as the antigen receptors on B (BCR) (6, 7) and T (TCR)
(4, 8) lymphocytes, as well as the Fc receptors (FcR) for IgG (FcRI,
Fc
RII, and Fc
RIII) (9-13) and IgE (Fc
RI) (14-16). Syk has
also been implicated in integrin-mediated signal transduction in B
lymphocytes, a monocytic cell line, and in platelet activation
(17-19). Upon ligation of these receptors, Syk is tyrosine
phosphorylated and catalytically activated. In contrast, the T cell
receptor associated protein tyrosine kinase, ZAP-70, which is
structurally homologous to Syk, has a more limited tissue distribution
(4, 20), and to date, it has only been demonstrated to mediate
signaling by two receptor complexes, the TCR (4) and CD16 (Fc
RIII)
on NK cells (13, 21).
Cross-linking of BCR and TCR induces B and T cell activation. The early
signaling pathways involve rapid tyrosine phosphorylation and
activation of Src and Syk family kinases. Optimal activation of Syk
family kinases, i.e. Syk and ZAP70, in COS cells requires Src family kinases (22, 23). Moreover, the activation of Src family
kinases temporally precedes that of Syk family kinases during T cell
activation (24), suggesting that Src family kinases may be the upstream
effectors of Syk family kinases in T cells. On the other hand, Couture
et al. (25) provided evidence for Syk-mediated activation of
Lck, a member of the Src family of kinases in T cells. A chicken B cell
line, DT40, has been used to examine the hierarchy of activation of Src
and Syk family kinases in B cells. This cell line does not express
detectable levels of many Src family members, including Src, Fyn, Lck,
Blk, Hck, or the Syk-related PTK, ZAP70 (26). Thus, the predominant
tyrosine kinases expressed in this cell line are Lyn and Syk (22, 26). DT40 cells deficient in Lyn and Syk (DT40Lyn and
DT40Syk
, respectively) were generated by gene
interruption (26). It was shown that BCR-induced tyrosine
phosphorylation and activation of PLC-
2 was abolished in
DT40Syk
cells, indicating Syk is required for this event.
In contrast, the phosphorylation of PLC-
2 was only modestly
decreased, and the activation of PLC-
2 was unaffected in
DT40Lyn
cells (22, 26). These data indirectly suggested
that Syk can be activated in the absence of Lyn and lead to activation of PLC-
2.
In this study, we compared DT40Syk and
DT40Lyn
cells with parental DT40 cells to determine the
requirements for Lyn and Syk for the activation of several downstream
effectors of BCR-induced signaling, including Ras GTPase activating
protein (Ras GAP) (27, 28), MAP kinase (29), and the serine/threonine
kinases, p90Rsk and p70S6k, involved in the regulation of ribosomal S6
protein that functions in the efficient translation of some proteins in mitogen-activated cells (30-33). Ras GAP regulates Ras by converting the active GTP bound form of Ras into the inactive GDP bound form and
may also function as an effector of Ras (34, 35). Activation of Ras can
lead to activation of MAP kinases (ERK1 and ERK2), which in turn can
lead to activation of p90Rsk. Alternatively, p90Rsk, as well as p70S6k,
can be activated independently of MAP kinases (36). By comparing
anti-Ig-induced responses in the parental and mutant DT40 cell lines,
we dissected the multiple signal transduction pathways that lead to the
activation of these downstream effectors. Together with previous
results, our data indicate that in addition to activation of signal
transduction pathways that include both Lyn and Syk and lead to
activation, for example of MAP kinase, cross-linking of the BCR also
results in activation of Syk that can occur independently of Lyn and
that is sufficient for the activation of p90Rsk. We also demonstrated that p70S6k is activated in B cells following cross-linking of the BCR.
Finally, either Lyn or Syk is sufficient for activation of p70S6k, or
BCR-induced activation of p70S6k does not require Syk or Lyn.
The chicken B cell line,
DT40, was cultured in RPMI 1640 supplemented with 10% heat-inactivated
fetal bovine serum, 1% chicken serum, 100 µg/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine.
DT40Lyn and DT40Syk
cells were cultured in
the same medium containing 2 mg/ml G418 (37). Wild type and mutants of
Syk were cloned into the expression vector pApuro (38) and transfected
into DT40Syk
cells by electroporation using a gene pulser
apparatus (Bio-Rad) at 330 V, 250 microfarad, and selected in the
presence of 0.5 µg/ml puromycin. Two independent transfectants of
each mutant that expressed comparable amounts of Syk by immunoblotting
were further selected by Southern blotting of genomic DNA (data not shown). All results are representative of at least three experiments using a minimum of two independent clones expressing wild type or each
mutant form of Syk.
For induction of tyrosine phosphorylation of Lyn, Syk, and Ras GAP, cells were stimulated with 10 µg/ml anti-chicken IgM mAb (M4) for 3 min. For activation of p90Rsk and p70S6k, cells in an exponential growth phase were serum starved for 8 and 12 h prior to stimulation, respectively. The indicated numbers of cells were stimulated with 10 µg/ml M4 anti-IgM monoclonal Ab or 100 nM phorbol ester (phorbol 12,13-dibutyrate (PDBu), Sigma) for 30 min at 37 °C. Cells were quickly chilled on ice and pelleted by centrifugation immediately after stimulation. Cell pellets were lysed in 500 µl of ice-cold lysis buffer (1 × PBS, 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml soy bean trypsin inhibitor, 20 µg/ml aprotinin, 100 µg/ml leupeptin, 1% Nonidet P-40, 0.1% deoxycholate, 10 mM NaF, 10 mM sodium pyrophosphate, and 100 mM sodium orthovanadate) for 15 min on ice. Cell lysates were clarified by centrifugation at 15,000 rpm for 10 min. Clarified cell lysates were normalized based on protein concentration as determined using the BCA® kit (Pierce), and equal amounts of protein were subjected to immunoprecipitation or immunoblotting for each lysate. For Ras GAP immunoprecipitation, the clarified cell lysates (equivalent to ~1 × 108 cells) were precleared with normal rabbit Ig overnight at 4 °C before polyclonal anti-Ras GAP Ab (Transduction Laboratories, KY) was added. For Lyn, Syk, p90Rsk, and p70S6k immunoprecipitation, the post-nuclear extracts (equivalent to ~5 × 107 cells) were incubated directly with polyclonal anti-Lyn Ab (a generous gift from Dr. J. Cambier, CO), anti-Syk Ab (a generous gift from Dr. J. Bolen, DNAX, CA), anti-p90Rsk, or anti-p70S6k (Santa Cruz Biotechnology, CA) at 4 °C overnight. Immune complexes were precipitated with 30 µl of protein A/G plus®-agarose beads (Santa Cruz Biotechnology) for p90Rsk immunoprecipitation or protein A-agarose beads (Life Technologies, Inc.) for all other immunoprecipitations for an additional 3-h incubation at 4 °C. For anti-Lyn and anti-Syk immunoprecipitation, the beads were washed twice with lysis buffer followed by two washes with PBS. For anti-Ras GAP, p90Rsk, and p70S6k immunoprecipitation, the precipitates were washed 5 times with RIPA buffer without SDS (1% Triton X-100, 1% deoxycholate, 158 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 100 µg/ml leupeptin, 100 µg/ml soy bean trypsin inhibitor) followed by two washes with lysis buffer and then PBS. Bound proteins were eluted by boiling in Laemmli buffer containing 0.4% dithiothreitol and resolved by 8% SDS-PAGE. Proteins with molecular mass less than 45 kDa were run off gels to obtain better separation of the proteins of interest from the Ab or to obtain increased resolution when determining shifts in mobility. The proteins were transferred to polyvinylidene difluoride membrane and subjected to immunoblotting. The blots were blocked with TNB buffer (30 mM Tris, pH 7.6, 75 mM NaCl, 3% bovine serum albumin) overnight at 4 °C. Polyclonal anti-Syk, anti-p90Rsk, anti-p70S6k Ab, monoclonal anti-Ras GAP Ab (Santa Cruz Biotechnology, CA), or monoclonal anti-phosphotyrosine Ab (4G10) was added at the concentrations suggested by the manufacturers. For anti-Ras GAP and phosphotyrosine immunoblotting, the blots were further incubated with 15 µg/ml polyclonal rabbit anti-mouse Ab for 1 h at 4 °C, whereas in p90Rsk immunoblotting, the blots were incubated with 15 µg/ml polyclonal rabbit anti-goat Ab for 1 h at 4 °C. The blots were then incubated with 1 µCi/ml iodinated protein A for 45 min at room temperature followed by autoradiography.
For immunoblotting of ERK1 and phosphorylated MAP kinase, cells were stimulated and lysed as described above. Cell lysates were normalized by BCA® kit (Pierce), and 100 µg of cell lysates (equivalent to ~2 × 106 cells/lane) were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane. The blots were blocked and blotted as described above using anti-ERK1 Ab (Transduction Laboratories, KY) or anti-phospho-MAP kinase Ab (New England Biolabs Inc., MA). The blots were developed as described above.
Protein Kinase AssaysAnti-Rsk immunoprecipitates were
prepared as above and washed once with kinase buffer (30 mM
Tris-HCl, pH 7.4, 10 mM MgCl2, 0.1 mM EGTA, 1 mM dithiothreitol, and 1 mg/ml
protein kinase A inhibitor (Santa Cruz Biotechnology)). The immune
complexes were resuspended in 12.5 µl of kinase buffer with 0.3 mg/ml
S6 peptide (Santa Cruz Biotechnology) and 5 µCi of
[32P]ATP (6000Ci/mmol), followed by incubation at
30 °C for 10 min. The reactions were terminated by adding 7.5 µl
of 3 × Laemmli sample buffer and boiling for 5 min. Samples were
resolved on an 18.5% polyacrylamide gel. Gels were dried and subjected
to autoradiography followed by quantitation using a PhosphorImager (Molecular Dynamics) and Image QuaNT software.
Anti-p70S6k immunoprecipitates were prepared as described above and
washed once with kinase buffer (50 mM MOPS, pH 7.2, 1 mM dithiothreitol, 30 µM ATP, 5 mM MgCl2, 1 mg/ml protein kinase A inhibitor).
The immune complexes were then resuspended in 12.5 µl of kinase
buffer containing 0.3 mg/ml S6 peptide and 5 µCi of
[-32P]ATP and incubated at 30 °C for 15 min. The
reactions were terminated by adding 7.5 µl of 3 × Laemmli
buffer and boiled for 5 min. Samples were then resolved on an 18.5%
polyacrylamide gel. The gel was then dried and subjected to
autoradiography.
The
relationship between Lyn and Syk in BCR-mediated activation has not
been clearly defined. In one study, anti-Ig-induced activation of
PLC-, a direct substrate of Syk (39, 40), was unaffected in a
Lyn-deficient cell line, DT40Lyn
, but abolished in a
Syk-deficient cell line, DT40Syk
(26). This result
suggested that the anti-Ig-induced activation of PLC-
required Syk
activation but could occur in the absence of Lyn. However, it has also
been suggested that activation of Syk is abolished in
DT40Lyn
cells, suggesting that Lyn is required for
activation of Syk (22). In view of this paradox, we tested the
hypothesis that Syk can be activated in the absence of Lyn by directly
comparing BCR-mediated signaling in DT40Syk
,
DT40Lyn
, and parental DT40 cells. Using an optimal
concentration of anti-Ig Ab (10 µg/ml M4), we found that the
anti-Ig-induced tyrosine phosphorylation of several species, including
proteins with approximate molecular masses of 55 and 65 kDa, was
defective in DT40Lyn
cells compared with parental DT40
cells (Fig. 1A). In contrast, the
anti-Ig-induced tyrosine phosphorylation of a different subset of
phosphoproteins including species with molecular masses of 87, 100, and
110 kDa was defective in DT40 Syk
cells (Fig.
1A). We have observed these differences consistently in many
independent experiments. Consistent with previous reports, the
anti-Ig-induced tyrosine phosphorylation of Lyn in
DT40Syk
cells was comparable with that in parental cells
(Fig. 1B). Furthermore, we detected anti-Ig-induced tyrosine
phosphorylation (Fig. 1C) and kinase activity (data not
shown) of Syk in DT40Lyn
cells, albeit to a lesser
extent, compared with parental DT40 cells (Fig. 1C). Taken
together with previous studies, these data indicate that although Lyn
may be an upstream effector of Syk in BCR-mediated signaling, Syk can
also clearly be phosphorylated via a Lyn-independent pathway in B
cells.
The Lyn-independent Activation of Syk Is Sufficient for Activation of p90Rsk
We then sought to establish whether the suboptimal
phosphorylation of Syk in DT40Lyn resulted in activation
of Syk and to identify potential downstream effectors in addition to
PLC-
that could be activated via a Lyn-independent but
Syk-dependent pathway. Although we have not yet identified the tyrosine phosphorylated proteins, pp87, pp100, and pp110, present
in DT40Lyn
cells but absent in DT40Syk
cells, we have established that these protein species are neither the
p85 or p110 subunits of PI3-kinase nor p120c-cbl (data not shown).
Since it has been suggested that BCR-induced tyrosine phosphorylation
leads to activation of a series of serine/threonine kinases (29), we
investigated whether the phosphorylation and activation of any
serine/threonine phosphorylated proteins/kinases requires Syk but could
occur in the absence of Lyn. One of the major serine/threonine kinases
activated in B cells is p90Rsk (29). p90Rsk is one member of a
serine/threonine kinase family that like the S6 kinase, p70S6k,
regulates the phosphorylation and activity of ribosomal S6 protein and
that is thought to be involved in translational control during the cell
cycle (41, 42). Based on mobility shift assays (Fig.
2A) and in vitro kinase assays
(Fig. 2, B and C), we found that ligation of
surface Ig induced activation of p90Rsk in parental DT40 cells as well
as DT40Lyn
cells. However, the anti-Ig-induced activation
of p90Rsk was abolished in DT40Syk
cells, indicating that
Syk is required for anti-Ig-induced activation of p90Rsk (Fig. 2,
B and C). The failure to activate p90Rsk in anti-Ig-stimulated DT40Syk
cells was not due to an
intrinsic defect in p90Rsk, since stimulation with phorbol ester (PDBu)
resulted in similar activation of p90Rsk in DT40Syk
cells, as observed in DT40Lyn
cells or parental DT40
cells. Thus, these data provide additional evidence supporting the
hypothesis that Syk can be functionally activated via a Lyn-independent
pathway that is sufficient for activation of the pathway leading to the
activation of p90Rsk.
p70S6 Kinase Is Activated in Anti-Ig-stimulated B Cells
To
date, two mitogen-induced S6 kinase families have been identified, the
Rsk family (including p90Rsk) and p70/85 S6 kinases. It was shown that
the members of these two families are regulated by different mechanisms
(25). It is well documented that cross-linking of BCR induces
activation of p90Rsk (29), but only CD38-induced activation of p70S6k
in B cells has been reported (43, 44). We sought to determine whether
p70S6k could also be activated by BCR-mediated signaling by comparing
the mobility of p70S6k on SDS-PAGE and kinase activity of p70S6k in
DT40 cells with that in DT40Syk and DT40Lyn
cells. Cross-linking of surface Ig induced a shift in mobility of
p70S6k on SDS-PAGE in parental DT40 cells (Fig. 3,
bottom panel). Furthermore, the shift in mobility
corrrelated with the activation of p70S6k kinase activity (Fig. 3,
top panel). Interestingly, both anti-Ig and PDBu-induced
activation of p70S6k were unaffected in DT40Syk
and
DT40Lyn
cells compared with parental cells, indicating
that either Lyn or Syk is sufficient for anti-Ig-induced activation of
p70S6k or that neither kinase mediates the activation of the signal
transduction pathway that leads to activation of p70S6k.
Tyrosine Phosphorylation of MAPK and Ras GTPase Activating Protein (Ras GAP) Is Dependent on Both Lyn and Syk
We next compared the
protein tyrosine kinases required for anti-Ig-induced activation of
MAPK and Ras GAP since the Ras/Raf/Mek/MAPK pathway has been implicated
in activation of p90Rsk in B cells (29). We measured the
anti-Ig-induced phosphorylation of MAPK in DT40Syk and
DT40Lyn
cells since activation of MAPK involves its
phosphorylation on tyrosine and threonine residues. Interestingly, we
observed that the anti-Ig-induced phosphorylation of MAPK, unlike
p90Rsk, is abolished in both DT40Syk
and
DT40Lyn
cells compared with parental cells (Fig.
4A). This result indicated that activation of
MAPK depends on Syk and Lyn, and thus the BCR-induced activation of
p90Rsk in the Lyn-deficient DT40 cells (Fig. 2) must be mediated by an
MAPK-independent pathway. These data do not, however, exclude the
possibility that MAPK can also mediate anti-Ig-induced activation of
p90Rsk in parental DT40 cells. Overall, these data are consistent with
previous reports that p90Rsk can be activated with or without
activation of MAPK in Xenopus oocytes (36).
We then examined the anti-Ig-induced tyrosine phosphorylation of Ras
GAP, which negatively regulates Ras by converting the GTP-bound Ras
(the active form) into the GDP-bound Ras (the inactive form) (34, 35).
It was previously shown that cross-linking of BCR induced the rapid
tyrosine phosphorylation of Ras GAP (27, 28). However, the upstream
effectors of this event in B cells were not determined. We found that
the anti-Ig-induced tyrosine phosphorylation of Ras GAP is ablated in
DT40Syk and DT40Lyn
cells, suggesting again
that both Syk and Lyn are required for this event (Fig.
4B).
To map the structural elements required
for the activation of Syk-dependent substrates, we
transfected DT40Syk cells with either wild type human Syk
cDNA, a mutant in which the tyrosine residues that are potential
sites for auto-phosphorylation were substituted with phenylalanines
(YY525/526FF) (45), or an enzymatically inactive mutant that encodes
for an arginine in place of lysine at position 402 in the catalytic
domain (K402R) (46). WTSyk and mutant forms of Syk were expressed in
DT40Syk
cells and selected based on comparable expression
of Syk as well as similar levels of expression of membrane IgM (data
not shown). Anti-Ig-induced activation and/or phosphorylation of the
Syk-dependent substrates, p90Rsk, MAPK, and Ras GAP, and
the profiles of total tyrosine phosphorylated proteins were compared.
The patterns of anti-Ig-induced tyrosine phosphorylation in
DT40Syk
cells transfected with WTSyk were restored to
that of the parental line (Fig. 5A). However,
the levels of tyrosine phosphorylation were increased in the WTSyk
transfectants due to expression of higher levels of recombinant Syk
compared with the levels of endogenous Syk produced in the parental
line (Fig. 5B, bottom panel). Therefore, the
following results obtained with DT40Syk
cells expressing
mutant forms of Syk are interpreted based on a comparison with DT40
cells expressing WTSyk rather than parental cells since they express
comparable levels of recombinant Syk to that of WT transfectants.
YY525/526FF completely reconstitutes the pattern of
BCR-induced tyrosine phosphorylation of DT40Syk cells
compared with WTSyk transfectants. However, the intensity of
BCR-induced tyrosine phosphorylation of several proteins in DT40Syk
expressing YY525/526FF was decreased compared
with WTSyk transfectants (Fig. 5A). These results suggest
that the putative autophosphorylation sites play a role in obtaining
optimal anti-Ig-induced tyrosine phosphorylation that is partially
overriden by the high levels of recombinant Syk expression in our
studies (Fig. 5B). The decrease in tyrosine phosphorylation
in DT40 cells expressing the YY525/526FF mutant appeared even more
pronounced in studies reported by others using different experimental
conditions (45). The enzymatically inactive mutant K402R failed to
restore anti-Ig-induced tyrosine phosphorylation in
DT40Syk
cells, suggesting the enzymatic activity of Syk
is required. This result also indicates that in contrast to
YY525/526FF, the defect in K402R cannot be overcome by
overexpression.
The level of tyrosine phosphorylation of Syk is increased in response
to B cell activation and is reportedly in part due to auto-phosphorylation (47). To investigate whether substitution of the
tyrosine residues at positions 525 and 526 affects the anti-Ig-induced
tyrosine phosphorylation of Syk, the lysates from DT40Syk
transfectants stimulated with anti-IgM Ab were subjected to
immunoprecipitation with anti-Syk Ab, and the immune complexes were
immunoblotted with anti-phosphotyrosine Ab. The wild type, YY525/526FF,
and K402R mutant forms of Syk were all tyrosine phosphorylated to comparable levels following cross-linking of BCR (Fig. 5B,
top panel). These data indicate that Syk is predominantly
trans-phosphorylated on tyrosines at sites other than the potential
autophosphorylated residues at positions 525 or 526.
The anti-Ig-induced activation and/or
phosphorylation of specific Syk-dependent substrates,
p90Rsk, MAPK, and Ras GAP, in DT40Syk cells expressing
WTSyk and mutant forms of Syk were examined. The anti-Ig-induced
mobility shift of p90Rsk on SDS-PAGE and activation of p90Rsk were
comparable in WTSyk and YY525/526FF transfectants, whereas they were
undetectable in vector and K402R transfectants (Fig.
6A). Moreover, we have consistently observed
high basal activity of p90Rsk in WTSyk transfectants compared with
DT40Syk
cells expressing mutant forms of Syk. This is
consistent with a higher basal level of total tyrosine phosphorylation
in WTSyk transfectants compared with other mutants, that might be due
to higher basal activity of WTSyk compared with that of mutant forms of
Syk. Similar to p90Rsk, the BCR-induced phosphorylation of MAPK was
restored in WTSyk and YY525/526FF transfectants but not in vector and
K402R transfectants (Fig. 6B). In contrast, the anti-Ig-induced tyrosine phosphorylation of Ras GAP is profoundly reduced in YY525/526FF transfectants and undetectable in vector and
K402R transfectants compared with that in WTSyk transfectants (Fig.
6C). Thus, the kinase activity of Syk is indispensable in the regulation of these molecules in BCR-mediated signaling, but the
putative autophosphorylation sites (YY525/526FF) are critical for the
regulation of Ras GAP among these substrates.
DT40 cells lack expression of Src, Fyn, Hck, Blk, and Lck but
express Lyn as the predominant Src family kinase (26). Furthermore, Syk, but not Zap70, is expressed in DT40 cells (26). This selective and
predominant expression of single members of the Src family and Syk
family of tyrosine kinases has made this cell line a useful model for
dissecting the roles of Lyn and Syk in BCR-mediated activation. The
high efficiency at which homologous recombination occurs in this cell
line also makes it particularly amenable for generating Syk- and
Lyn-deficient DT40 cells to address this issue (22, 26). Initially, it
was reported that although activation of PLC- was dependent on Syk,
the activation of PLC
was not defective in Lyn-deficient cells (26).
Paradoxically, it was subsequently suggested that Lyn is required for
BCR-mediated activation of Syk (22). In this study, we demonstrated
that the profile of anti-Ig-induced tyrosine phosphorylation in
DT40Lyn
cells was different from and included species
that were not detectable in DT40Syk
cells. Moreover, Syk
was tyrosine phosphorylated in the absence of Lyn, albeit to a lesser
extent compared with parental cells, indicating that Syk can be
activated via at least two independent pathways, one of which
apparently does not require Lyn in DT40 cells. These apparently
contradictory results might be attributed to the use of a higher
concentration of the monoclonal anti-IgM Ab (M4) to induce BCR
cross-linking and to the higher numbers of cells used in our analysis
compared with previous reports, both of which may be required for
detection of the low levels of anti-Ig-induced tyrosine phosphorylation
of Syk in DT40Lyn
cells.
Our data indicate that the serine/threonine kinase, p90Rsk, as well as
PLC- (26), can be stimulated in DT40Lyn
cells but not
in DT40Syk
cells. Thus, Syk is uniquely required for
anti-Ig-induced activation of p90Rsk, supporting the hypothesis that
Syk can be functionally activated in the absence of Lyn and lead to the
activation of p90Rsk. That Syk can be activated without the activation
of Lck in T cells has also recently been reported (48). Activation of
Lck in Jurkat T cells requires CD45, a receptor protein tyrosine phosphatase (49, 50). However, Chu et al. (48) recently demonstrated that Syk can be activated in CD45-deficient Jurkat cells
by stimulation with anti-CD3. Similar results were recently reported in
a CD45-deficient B cell line in which the activation of Lyn is
CD45-dependent (51). The Lyn-independent activation of Syk
in DT40 cells might be due to autophosphorylation or other kinases
previously implicated in the activation of B cells, such as Src family
members like Fgr or a member of the Tec family of PTK such as Btk (52,
53). However, the role of Btk in activation of Syk after BCR
cross-linking was demonstrated to be minor since Syk was tyrosine
phosphorylated and activated in Btk-deficient DT40 cells to an extent
comparable with that observed in parental DT40 cells (53). Whether
these cells express Fgr and other kinases that can potentially activate
Syk in the absence of Lyn after ligation of BCR will require further
investigation.
Cross-linking of the BCR leads to activation of Ras/Raf/Mek/MAPK/p90Rsk
pathway (29). Consistent with previous results, we demonstrated that
p90Rsk can be activated in DT40 cells after cross-linking of BCR. In
addition, we showed that Syk is uniquely and absolutely required for
the anti-Ig-induced activation of p90Rsk since BCR-mediated activation
of p90Rsk is unaffected in DT40Lyn cells but ablated in
DT40Syk
cells. However, when we examined the potential
mediators of the Syk-dependent activation of p90Rsk in
DT40Lyn
cells, such as MAPK, we found that in contrast to
p90Rsk, the anti-Ig-induced activation of MAPK was abolished in the
absence of Lyn or Syk. These results indicate that Lyn and Syk are
indispensable for BCR-induced activation of MAPK. They also indicate
that the anti-Ig-induced activation of Syk in DT40Lyn
cells leads to activation of a MAPK-independent signal transduction pathway that results in activation of p90Rsk (Fig. 7).
Similar alternative pathways of activation of p90Rsk have been
described in other cell types (36). In contrast to p90Rsk, a role for p70S6k in BCR-mediated signaling has not previously been reported. In
this study, we demonstrated that ligation of anti-Ig induced activation
of p70S6k in B cells. We further showed that the anti-Ig-induced activation of p70S6k is unaffected in the absence of Syk or Lyn, suggesting either Lyn or Syk is each sufficient for activation of
p70S6k or that neither kinase mediates this response (Figs. 3 and 7).
Our results also indicate that the regulation of p70S6k by protein
tyrosine kinases in B cells is distinct from that of p90Rsk. These
differences might result in activation of distinct mediators coupling
the protein tyrosine kinases to p70S6k or p90Rsk as shown in other cell
types (31, 33, 54-58). For example, treatment with the
immunosuppressant, rapamycin, selectively inhibited the activation of
p70S6k, whereas it had no effect on the activation of p90Rsk in several
studies (41, 59, 60). Furthermore, whereas activation of MAPK in
fibroblasts led to the activation of p90Rsk, activation of PI3-kinase
in platelet-derived growth factor-stimulated fibroblasts correlated
with activation of p70S6k (57, 58). Our preliminary data also showed
that treatment with wortmannin, an inhibitor of PI3-kinase, inhibited
anti-Ig-induced activation of p70S6k but not p90Rsk (data not shown).
In addition, mutations of the PI3-kinase binding sites of the
platelet-derived growth factor receptor abolished the activation of
both PI3-kinase and p70S6k, providing evidence for a role for
PI3-kinase in the activation of p70S6k (Fig. 7) (57, 61).
Alternatively, it was shown that PKC can also mediate activation of
p70S6K since treatment of DT40 cells and other cell types with
PDBu activates p70S6k (Fig. 7) (62). The mechanism of
BCR-mediated activation of p70S6k will be the focus of future
studies.
In contrast to the downstream activation of serine/threonine kinases,
the proximal events after cross-linking of BCR involve the rapid
tyrosine phosphorylation of several substrates, including Ras GAP.
However, the role for distinct protein tyrosine kinases in the
phosphorylation of specific substrates had not previously been
delineated in B cells. Ras GAP physically associates with the Src
family kinases, Lyn, Fyn, and Yes, in thrombin-activated platelets,
suggesting a role for these kinases in the regulation of Ras GAP(63).
Using DT40Lyn and DT40Syk
cells, we
demonstrated that both Lyn and Syk are required for anti-Ig-induced
tyrosine phosphorylation of Ras GAP. To date, there is no evidence to
show that the GTPase activating activity of Ras GAP correlates with
this increase in tyrosine phosphorylation. However, tyrosine
phosphorylation of Ras GAP in activated B cells increased its
association with two tyrosine phosphorylated species, pp190 and pp62,
which were identified as a transcription repressor and a RNA binding
protein, respectively (64, 65).
The structural elements of Syk required for the regulation of the
Syk-dependent substrates, Ras GAP, MAPK, and p90Rsk, were examined. We demonstrated that the kinase activity of Syk is required for the regulation of each of these proteins and for reconstitution of
the total profile of anti-Ig-induced tyrosine phosphorylation (Figs. 5
and 6). Moreover, mutations of the putative autophosphorylation sites
of Syk (YY525/526FF) in B cells resulted in suboptimal tyrosine phosphorylation of some cellular proteins including Ras GAP after anti-Ig stimulation compared with wild type Syk, which is consistent with previous reports (45). However, the anti-Ig-induced
phosphorylation and/or activation of MAPK and p90Rsk in
DT40Syk cells expressing YY525/526FF were comparable with
wild type Syk. These data suggest the differential requirement for the
autophosphorylation sites of Syk for activation of different downstream
substrates.
In summary, we conclude that BCR-mediated activation of Syk can be induced via at least two different pathways in B cells, one of which does not require Lyn. Anti-Ig-activated Syk then evokes multiple signal transduction pathways leading to activation and/or phosphorylation of Ras GAP, MAPK, and p90Rsk. The kinase activity of Syk was found to be indispensable in transducing BCR-mediated signaling to Ras GAP, MAPK, and p90Rsk, whereas the activation of p70S6k can occur independently of Syk. These findings provide an important link between BCR proximal tyrosine kinases and downstream effectors that are likely to play an important role in activation-dependent gene expression in B cells.
The authors thank Dr. Joseph Bolen for the generous gift of anti-Syk antisera. We are grateful to Drs. Paul Stein and Aili Lazzar for many helpful discussions and reading the manuscript.