From the Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aggregation of the B-cell antigen receptor leads
to the activation of the 72-kDa Syk protein-tyrosine kinase and the
phosphorylation of tubulin on tyrosine. To explore the requirement of
Syk catalytic activity for tubulin phosphorylation, tubulin was
isolated from cytosolic fractions from anti-IgM-activated B-cells
(DT40) that lacked endogenous Syk and immunoblotted with
anti-phosphotyrosine antibodies. Tubulin was not
tyrosine-phosphorylated in Syk Aggregation of the B-cell antigen receptor
(BCR)1 complex leads to a
host of intracellular events culminating in cellular proliferation and
antibody production in mature B-cells. The earliest events to occur
after aggregation are the rapid induction of protein-tyrosine phosphorylation and the subsequent production of inositol
1,4,5-trisphosphate, release of calcium from intracellular stores, and
activation of the mitogen-activated protein kinase pathway (1).
The BCR, which is composed of a membrane-bound surface immunoglobulin
and associated Ig The recruitment of Syk to the antigen receptor activates and positions
the kinase at the plasma membrane, where it can interact with and
phosphorylate substrates that are important for receptor-mediated signaling. Tyrosine-phosphorylated and activated Syk is also released from the antigen receptor, which may allow interactions with additional cellular substrates (6). One potential cytosolic substrate with which
Syk interacts is Antibodies and Reagents--
DT40 and Syk Expression of Syk(K396R) in DT40 Cells--
Generation of a
cDNA for Myc epitope-tagged Syk has been described previously (7).
To generate kinase-deficient Myc-tagged Syk (Syk(K396R)) cDNA,
site-directed mutagenesis was carried out using the unique site
elimination method described by Deng and Nickoloff (11), and the
mutation was confirmed by DNA sequencing. Syk Cellular Fractionation--
Cytosolic fractions from chicken
DT40 and murine Bal17 B-cells (5-10 × 106
cells/sample) were obtained as described previously (6). Cells were
incubated on ice in the presence or absence of affinity purified anti-IgM antibodies (25-50 µg/ml) for 10 min or as indicated, prior
to lysis.
Immunoprecipitation and in Vitro Kinase Assay--
Proteins were
immunoprecipitated by incubation with anti- Colchicine-Agarose Chromatography--
Cytosolic fractions
prepared as above were incubated with colchicine-agarose for 45 min at
4 °C. The resin was washed three times in homogenization buffer.
Proteins were eluted by addition of SDS sample buffer and separated by
SDS-PAGE.
Taxol Treatment of Cells--
Bal17 B-cells (5-10 × 106 cells/sample) were treated with 7.5 µg/ml Taxol or an
equal volume of Me2SO as a vehicle control for 2.5 h
at 37 °C.
Tubulin Associates with Syk in B-cell Lysates--
To explore the
potential interactions of B-cell tubulin with endogenous
protein-tyrosine kinases, tubulin was immunoprecipitated with an
anti-tubulin monoclonal antibody from the soluble fraction of lysates
prepared from unstimulated and anti-IgM-activated B-cells. The
resulting immune complexes were then incubated with
[
A small amount of tubulin can be coimmunoprecipitated with Syk in
anti-Syk immune complexes prepared from the soluble fractions of
anti-IgM activated B cells (6), suggesting that Syk is the kinase most
likely to be associated with soluble tubulin. To enhance our ability to
detect tubulin-associated Syk, Syk Syk-Tubulin Association Is Independent of Tubulin
Phosphorylation--
To determine whether Syk-catalyzed tubulin
phosphorylation was required for Syk association, Syk Syk Is Required for the Tyrosine Phosphorylation of
Tubulin--
The lack of tubulin phosphorylation in
Syk(K396R)-expressing cells suggested that Syk was required for the
tubulin phosphorylation that is observed in B-cells following antigen
receptor engagement (6). To confirm this, the receptor-stimulated
phosphorylation of tubulin was compared in wild-type DT40 cells
expressing the normal level of endogenous Syk and Syk
Densitometric analyses of anti-tubulin and anti-phosphotyrosine
immunoblots were used to determine the extent of phosphorylation of
cytosolic tubulin by comparing tubulin isolated from cytosolic extracts
to purified tubulin phosphorylated in vitro to known stoichiometries using Syk. The results demonstrated that in
unstimulated cells, approximately 1.2% of cytosolic tubulin is
tyrosine-phosphorylated, whereas in cells stimulated with anti-IgM
antibodies, approximately 4.7% of cytosolic tubulin is
tyrosine-phosphorylated. These levels of tubulin tyrosine
phosphorylation are similar to those observed by Ley et al.
(14) in the Jurkat T cell line.
Multiple Tyrosine-phosphorylated Proteins Bind
Colchicine-Agarose--
Immunoblot analysis with anti-phosphotyrosine
antibodies of colchicine-agarose-associated proteins from wild-type
DT40 B-cells revealed three phosphotyrosine-containing proteins, in
addition to tubulin, the appearance of which was dependent on both
receptor cross-linking and the expression of endogenous Syk (Fig. 2).
The apparent molecular weights of these proteins, which also appear as
a subset of the tyrosine-phosphorylated proteins seen in
colchicine-agarose precipitations from Bal17 murine B-cells (see Fig.
4A), were 110,000, 90,000, and 74,000. These data suggested
that Syk substrates other than tubulin were associating with the
colchicine-agarose affinity resin.
The apparent variation in recovery of phosphoproteins between DT40 and
Bal17 B-cells was reproducible and may have resulted from the decreased
expression of protein-tyrosine kinases, specifically of the Src family,
in the DT40 cell line. In addition to expressing only one Src family
kinase (Lyn) (13), we have observed that DT40 B-cells express lower
levels of Syk compared with other B-cell lines.
Several experiments were conducted to explore the specificity of the
interaction of these tyrosine-phosphorylated proteins with
colchicine-agarose. The inclusion of an excess of colchicine blocked
the interaction of all of the tyrosine-phosphorylated proteins with
colchicine-agarose, indicating that their interaction with the resin
was specific for the immobilized ligand (data not shown). In addition,
no binding of tyrosine-phosphorylated proteins to either phenyl-agarose
or butyl-agarose was observed, indicating that the phosphoproteins were
not bound due to nonspecific, hydrophobic interactions (data not
shown). To determine whether the binding of these phosphoproteins to
colchicine-agarose occurred via their specific interaction with
tubulin, immunoprecipitations using a monoclonal anti-tubulin antibody
were performed in parallel with adsorption to colchicine-agarose. Bound
proteins were separated by SDS-PAGE and analyzed by
anti-phosphotyrosine immunoblotting. As shown in Fig.
3A, both colchicine-agarose
and anti-tubulin immune complexes contained a comparable set of
phosphotyrosine-containing proteins. The recovery of tubulin-associated
proteins is enhanced by the use of colchicine-agarose, which binds a
higher fraction of the available soluble tubulin than does the
monoclonal antibody. Western blotting analysis of immunoprecipitated
tubulin bands in Fig. 3A, lanes 3 and 4, is
complicated by their comigration with the heavy chain of the
anti-tubulin monoclonal antibody.
To further test this specificity, cells were pretreated with the
microtubule-stabilizing agent Taxol to promote the polymerization of
soluble tubulin. Cytosolic fractions were prepared from
anti-IgM-activated control and Taxol-treated cells and adsorbed to
colchicine-agarose. As shown in Fig. 3C, Taxol pretreatment
essentially removed all detectable tubulin from the cytosolic fraction.
This in turn led to a dramatic decrease in the level of
colchicine-agarose-associated phosphoproteins that could be recovered
(Fig. 3B).
Tubulin Associates with Cbl and Vav--
The 120-kDa
proto-oncogene product Cbl becomes prominently tyrosine-phosphorylated
in response to BCR aggregation and has been shown to associate with Syk
in vivo (15, 16). To determine whether the
tubulin-associated, 110-kDa phosphoprotein corresponded to Cbl,
colchicine-agarose binding proteins prepared from Bal17 B-cells were
probed by immunoblotting with anti-Cbl antibodies. As shown in Fig.
4 (lanes 3 and 4),
Cbl was constitutively present in the colchicine-agarose-bound
fraction.
It has been previously reported that the 95-kDa tyrosine kinase
substrate Vav associates with both tubulin and ZAP-70 in Jurkat T-cells
(17). To determine whether the 90-kDa protein that was present in
anti-phosphotyrosine immunoblots of colchicine-agarose adsorbed
proteins represented Vav, the immunoblot was reprobed using anti-Vav
antibodies. As shown in Fig. 4 (lanes 5 and 6), Vav also was found associated with the colchicine-agarose-bound fraction. Again, as with Cbl, the presence of Vav is constitutive.
To determine the relative stoichiometries of Cbl, Syk, and Vav
associated with cytosolic tubulin, immunoblotting of total cytosolic
fractions and colchicine-agarose fractions was performed, followed by
densitometric analysis. The results demonstrated that approximately
25% of cytosolic Cbl, 22% of cytosolic Syk and 36% of cytosolic Vav
associate with cytosolic tubulin in B-cells.
Phosphorylation of Tubulin-associated Proteins, but Not of Tubulin,
Requires the Interaction of Syk with the Antigen
Receptor--
Substitution of Tyr-130 with Glu produces a
constitutively active form of Syk with a significantly reduced affinity
for the BCR and a reduced ability to support receptor-mediated tyrosine phosphorylation of multiple cellular substrates (7). To determine whether Syk(Y130E) could associate with tubulin, cytosolic fractions were prepared from Syk
The phosphorylation of colchicine-agarose-binding proteins was compared
in Syk A small amount of tubulin can be coimmunoprecipitated with Syk in
anti-Syk immune complexes prepared from the soluble fractions of
anti-IgM activated B-cells (6), indicating that these proteins are able
to interact with one another. In the reciprocal experiment, antibodies
to tubulin coimmunoprecipitate an endogenous protein-tyrosine kinase
activity that catalyzes the tyrosine phosphorylation of tubulin (Fig.
1A). Low levels of a 72-kDa phosphoprotein can be seen in
the anti-tubulin phosphorylation assays, but the low levels of Syk in
these immune complexes proved difficult to detect by Western blotting
with the available anti-Syk antibodies. Tubulin-associated Syk,
however, could be detected easily when isolated from a
Syk It should be noted that although tubulin is the predominant
phosphoprotein seen in Fig. 1A, longer exposures did reveal
the presence of a number of other phosphoproteins, including several between 66 and 205 kDa. The absence of a robust Syk autophosphorylation is not surprising, as we have previously observed significant decreases
in Syk autophosphorylation in vitro in the presence of a
good substrate (18). This also is likely to explain the absence of
other prominently phosphorylated proteins, because it is likely that
Syk is the predominant tyrosine kinase in these immune complexes and
that tubulin is the best Syk substrate present.
Two additional hematopoietic cell tyrosine kinases, Fyn (19) and ZAP-70
(17), have been reported to bind tubulin. The interaction of Fyn with
tubulin is mediated by the Fyn SH2 domain and requires the prior
tyrosine-phosphorylation of tubulin (19). However, a similar mechanism
of association is unlikely to mediate the Syk-tubulin interaction,
because a catalytically inactive form of Syk retains its tubulin
binding capacity despite its inability to catalyze tubulin
phosphorylation (Fig. 1). Also, the interactions of Syk(WT) with
tubulin in Syk(WT)-expressing DT40 cells (Fig. 1B) and of
ZAP-70 with tubulin in Jurkat T-cells (17) are independent of receptor
engagement. Likewise, a mutant form of Syk (Syk(Y130E) that exhibits a
much reduced affinity for the phosphorylated BCR (7) retains its
ability to bind tubulin (Fig. 5A). Thus, tubulin phosphorylation is not a prerequisite for Syk binding. Tubulin, however, is capable of binding to the SH2 domains of Grb2, the p85
subunit of phosphatidylinositide 3-kinase and phospholipase C- The nature of the Syk-tubulin interaction is also reflected in the
manner in which tubulin becomes phosphorylated on tyrosine. Previous
studies implicated Syk in the in vivo phosphorylation of
tubulin, because the pretreatment of B-cells with the Syk-selective inhibitor piceatannol abrogated the receptor-stimulated tyrosine phosphorylation of tubulin (6). We have now extended these findings,
using both Syk We observed at least three protein substrates that become
phosphorylated on tyrosine following receptor engagement and that co-isolate with soluble tubulin by affinity chromatography on colchicine-agarose (Fig. 2) or by immunoprecipitation with anti-tubulin antibodies (Fig. 3A). These include Cbl, Vav (Fig. 4), and
one additional protein of 74,000 Da that remains to be identified. Both
Cbl and Vav are known to be phosphorylated by tyrosine kinases activated downstream of immune recognition receptors (15, 16, 21-26).
The tyrosine phosphorylation of tubulin is distinguished from the
phosphorylation of other proteins, such as Cbl and Vav, which require
Syk to be activated by receptor cross-linking. Unlike tubulin, these
tubulin-associated proteins are not constitutively phosphorylated in
cells expressing Syk(Y130E) (Fig. 6), and this is true in general for
the bulk of tyrosine-phosphorylated proteins seen in whole cell lysates
(7). If Syk is directly responsible for the phosphorylation of Cbl and
Vav, then this difference might be accounted for by differences in the
mechanisms by which Syk interacts with its various substrates. Because
the recovery of these proteins by affinity chromatography was blocked
by free colchicine and required the presence of soluble tubulin dimers in the lysate (Fig. 3B), it appears that some substrates for
receptor-activated protein-tyrosine kinases share with Syk the ability
to interact with tubulin or tubulin-associated proteins.
The clustering of Syk family kinases at the site of aggregated antigen
receptors promotes the phosphorylation of tyrosine residues that serve
as putative docking sites for SH2 domain- or phosphotyrosine-binding
domain-containing proteins. For example, ZAP-70 bound to proteins
containing multiple ITAMs autophosphorylates on sites that interact
with Fyn, Lck, GAP and Abl (27). Both Cbl and Vav possess multiple
domains that can participate in protein-protein interactions as well.
In addition to several proline-rich, SH3-binding motifs and multiple
tyrosine phosphorylation sites, Cbl has a novel phosphotyrosine binding
domain that interacts with Syk and ZAP-70 in an
activation-dependent manner (28). Vav, a potential guanine
nucleotide exchange factor for the Ras-related GTP-binding protein,
Rac-1 (29), contains a guanine nucleotide exchange factor domain
(similar to the exchange factor Dbl), a pleckstrin homology domain, a
number of phosphorylatable tyrosine residues, and an SH2 domain flanked
by two SH3 domains. Vav interacts with activated, phosphorylated Syk
and ZAP-70 in B- and T-cells via its SH2 domain (30, 31). Thus, the
interactions of Cbl and Vav with active Syk may require the proper
phosphorylation of Syk to occur at the site of aggregated antigen
receptors, whereas the interaction of tubulin with Syk occurs
independently of the phosphorylation of either the enzyme or the substrate.
Although no description prior to this report has been made of an
association between Cbl and tubulin, an interaction of Vav with tubulin
has been described previously in T-cells (17). In T-cells, both ZAP-70
and Vav bind tubulin independently, as well as binding to one another
(17). Interestingly, in RBL-2H3 cells, cross-linking studies have
revealed that a fraction of Vav exists in soluble lysates in large
complexes of up to 300 kDa that also contain Syk, Grb2, Raf-1, Mek-1,
and Erk-2 (32). It was not determined whether these complexes also
contained tubulin, but several of the components of this complex
(i.e. Vav, Syk, Grb2, and Erk-2) have been previously
reported to interact with tubulin (6, 17, 20, 33). Also in RBL-2H3 mast
cells, Cbl associates constitutively with Syk, and upon engagement of Fc The physiological relevance of the association of Syk, Vav, and Cbl
with tubulin is as yet uncertain. Tubulin can form protein-protein interactions with a wide variety of potential participants of hematopoietic cell signal transduction pathways ranging from receptors (e.g. the Ig- B-cells.
Phosphorylation could be restored by the expression of wild-type, but
not catalytically inactive, Syk. However, both catalytically inactive
and wild-type Syk were capable of constitutive association with
tubulin, indicating that tubulin phosphorylation is not required for
this interaction. Anti-phosphotyrosine antibody immunoblotting of
proteins adsorbed to colchicine-agarose revealed the presence of three
major tubulin-associated phosphoproteins of 110, 90, and 74 kDa, the
phosphorylation of which was dependent on Syk expression. The proteins
of 110 and 90 kDa were identified as Cbl and Vav, two proto-oncogene
products known to become prominently phosphorylated following receptor
engagement. Both proteins were shown to be constitutively associated
with tubulin.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
and Ig
chains, lacks intrinsic tyrosine kinase
activity (2). Transduction of signals is accomplished by activation of
intracellular protein-tyrosine kinases, including members of the Src
family (Blk, Fyn, Lck, and Lyn) (3-5) and the Syk family (Syk) (1).
Activation of Syk requires the presence of conserved amino acid
sequences, the immunoreceptor tyrosine-based activation motif (ITAM),
in the cytoplasmic tails of Ig
and Ig
. Each ITAM is composed of
two YXX(L/I) cassettes separated by a 6-8-amino acid spacer
(1). Phosphorylation of the ITAM tyrosines, presumably by Src family
kinases, provides a docking site for the tandem pair of SH2 domains
found in the amino-terminal half of Syk.
-tubulin (6). To further explore this interaction,
we have isolated tubulin-containing cytosolic complexes from B-cells by
affinity chromatography and analyzed proteins bound to them. In this
report, we provide evidence that Syk associates with tubulin in a
manner that is independent of tubulin phosphorylation. In addition to
Syk, cytosolic tubulin is constitutively associated with at least three
tyrosine kinase substrates, which include the proto-oncogene products
Cbl and Vav.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
DT40
chicken B-cells were obtained from Dr. Tomohiro Kurosaki.
Syk
DT40 cell lines expressing Syk(WT), Syk(Y130E), and
Syk(Y130F) were described previously (7). Syk(K396R) is described
below. The 9E10 anti-Myc hybridoma cell line was purchased from the
American Type Culture Collection, and the ascites fluid was prepared by the Purdue University Cancer Center Antibody Production Facility. The
following antibodies were also used in this report: goat anti-chicken IgM and goat anti-human IgM antibodies (Bethyl Laboratories), goat
anti-mouse IgM antibodies (Cappel), anti-phosphotyrosine monoclonal
antibody RC20H (Transduction Laboratories), anti-Vav antibody (Upstate
Biotechnology), anti-Cbl antibody (Upstate Biotechnology and Santa Cruz
Biotechnology), anti-
-tubulin monoclonal antibody (a kind gift of
Dr. David Asai, Purdue University) and polyclonal anti-phosphotyrosine
and anti-Syk antibodies (prepared as described previously (8, 9)).
Colchicine-agarose was prepared as described (10).
DT40 cells
were electroporated with 25 µg of Syk(K396R) cloned into the
XhoI site of the pGEM/EPB expression vector (12), along with
2.5 µg p3'SS (Stratagene), which contains the hygromycin resistance
gene. Cells were selected in RPMI 1640 medium containing 2 mg/ml
hygromycin and screened for Syk expression by immunoblotting. The
absence of Syk(K396R) catalytic activity was confirmed by an in
vitro kinase assay (data not shown).
-tubulin or anti-Myc
antibodies coupled to protein G plus agarose (Oncogene Science) or
protein A-Sepharose, respectively. Immune complexes were washed with
homogenization buffer three times. In vitro kinase assays
were performed as described elsewhere (8). Phosphotyrosine-containing
proteins were separated by SDS-PAGE, transferred to Immobilon-P
(Millipore), and visualized by autoradiography following incubation of
the membrane with 1 N KOH at 55 °C for 2 h to
reduce the levels of phosphoserine and phosphothreonine.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-32P]ATP. As illustrated in Fig.
1A, tubulin was the
predominant substrate present in the anti-tubulin immune complexes.
Tubulin-associated phosphate was stable to treatment with 1N KOH at
55 °C for 2 h, suggesting that phosphorylation occurred
predominantly on tyrosine.
View larger version (27K):
[in a new window]
Fig. 1.
Tubulin coprecipitates with Syk from the
cytosolic fraction of B-cells, and this interaction is independent of
tubulin tyrosine phosphorylation. A, Bal17 B-cells were
left unstimulated ( ) or stimulated (+) with anti-IgM antibodies.
Cells were Dounce homogenized, and tubulin was immunoprecipitated from
the cytosolic fractions with anti-tubulin antibodies. In
vitro kinase assays were performed on the resultant
immunoprecipitates, and proteins were separated by SDS-PAGE and
transferred to Immobilon-P. The membrane was subsequently treated with
1 N KOH to remove phosphoserine and phosphothreonine, and
autoradiography was performed. The migration position of tubulin is
indicated by an arrow. B, Syk(WT) (lanes
1 and 2) or Syk(K396R) (lanes 3 and
4) stably transfected cell lines were left unstimulated (
)
or stimulated (+) with anti-IgM antibodies. Cytosolic fractions were
incubated with colchicine-agarose. After separation of bound proteins
by SDS-PAGE and transfer to Immobilon-P, the blot was probed with
anti-Syk antibodies. The migration position of Syk is indicated.
C, Syk(WT)-expressing (lanes 1 and 2)
or Syk(K396R)-expressing (lanes 3 and 4) DT40
cells were treated as in B, and the resultant blot was
probed with anti-phosphotyrosine antibodies. The migration position of
tubulin is indicated.
DT40 chicken B-cells
(13) were transfected with a cDNA encoding wild-type murine Syk
(Syk(WT)) (7). These cells express an elevated level of Syk protein
that is readily detectable by immunoblotting with anti-Syk antibodies.
Cytosolic fractions were prepared from control and anti-IgM-treated
cells and adsorbed to colchicine-agarose to isolate tubulin and
tubulin-associated proteins. Colchicine-binding proteins were separated
by SDS-PAGE and analyzed by immunoblotting with anti-Syk antibodies. As
shown in Fig. 1B, Syk(WT) could be readily detected in the
bound fraction. Receptor cross-linking had no detectable effect on the
amount of tubulin-associated Syk(WT). The amount of tubulin bound to
the affinity resin, as determined by immunoblotting with anti-tubulin
antibodies, also was not detectably altered by receptor engagement
(data not shown).
DT40 cells were prepared that expressed a catalytically inactive form
of Syk (Syk(K396R)), in which an essential lysine in the catalytic
domain was replaced by arginine. The ability of Syk(K396R) to bind
tubulin was assessed by immunoblot analysis of
colchicine-agarose-binding proteins prepared from the soluble fractions
of the transfected cells. As shown in Fig. 1B, Syk(K396R)
still associated with tubulin. Immunoblotting of the affinity-purified
tubulin with anti-phosphotyrosine antibodies indicated that tubulin was
not phosphorylated on tyrosine in cells expressing only a catalytically
inactive form of Syk (Fig. 1C).
DT40 cells. Colchicine-binding proteins isolated from the cytosolic fractions of control and anti-IgM-treated cells were separated by
SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine antibodies. As shown in Fig. 2, the
phosphorylation of tubulin on tyrosine was completely abrogated in the
absence of Syk expression. Immunoblot analysis with anti-tubulin
antibodies confirmed the presence of equivalent amounts of tubulin in
the samples prepared from the wild-type and Syk
cells
(data not shown).
View larger version (42K):
[in a new window]
Fig. 2.
Syk expression is required for the tyrosine
phosphorylation of tubulin in B-cells. Wild-type (lanes
1 and 2) or Syk (lanes 3 and
4) DT40 chicken B-cells were left unstimulated (
) or
stimulated (+) with anti-IgM antibodies. Cells were Dounce homogenized,
and cytosolic fractions were incubated with colchicine-agarose. Bound
proteins were separated by SDS-PAGE, transferred to Immobilon-P, and
immunoblotted with anti-phosphotyrosine antibodies. The migration
position of tubulin is indicated.
View larger version (41K):
[in a new window]
Fig. 3.
Binding of tyrosine-phosphorylated proteins
to colchicine-agarose is mediated specifically through their
association with tubulin. A, cytosolic fractions
prepared from unstimulated ( ) or anti-IgM-stimulated (+) DT40 cells
were incubated with colchicine-agarose (lanes 1 and
2) or anti-tubulin antibodies immobilized on protein
G-agarose (lanes 3 and 4). The blot was probed
with anti-phosphotyrosine antibodies. B, Bal17 cells were
left untreated (lanes 1-3) or treated with Taxol
(lanes 4-6) and then stimulated with anti-IgM antibodies
for the times indicated. Cytosolic fractions from these cells were
incubated with colchicine-agarose, and bound proteins were separated by
SDS-PAGE. The resultant blot was probed with anti-phosphotyrosine
antibodies. C, Bal17 cells were left untreated (
) or
treated with Taxol (+). Cytosolic fractions from these cells were
incubated with colchicine-agarose, and bound proteins were separated by
SDS-PAGE. The resultant blot was probed with anti-tubulin
antibodies.
View larger version (34K):
[in a new window]
Fig. 4.
Cbl and Vav associate with soluble tubulin in
B-cells. Bal17 murine B-cells were left unstimulated ( ) or
stimulated (+) with anti-IgM antibodies. Cytosolic fractions were
incubated with colchicine-agarose, and bound proteins were separated by
SDS-PAGE. Membranes were probed with anti-phosphotyrosine antibodies
(lanes 1 and 2), anti-Cbl antibodies (lanes
3 and 4), or anti-Vav antibodies (lanes 5 and 6).
DT40 cell lines expressing
equivalent levels of Syk(WT) or Syk(Y130E) and adsorbed to
colchicine-agarose. Bound proteins were separated by SDS-PAGE and
immunoblotted with anti-Syk antibodies (Fig.
5A) and then stripped and
reprobed with anti-tubulin antibodies (Fig. 5B). These data
indicate that the mutation at Tyr-130, which strongly affects Syk-BCR
interactions, has little or no effect on the Syk-tubulin interaction.
View larger version (27K):
[in a new window]
Fig. 5.
Efficient interaction of Syk with the BCR is
not required for the interaction of Syk and tubulin. A,
Syk(WT) (lanes 1 and 2) or Syk(Y130E)
(lanes 3 and 4) stably transfected cell lines
were left unstimulated ( ) or stimulated (+) with anti-IgM antibodies.
Cytosolic fractions were incubated with colchicine-agarose. After
separation of bound proteins by SDS-PAGE and transfer to Immobilon-P,
the blot was probed with anti-Syk antibodies. The migration position of
Syk is indicated. B, the blot shown in A was
reprobed with anti-tubulin antibodies. The migration position of
tubulin is indicated.
DT40 B-cells expressing either Syk(WT) or the
Syk(Y130E) mutant (Fig. 6). In
Syk(WT)-expressing cells, receptor engagement stimulated the
phosphorylation of the three tubulin-associated proteins. In contrast,
receptor cross-linking had little effect on the phosphorylation of
tubulin-associated proteins in the Syk(Y130E)-expressing cells.
View larger version (50K):
[in a new window]
Fig. 6.
Phosphorylation of tubulin-associated
proteins, but not of tubulin, requires the interaction of Syk with the
antigen receptor. Syk(WT) (lanes 1 and 2) or
Syk(Y130E) (lanes 3 and 4) stably transfected
cell lines were left unstimulated ( ) or stimulated (+) with anti-IgM
antibodies. Cytosolic fractions were incubated with
colchicine-agarose. After separation of bound proteins by
SDS-PAGE and transfer to Immobilon-P, the blot was probed with
anti-phosphotyrosine antibodies.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
DT40 B cell line in which murine Syk was expressed at
an elevated level (Figs. 1B and 5A). This
association permits the in vitro phosphorylation of tubulin
by Syk on a single tyrosine that lies near the carboxyl terminus of the
subunit (6).
1 at
regions distinct from the phosphotyrosine binding sites (20). Because
the amino termini of Syk and ZAP-70 each contain a tandem pair of SH2
domains, an SH2 domain-mediated interaction of these kinases with
tubulin in a phosphotyrosine-independent manner remains a possibility.
DT40 cells and Syk
cells
stably expressing a catalytically inactive form of Syk, to show that
Syk catalytic activity is indeed required for tubulin phosphorylation
(Figs. 1C and 2). Thus, the phosphorylation state of tubulin
on tyrosine in B-cells is an indicator of the presence of
tubulin-associated, active Syk kinase. In cells expressing normal
levels of endogenous Syk, there is little or no tubulin phosphorylation
in the absence of receptor cross-linking (Fig. 2). In cells
overexpressing Syk(WT), the basal level of tubulin phosphorylation is
somewhat elevated, and this increases with receptor cross-linking (Fig.
1C). In cells overexpressing Syk(Y130E), a form of Syk that
is constitutively active and fails to bind to the antigen receptor (7),
tubulin is constitutively phosphorylated on tyrosine and this
phosphorylation is not enhanced further by receptor cross-linking (Fig.
6).
RI receptors it becomes phosphorylated on tyrosine (16). Like the
Vav-ZAP-70 interaction, it is possible that Syk and Cbl may also
interact both directly and indirectly through the mutual binding of tubulin.
and Ig-
components of the BCR in B-cells
(34) and CD2 in T-cells (35)) to downstream signaling molecules, such
as ZAP-70 (17), Fyn (19), Syk (6), GSK-3 (36), c-Mos (37),
mitogen-activated protein kinase (33), Cdc2 (38), Cdk5 (39), Vav (17),
Cbl (this report), phosphoinositide 3-kinase (40), Grb2, phospholipase
C-
1 (20), neurofibromin (41), several G protein
subunits (42),
and protein phosphatase 2A (43). The frequent reports of
tubulin-associated signaling molecules suggests that tubulin or
microtubules might function in part to promote the formation of
signaling complexes or perhaps aid in the intracellular localization of
these signaling molecules.
![]() |
FOOTNOTES |
---|
* This research was supported by Public Health Service Grant CA37372 awarded by the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Public Health Service Predoctoral Fellowship 3 F31
GM18062-01S1 awarded by the National Institutes of Health.
§ To whom correspondence should be addressed: Dept. of Medicinal Chemistry and Molecular Pharmacology, Hansen Life Science Research Bldg., Purdue University, West Lafayette, IN 47907. Tel.: 765-494-1457; Fax: 765-494-9193; E-mail: geahlen{at}pharmacy.purdue.edu.
The abbreviations used are: BCR, B-cell antigen receptor; SH2, Src homology 2; ITAM, immunoreceptor tyrosine-based activation motif; PAGE, polyacrylamide gel electrophoresis; WT, wild-type.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|