(Received for publication, September 11, 1995; and in revised form, December 20, 1995)
From the
Syk (p72) is a 72-kDa, nonreceptor,
protein-tyrosine kinase that becomes tyrosine-phosphorylated and
activated in B lymphocytes following aggregation of the B-cell antigen
receptor. To explore the subcellular location of activated Syk,
anti-IgM-activated B-cells were fractionated into soluble and
particulate fractions by ultracentrifugation. Activated and
tyrosine-phosphorylated Syk was found predominantly in the soluble
fraction and was not associated with components of the antigen
receptor. Similarly, the activated forms of Syk and its homolog,
ZAP-70, were found in soluble fractions prepared from
pervanadate-treated Jurkat T-cells. A 54-kDa protein that
co-immunoprecipitated with Syk from the soluble fraction of activated
B-cells was identified by peptide mapping as
-tubulin.
-Tubulin was an excellent in vitro substrate for Syk and
was phosphorylated on a single tyrosine present within an acidic
stretch of amino acids located near the carboxyl terminus.
-Tubulin was phosphorylated on tyrosine in intact cells following
aggregation of the B-cell antigen receptor in a reaction that was
inhibited by the Syk-selective inhibitor, piceatannol. Thus, once
activated, Syk releases from the aggregated antigen receptor complex
and is free to associate with and phosphorylate soluble proteins
including
-tubulin.
Syk (p72) is a 72-kDa cytoplasmic
protein-tyrosine kinase that is expressed in a variety of hematopoietic
cells where it participates in signal transduction cascades initiated
by the engagement of the cell surface receptors with which it
associates. Syk is activated in B lymphocytes by aggregation of the
B-cell antigen receptor (BCR)(
)(1) ; in platelets by
thrombin (2) or by integrin ligation(3) ; in mast cells
by aggregation of Fc
RI receptors(4) ; in monocytes by
cross-linking Fc
RI and Fc
RII
receptors(5, 6) ; in macrophages by engagement of the
Fc
RIIIA receptor(7) ; in T-cells by cross-linking the
T-cell antigen receptor (TCR)(8, 9) ; in peripheral
blood lymphocytes by interleukin-2 (10) ; and in granulocytes
in response to granulocyte colony-stimulating factor(11) .
The activation of Syk in response to the engagement of multichain
immune recognition receptors (12) has been studied in the
greatest detail. The aggregation of these receptors leads to the
phosphorylation of multiple cellular proteins on tyrosine, initiating
signaling cascades resulting in increased inositol 1,4,5-trisphosphate
production and calcium mobilization (for reviews, see (13, 14, 15, 16) ). That Syk is an
important mediator of receptor signaling is indicated by an absence of
signaling in Syk DT40 chicken B-cells(17) ,
an inhibition of Fc
RI-mediated signaling in mast cells by a
Syk-selective inhibitor(18) , and a blockage in B-cell
development in Syk
``knockout'' mice at
stages that normally require signals to be sent through the BCR or
pre-BCR(19) .
The efficient activation of Syk requires the presence on receptor components of an immunoreceptor tyrosine activation motif (ITAM), consisting of two YXXL/I cassettes separated by 6-8 amino acids (20) and the presence of at least one member of the Src family of protein-tyrosine kinases. Receptor aggregation leads to the rapid phosphorylation of the ITAM, presumably by Src family kinases, establishing a binding site for the two tandem, amino-terminal, SH2 domains of Syk. This occupancy allows the kinase to catalyze an autophosphorylation reaction, which leads to an increase in its intrinsic activity(21) .
Many of the proteins
phosphorylated in response to receptor aggregation are likely to be
substrates for Syk or for Syk-activated kinases. However, little is
known regarding the nature of the substrates that are phosphorylated by
activated Syk and where in the cell these protein-substrate
interactions occur. Whether these interactions occur while Syk is
associated with the receptor, for example, has not been explored in
detail. To begin examining such questions, we have used cellular
fractionation techniques to characterize the cytosolic and
membrane-associated pools of Syk. In this report, we show that Syk
activated by the aggregation of the BCR is found almost exclusively in
the cytosolic fraction of activated B-cells. This activated, cytosolic
Syk associates with and phosphorylates tubulin. In vitro,
-tubulin is an excellent substrate for Syk and becomes
phosphorylated on a tyrosine residue located near the carboxyl
terminus.
To prepare GST-p35, the GST-Syk-expressing transfer vector pNTXSyk was digested with XhoI (just upstream of the translational start site) and ApaI (codon for proline 326). The vector fragment was gel-purified, blunt-ended with mung bean nuclease, and recircularized to generate pNTXp35. Sequencing of the fusion junction confirmed that the open reading frame had been maintained. Sf9 cell co-transfection and plaque purification were carried out as described above for GST-Syk.
For some experiments, GST-Syk immobilized on glutathione-Sepharose (see above) was used in place of the antibody-protein A-Sepharose complex.
One-dimensional peptide mapping of phosphoproteins was performed essentially as described by Cleveland et al.(29) . Phosphopeptides excised from SDS-polyacrylamide gels were reelectrophoresed on a 15% gel in the presence of 17 ng of Staphylococcus aureus V8 protease.
Figure 1:
Activated Syk appears in the cytosolic
fraction of anti-IgM-activated B-cells. Human DG75 B-cells were
activated with anti-IgM antibodies for 0, 4, 10, or 15 min. Cells were
lysed by Dounce homogenization and separated into particulate and
soluble fractions by ultracentrifugation. Proteins were
immunoprecipitated with polyclonal rabbit anti-Syk antiserum (lanes
2-5 and 7-10) or with preimmune serum (P), (lanes 1 and 6). The resulting immune
complexes were incubated with [-
P]ATP to
allow Syk autophosphorylation, which was visualized by autoradiography
following the separation of proteins by SDS-PAGE (Panel A).
Alternatively, Syk present in immune complexes was visualized by
Western blotting with anti-Syk antibodies (Panel B). The large
band at 50 kDa in Panel B represents the heavy chain of rabbit
IgG.
Figure 4:
A 54-kDa kinase substrate
co-immunoprecipitates with Syk from the cytosolic fractions of
anti-IgM-activated B-cells. A, Bal17 murine B-cells were
either untreated (lane 1) or were stimulated with anti-IgM
antibodies for 15 s (lane 2), 45 s (lane 3), 2 min (lane 4), or 10 min (lane 5). Cytosolic fractions
were prepared and Syk was immunoprecipitated with anti-Syk antibodies.
The immune complexes were incubated with
[-
P]ATP, separated by SDS-PAGE, transferred
to membranes, and subjected to autoradiography after incubation with
KOH. B, particulate (P) or soluble (S)
fractions from unactivated (lanes 2 and 4) or
anti-IgM-activated (lanes 3 and 5) Bal17 B-cells were
incubated with a GST-Syk fusion protein linked to
glutathione-Sepharose. Bound proteins were incubated with
[
-
P]ATP. Lane 1, GST-Syk
alone.
Syk protein present in the anti-Syk peptide immune complexes was detected by Western blotting with anti-GST-p35 antibodies following the separation of the antigen-antibody complexes by SDS-PAGE (Fig. 1B). The cross-linking of surface IgM had no appreciable effect on the amount of Syk recovered from either the particulate or soluble fractions.
Figure 2:
Tyrosine-phosphorylated Syk appears only
in the cytosolic fraction of anti-IgM-activated B-cells. DG75 human
B-cells were activated with anti-IgM antibodies for 0, 4, 10, or 15
min. Cells were lysed by Dounce homogenization and separated into
particulate and soluble fractions by ultracentrifugation.
Phosphotyrosine-containing proteins were immunoprecipitated with
anti-phosphotyrosine antibodies. Syk was visualized in the resulting
immune complexes by autophosphorylation following incubation with
[-
P]ATP (Panel A) or by Western
blotting with anti-Syk antibodies (Panel B). P = preimmune serum. The large band at 50 kDa in Panel B represents the heavy chain of rabbit
IgG.
The Syk protein recovered from the cytosolic fraction by immunoprecipitation with anti-phosphotyrosine antibodies and detected either by autophosphorylation (Fig. 2A) or Western blotting (Fig. 2B) migrated on SDS-PAGE as a single, prominent band and co-migrated with the upper band of the doublet observed in anti-Syk immune complex kinases assays (Fig. 1A).
To determine if cytosolic
Syk was also activated in murine B-cells, anti-Syk immune complexes
were prepared from the particulate and soluble fractions of untreated
or anti-IgM-activated Bal17 cells and incubated with buffer containing
[-
P]ATP. As shown in Fig. 3A, only the activity of Syk recovered from the
cytosolic fraction was markedly affected by receptor cross-linking. The
Ig-
component of the murine BCR, which migrates on
SDS-polyacrylamide gels with an apparent molecular weight of 34,000,
could be observed co-immunoprecipitating with Syk only in samples
prepared from the particulate fraction (Fig. 3A).
Figure 3:
Activated Syk appears in the cytosolic
fraction of B-cells or T-cells treated with pervanadate. A,
Syk was immunoprecipitated with anti-Syk antibodies from particulate (lanes 1 and 2) or soluble (lanes 3 and 4) fractions prepared from untreated(-) or
anti-IgM-activated (10 min) (+) murine Bal17 B-cells. The
resulting immune complexes were incubated in the presence of
[-
P]ATP. Phosphoproteins were separated by
SDS-PAGE, transferred to poly(vinylidene fluoride) membranes, treated
with 1 N KOH, and visualized by autoradiography. The migration
position of the Ig-
component of the mouse BCR is indicated by the open arrow. B, Syk was immunoprecipitated with anti-Syk
antibodies from particulate (P) (lanes 1 and 2) or soluble (S) (lanes 3 and 4)
fractions prepared from untreated(-) or pervanadate (PV)-treated (+) Bal17 murine B-cells and visualized by
autophosphorylation with [
-
P]ATP. C, Syk (lanes 1-4) or ZAP-70 (lanes 5 and 6) was immunoprecipitated from the soluble fractions
of untreated(-) or pervanadate-treated (+) Bal17 B-cells (lanes 1 and 2) or Jurkat T cells (lanes
3-6) with anti-Syk (lanes 1-4) or anti-ZAP-70 (lanes 5 and 6) antibodies. Kinases were visualized
in the resulting immune complexes by
autophosphorylation.
Figure 5:
Syk
associates with and phosphorylates tubulin. A, Syk was
immunoprecipitated with anti-Syk antibodies from the cytosolic fraction
of anti-IgM-activated Bal17 B-cells and incubated with
[-
P]ATP to detect the 54 kDa Syk-associated
protein (lane 1). Recombinant Syk and purified tubulin (lane 2), purified tubulin alone (lane 3) or
recombinant Syk alone (lane 4) were incubated in vitro with [
-
P]ATP, separated by SDS-PAGE
and subjected to autoradiography. B, phosphopeptide map
generated by the partial proteolysis of the 54-kDa, Syk-associated
substrate (lane 1) and of in vitro phosphorylated
tubulin (lane 2). C, Syk was immunoprecipitated with
anti-Syk antibodies from the cytosolic fractions of anti-IgM-activated (lanes 2 and 4) or unactivated (lanes 1 and 3) Bal17 B-cells that had been pretreated with (lanes 3 and 4) or without (lanes 1 and 2)
Taxol. Syk and associated substrates were visualized by
autophosphorylation with [
-
P]ATP. The
migration position of tubulin is indicated by the arrow.
Tubulin phosphorylated in vitro by Syk co-migrated by SDS-PAGE with the 54-kDa Syk-associated substrate (Fig. 5A). Phosphopeptide mapping by partial proteolysis (29) of each protein gave rise to a single, prominent phosphopeptide that co-migrated on 15% SDS-polyacrylamide gels (Fig. 5B), suggesting that they represented the same or highly related proteins.
To support the identification of the 54-kDa substrate as tubulin, we examined the effect of Taxol on its recovery from cytosolic fractions of activated Bal17 B-cells. Taxol stabilizes microtubules and promotes the polymerization of unassembled tubulin(36, 37, 38) . Syk was immunoprecipitated with anti-Syk antibodies from the cytosolic fractions of anti-IgM-activated and unactivated Bal17 cells or Taxol-pretreated Bal17 cells and detected by autophosphorylation. As shown in Fig. 5C, the 54-kDa protein co-immunoprecipitated with Syk from the cytosolic fraction of anti-IgM-activated Bal17 cells, but not from activated cells that had been pretreated with Taxol.
Figure 6:
Phosphorylation of tubulin in vitro by Syk. Equimolar amounts of tubulin dimer (), the
cytoplasmic domain of band 3 (cfb3) (
), and myelin basic
protein (MBP) (
) were phosphorylated in vitro without (lane 1) or with (lanes 2-5)
recombinant Syk for 0.5, 2, 5, or 10 min. In Panel A,
phosphoproteins were detected by exposure of SDS-polyacrylamide gels to
x-ray film for 15 min except for the lower right panel, which
was exposed for 12 h. For Panel B, radiolabeled proteins were
excised from the gel and the amount of
P incorporated was
quantified by liquid scintillation
spectrometry.
Tubulin exists as a heterodimer of
tightly associated and
subunits that exhibit about 40%
sequence similarity(43) . To further examine the substrate
specificity of Syk, phosphotubulin was separated by SDS-PAGE under
conditions that allow separation of the distinct
and
subunits(28) . As shown in Fig. 7A, Syk
selectively catalyzed the phosphorylation of the
-tubulin subunit.
Figure 7:
Syk phosphorylates -tubulin on a
tyrosine located near the carboxyl terminus. A, purified chick
brain tubulin was phosphorylated in vitro with
[
-
P]ATP using recombinant Syk and was
separated by SDS-PAGE under conditions that resolve the
and
monomers. Phosphotubulin detected by autoradiography is shown in lane 1, tubulin protein in lane 2. B, tubulin was
phosphorylated in vitro by GST-Syk and then treated with
carboxypeptidase A for 10 (lane 1), 20 (lane 2), or
50 (lane 3) min to remove the carboxyl-terminal tyrosine.
Phosphotubulin was detected by autoradiography following SDS-PAGE. C, In vitro phosphorylated tubulin was treated with
subtilisin for 0 (lane 1), 5 (lane 2), 10 (lane
3), 20 (lane 4), or 30 (lane 5) min to remove 4
kDa of the carboxyl terminus. The resulting 50-kDa amino-terminal
fragment detected by staining with Coomassie Blue is indicated by the arrow. The resulting autoradiogram is shown in the lower
panel.
Cleavage of - and
-tubulin with subtilisin generates
50,000-kDa intermediates that lack the acidic carboxyl
termini(44) . To explore the location of the site of
phosphorylation on
-tubulin, purified tubulin was phosphorylated in vitro with recombinant GST-Syk and then incubated with
subtilisin for varying periods of time. As illustrated in Fig. 7C, no radiolabel was associated with the 50-kDa
fragments of
- and
-tubulin generated by digestion with
subtilisin.
The 4-kDa carboxyl-terminal region of -tubulin
contains two potential sites of tyrosine phosphorylation, one internal
site and a second at the extreme carboxyl terminus. To differentiate
between these possible sites, tubulin phosphorylated in vitro by recombinant GST-Syk was incubated with carboxypeptidase A for
varying periods of time. As shown in Fig. 7B, while
carboxypeptidase A digestion generated slightly faster migrating forms
of phosphotubulin, it did not rapidly catalyze removal of the
radiolabeled amino acid (Fig. 7B). This indicates that
the principal site of phosphorylation on
-tubulin is not at the
extreme carboxyl terminus.
Figure 8:
Tubulin is phosphorylated on tyrosine in
intact cells following activation. A and B, Bal17
B-cells were stimulated with anti-IgM antibodies for 0 (lanes
1, 4, and 7), 0.75 (lanes 2, 5, and 8) or 10 (lanes 3, 6, and 9) min. Soluble lysates were adsorbed to colchicine-Sepharose.
Phosphotyrosine-containing proteins were identified by Western blotting
with anti-phosphotyrosine antibodies (Panel A). Tubulin was
identified by Western blotting with anti--tubulin antibodies (Panel B). Lanes 1-3 represent crude soluble
extracts; lanes 4-6, unadsorbed proteins; and lanes
7-9, proteins bound to colchicine-Sepharose. C,
DG75 B cells were stimulated for 0 (lane 1), 2 (lane
2), 10 (lane 3), or 15 (lane 4) min with
anti-IgM antibodies. Cytosolic extracts were adsorbed to
colchicine-Sepharose. Bound proteins were separated by SDS-PAGE and
those containing phosphotyrosine were detected by Western blotting with
anti-phosphotyrosine antibodies. Lane 5 shows the migration
position of
-tubulin as detected by Western blotting with
anti-
-tubulin. D, Bal17 B-cells were pretreated for 60
min without (lanes 1-3) or with (lanes
4-6) 15 µg/ml piceatannol and then activated for 0 (lanes 1 and 4), 0.75 (lanes 2 and 5), or 10 (lanes 3 and 6) min with anti-IgM antibodies.
Phosphotyrosine-containing proteins in the soluble fraction that bound
to colchicine-agarose were detected by Western blotting with
anti-phosphotyrosine antibodies.
Similar results
were observed using the human DG75 B-cells. In this experiment, the
and
monomers were separated during electrophoresis.
Receptor cross-linking primarily led to increases in the
phosphorylation of the
subunit.
To further establish a role
for Syk in the phosphorylation of tubulin in intact cells, we examined
the ability of piceatannol to block anti-IgM-stimulated tubulin
phosphorylation. At low concentrations, piceatannol selectively
inhibits the receptor-mediated activation of Syk as compared to the Src
family kinases in mast cells (18) and B-cells. ()Bal17 B-cells were pretreated for 1 h with 15 µg/ml
piceatannol or with Me
SO carrier alone (final
concentration, 0.15%) prior to activation with anti-IgM antibodies. As
shown in Fig. 8D, the time-dependent phosphorylation of
tubulin in Bal17 B-cells in response to receptor cross-linking was
inhibited by pretreatment with piceatannol.
In lymphoid cells, the Src family kinases are localized to the particulate fraction where they are anchored by protein-protein and protein-membrane interactions mediated, in part, by post- and co-translational acylations and the presence of SH3 domains (for review, see (45) ). Syk, which lacks both an SH3 domain and a consensus sequence for N-myristoylation, is distributed more evenly between the particulate and soluble fractions in B-cells (Fig. 1). The distribution observed in B-cells is similar to that reported previously for peripheral blood lymphocytes(46) . Both Syk and Src family kinases are activated when transmembrane antigen receptors are aggregated by extracellular polyvalent ligands or antireceptor antibodies. The activated Src family kinases remain associated with membrane or cytoskeletal components present in the particulate fraction where they are presumably poised to phosphorylate physiologically relevant substrates also localized to these regions (e.g. components of the antigen receptor complex). Activated Syk, on the other hand, appears predominantly in the soluble fraction of B-cell lysates (Fig. 2). Syk, activated in peripheral blood lymphocytes in response to interleukin-2, is also localized predominantly to the cytosolic fraction(46) . Thus, Syk may be positioned within the cell to interact with and phosphorylate a distinct subset of proteins distinguished from those of the Src family kinases, in part, by their subcellular location.
Biochemical and
genetic evidence indicates that the BCR-mediated activation of Syk
proceeds via the association of its tandem, amino-terminal, SH2 domains
with the tyrosine-phosphorylated ITAM motifs of Ig- or
Ig-
(21, 22, 47) . Thus, Syk would be
expected to be recruited to the antigen receptor complex following its
aggregation and phosphorylation. This recruitment of Syk to the
receptor, however, does not measurably alter the overall subcellular
distribution of the kinase between particulate and soluble fractions (Fig. 1). Following the interaction of Syk with the
phosphorylated ITAM or with dually phosphorylated peptides modeled on
the ITAMs of Ig-
or Fc
RI-
, Syk is activated as it
becomes phosphorylated on tyrosine(21, 48) . In the
intact B-cell, subcellular fractionation studies indicate that this
tyrosine-phosphorylated Syk is almost exclusively confined to the
soluble fraction (Fig. 2). This is most easily seen in extracts
prepared from human B-cells where enhanced tyrosine phosphorylation
leads to a shift in the electrophoretic mobility of Syk to a slower
migrating species (Fig. 1). This slower migrating form of Syk is
the major form that is immunoprecipitated with anti-phosphotyrosine
antibodies (Fig. 2). Immunoprecipitates of soluble,
tyrosine-phosphorylated Syk do not contain the co-immunoprecipitated
Ig-
that is visible in anti-Syk immune complexes isolated from the
membrane fraction of activated B-cells (Fig. 3A). These
observations suggest a model whereby Syk interacts only transiently
with the aggregated receptor and is released from the receptor when the
kinase becomes phosphorylated on tyrosine. This phosphorylation could
occur as a consequence of the actions of Src-family kinases or from Syk
autophosphorylation. Increases in the intrinsic activity of Syk
accompany this phosphorylation such that the activated Syk is also
found predominantly in the soluble fraction ( Fig. 1and Fig. 3).
Syk is also activated in B-cells treated with
pervanadate, a potent protein tyrosine phosphatase inhibitor (Fig. 3). In B-cells, vanadate, which also inhibits protein
tyrosine phosphatases, induces the hyperphosphorylation of the Ig-
and Ig-
components of the BCR (49) and pervanadate likely
functions in a similar fashion. This pervanadate-activated Syk does not
remain associated with the phosphorylated antigen receptor complex and
is found primarily in the soluble fraction (Fig. 3). ZAP-70, a
Syk homolog that is expressed predominantly in T-cells, also appears in
a soluble, activated form when a Jurkat T-cell line that expresses both
Syk and ZAP-70 is treated with pervanadate (Fig. 3). This
activation of ZAP-70 is blocked in J.CaM1 cells, (
)a
Jurkat-derived cell line that lacks the functional Lck needed to
phosphorylate components of the TCR/CD3 complex(50) . Thus,
ZAP-70 is likely released from the T-cell receptor following its
activation and phosphorylation on tyrosine in a manner similar to that
observed for Syk and the BCR.
Soluble, activated Syk would be poised to interact with and phosphorylate a subset of protein-tyrosine kinase target proteins present in the cytosol of activated cells. A key to the nature of one of these potential substrates was the observation that a 54-kDa protein co-immunoprecipitated with Syk and became phosphorylated on tyrosine in anti-Syk immune complexes. This protein was found in both anti-Syk immune complexes and among proteins adsorbed to an immobilized GST-Syk fusion protein (Fig. 4). The 54-kDa protein was identified as tubulin by peptide mapping and by its loss from the cytosolic fraction of activated cells in which the polymerization of tubulin into microtubules was forced by pretreatment with Taxol (Fig. 5).
Tubulin is an excellent in vitro substrate
for Syk. Syk preferentially catalyzes the phosphorylation of the
-tubulin monomer on a single tyrosine residue localized to a
region of the protein near the carboxyl terminus. This site lies within
a carboxyl-terminal peptide that is released from
-tubulin
following limited proteolytic digestion with subtilisin that has the
sequence FSEAREDMAALEKDYEEVGVDSVEGEGEEEGEEY(44) (sequence given for the
major brain forms of mammalian and chicken
-tubulin)(43, 51) . This peptide contains two
tyrosine residues, one of which is located at the extreme carboxyl
terminus and has been reported to be an in vitro site of
phosphorylation catalyzed by the insulin receptor based on its rapid
release in response to carboxypeptidase A(52) . Treatment of
Syk-phosphorylated tubulin with carboxypeptidase A, however, has little
effect on its state of phosphorylation (Fig. 7B),
indicating that the tyrosine 20 residues upstream from the carboxyl
terminus, Tyr
, rather than the carboxyl-terminal
tyrosine, is the major site of phosphorylation. Other protein-tyrosine
kinases have been reported to phosphorylate tubulin, but the pattern of
phosphorylation generally differs from that observed with Syk. For
example, Src phosphorylates
- and
-tubulin with nearly equal
efficiency in vitro(53) and in nerve growth cone
membranes(54) , while the epidermal growth factor receptor
preferentially phosphorylates
-tubulin(53) .
In our
hands, Syk demonstrates a highly restricted substrate specificity in vitro(55) . The site of Syk phosphorylation on
-tubulin is similar in sequence to the sites of phosphorylation
previously identified on the erythrocyte anion transport channel (band
3) (56) and glycogen synthase(57) . An alignment of the
phosphorylation sites identified on these three substrates indicate a
strong preference for acidic amino acid residues located both proximal
and distal to the site of tyrosine phosphorylation:
-tubulin,
Met-Ala-Ala-Leu-Glu-Lys-Asp-Tyr-GluGlu-Val-Gly; band 3,
Met-Glu-Glu-Leu-Asn-Asp-Asp-Tyr-Glu-Asp-Asp-Met-; glycogen
synthase, Pro-Glu-Glu-Asp-Gly-Glu-Arg-Tyr-Glu-Asp-Glu-Glu-.
-Tubulin, which also contains a conserved tyrosine residue in an
analogous position(43) , lacks the surrounding acidic residues
that are important for substrate recognition by Syk and is not readily
phosphorylated by Syk in vitro (Fig. 7). Sites of Syk
phosphorylation that have been sequenced for histone H2B (58) and myelin basic protein (41) do not fit this
general motif. However, myelin basic protein is a comparatively poor
substrate for Syk (Fig. 6) and is phosphorylated at a rate much
slower than that observed for the phosphorylation of band 3 or
-tubulin.
Tubulin is also phosphorylated on tyrosine in intact
B-cells following aggregation of the BCR (Fig. 8). This
phosphorylation occurs under conditions in which Syk is activated and
occurs primarily on the -subunit. Preferential phosphorylation of
-tubulin is consistent with the demonstrated substrate specificity
of the kinase in vitro (Fig. 7). The preincubation of
B-cells with piceatannol at concentrations in which the activation of
Syk is preferentially inhibited blocks receptor-mediated
phosphorylation of tubulin, suggesting that Syk is the kinase primarily
responsible for the tyrosine phosphorylation of tubulin following
receptor aggregation. The observation that tubulin is phosphorylated in
activated lymphoid cells is similar to that reported previously by Ley et al.(59) who found that
-tubulin is
tyrosine-phosphorylated in human T lymphocytes in response to
engagement of the T-cell antigen receptor. The phosphorylated
-tubulin was restricted to the 45% of tubulin that is not
polymerized in Jurkat T-cells(59) . TCR engagement leads to the
activation of ZAP-70, a Syk homolog found in T-cells that has a
substrate specificity similar to that of Syk. When Jurkat T-cells are
treated with pervanadate, activated ZAP-70 appears in the soluble
fraction (Fig. 3) where the unpolymerized
-tubulin would be
expected to be located. Thus, ZAP-70 would be a good candidate for the
T-cell
-tubulin kinase.
Determinations of the effect that
tyrosine-phosphorylation has on the properties of tubulin and the role
that this plays in B-cell function must await further experimentation.
Tubulin phosphorylated on the carboxyl-terminal tyrosine by the insulin
receptor fails to polymerize into microtubules(52) . Similarly,
- and
-tubulin phosphorylated near the carboxyl terminus by a
Ca
/calmodulin-dependent protein kinase fails to
polymerize(60) . An inhibitory effect of phosphorylation on the
incorporation of tubulin into microtubules is also consistent with the
observation that
-tubulin, phosphorylated in response to ligation
of the TCR, is found exclusively in the cytosolic fractions of Jurkat
T-cells(59) . Thus, tyrosine phosphorylation may play a role in
altering the microtubule/tubulin monomer equilibrium by lowering the
pool of tubulin monomers available for polymerization. The acidic
carboxyl-terminal region of tubulin contains the sites of interaction
with the microtubule-associated proteins (MAPs) that regulate
microtubule assembly and function(61, 62) . Through
the use of synthetic peptides, the site of interaction of MAP2 and tau
with
-tubulin has been narrowed to a 12 amino acid region,
KDYEEVGVDSVE, that encompasses Tyr
(61) .
Antibodies prepared against a peptide corresponding in sequence to this
region block MAP-induced microtubule assembly and depolymerize
preformed microtubules(62) . Thus, phosphorylation in this
region of
-tubulin might reasonably be expected to alter its
association with one or more MAPs. The binding of the acidic amino
terminus of erythrocyte band 3 to glycolytic enzymes is inhibited by
tyrosine phosphorylation(56) . By analogy to band 3, the
phosphorylation of
-tubulin may likewise block its ability to
interact with MAPs. Alternatively, tyrosine-phosphorylated tubulin
could serve as a binding site for signaling molecules that possess SH2
domains and could serve a role in mediating their redistribution within
the cell following receptor aggregation. Experiments to explore these
possibilities are currently under way.