(Received for publication, August 14, 1995; and in revised form, October 11, 1995)
From the
Two molecules involved in signal transduction via the T cell antigen receptor, namely the protein-tyrosine kinase ZAP-70 and the proto-oncoprotein Vav, were found to be constitutively associated with tubulin in Jurkat T cells. Both were able to bind to tubulin independently of one another, as determined by transient transfection into COS-7 cells. The ZAP-70 associated with tubulin was preferentially tyrosine-phosphorylated after T cell antigen receptor stimulation of Jurkat T cells, suggesting that this interaction was functionally significant. Vav was also found to co-immunoprecipitate with ZAP-70 from cell extracts depleted of tubulin. This raised the possibility that Vav might be a substrate for ZAP-70 protein-tyrosine kinase activity. However, tyrosine phosphorylation of Vav preceded that of ZAP-70, indicating that Vav was unlikely to be a downstream target of ZAP-70. The association of ZAP-70 and Vav with tubulin implies that the microtubules may be involved in the signaling function of these two molecules, perhaps by targeting them to their appropriate intracellular location.
Stimulation of the T cell antigen receptor (TCR) ()initiates a cascade of signal transduction events, the
most proximal of which is the induction of PTK activity, which is
essential for the signaling process(1) . This involves the
cytoplasmic tails of the CD3 complex (
) and the
chain of the TCR becoming phosphorylated on specific tyrosine residues
within ITAMs(1) . Studies in mutant Jurkat T cells and
transfected COS cells have indicated that the Src family
protein-tyrosine kinase, Lck, is required for tyrosine phosphorylation
of the TCR immunoreceptor tyrosine-based activation
motifs(2, 3) . Biochemical and genetic experiments
have also indicated a role for the Src family PTK, Fyn, in TCR
signaling(4) . However, its intracellular localization suggests
that its function may be distinct from that of Lck(5) .
ITAM phosphorylation results in recruitment of the Syk family PTKs, ZAP-70 and Syk, to the TCR via the binding of their two SH2 domains (6, 7, 8, 9) . ZAP-70 and Syk are then tyrosine-phosphorylated themselves, which for ZAP-70 has been shown to activate its kinase activity(10) , and tyrosine phosphorylation of multiple intracellular proteins is induced(3, 6) . An essential role for ZAP-70 in TCR signaling and T cell development has been revealed by genetic studies(11, 12) .
TCR stimulation induces tyrosine
phosphorylation of a number of other intracellular proteins besides TCR
subunits and ZAP-70(13) . The identity of many of these
proteins is not known, and the functional consequences of the majority
of the tyrosine phosphorylations are unclear. However, several
phosphotyrosyl proteins have been identified which appear to play an
important role in the signaling process. These include phospholipase C
1, ERK mitogen-activated protein kinases, and Vav(14) .
Tyrosine phosphorylation of phospholipase C
1 increases its
catalytic activity, resulting in the hydrolysis of phosphatidylinositol
4,5-bisphosphate to inositol 1,4,5-trisphosphate and
diacylglycerol(14, 15) . These second messengers, in
turn, induce the mobilization of cytoplasmic calcium and the activation
of protein kinase C, respectively. The phosphorylation and activation
of the ERK by the TCR induces the phosphorylation of a number of
transcription factors that are important in the induction of gene
transcription(16) . The role of Vav is unknown. However, gene
targeting experiments have indicated that it is required for optimal
signaling via the TCR (17, 18, 19) . Sequence
homology suggests that Vav is a GDP/GTP exchange protein for a small
G-protein of the Rho/Rac subfamily(20) , but its identity is
presently unclear.
This laboratory has demonstrated previously that
-tubulin is constitutively tyrosine-phosphorylated in human T
cells(21) . In this study, it is shown that TCR stimulation of
Jurkat T cells induced the tyrosine phosphorylation of two proteins
that co-precipitated with tubulin. These proteins were identified as
ZAP-70 and Vav and suggest a role for the microtubule cytoskeleton in
the signaling functions of these two proteins.
ZAP-70 cDNA (from Arthur Weiss, Howard Hughes Medical Institute, San Francisco, CA) and vav cDNA were subcloned into the eukaryotic expression vector pcDNA3neo (InVitrogen) for COS-7 transfection experiments.
Immunoprecipitation of proteins from cell lysates and Western blotting was carried out as described previously(21) . To analyze the interaction of ZAP-70 with Vav SH2 domain, 5 µg of GST-SH2(Vav) fusion protein or GST were prebound to 10 µl of glutathione-Sepharose (Pharmacia) and were washed in IPB, prior to addition to cell lysates. Polyvinylidene difluoride membranes were stripped of bound antibody using the Amersham ECL protocol in experiments in which blots were probed for multiple antigens.
Tyrosine phosphorylation of two proteins which
co-precipitated with -tubulin was strongly induced after CD3
stimulation in Jurkat T cells (Fig. 1a). These proteins
had relative molecular massesS of 70 and 100 kDa. On longer exposure,
constitutive tyrosine-phosphorylated
-tubulin (a doublet with an
approximate relative molecular mass of 55 KDa) was detected, as
reported previously (21) . In some experiments, a weak Tyr(P)
band was also detected at 75 kDa. Immunoprecipitation with other
anti-
-tubulin antibodies and an anti-
-tubulin antibody
produced qualitatively similar results (data not shown). Comparison of
anti-
tubulin immunoprecipitates with anti-Tyr(P)
immunoprecipitates indicated that the interaction between tubulin and
the two Tyr(P) proteins was highly selective (Fig. 1a).
Figure 1:
Co-precipitation of ZAP-70 and Vav with
tubulin. a, Jurkat T cells were stimulated for 2 min with OKT3
antibody (+) or left unstimulated(-). Cell extracts were
then immunoprecipitated with anti-Tyr(P) (PTyr) or
anti--tubulin (
-Tub) antibodies. Immunoprecipitated
proteins were Western blotted and probed sequentially for Tyr(P), Vav
and ZAP-70, as indicated on the right of the panels. b, cell extracts from unstimulated(-) or OKT3-stimulated
(+) Jurkat T cells were immunoprecipitated with anti-Vav (Vav) or anti-ZAP-70 (ZAP) antisera. The immunizing
peptides to which the ZAP-70 and Vav antisera were raised were added to
half of the lysates (+P), to confirm the specificity of
immunoprecipitation. Western blots were probed sequentially for
-tubulin,
-tubulin, ZAP-70, and Vav, as indicated. c, COS-7 cells were transfected with the pcDNA3 expression
vector containing ZAP-70 cDNA (ZAP), Vav cDNA or no insert (pC3).
-Tubulin was immunoprecipitated from extracts of
the transfected COS-7 cells, Western blotted, and probed sequentially
for ZAP-70, Vav, or
-tubulin, as indicated on the left-hand
side of the panels.
A panel of antibodies was used to investigate by immunoblotting
whether the tubulin-associated proteins corresponded to previously
identified Tyr(P) proteins. This analysis indicated that both ZAP-70 (9) and Vav (22) co-precipitated with tubulin in
Jurkat T cells (Fig. 1a) and also in human T
lymphoblasts (data not shown). These proteins had identical mobilities
to the 70- and 100-kDa tubulin-associated Tyr(P) proteins,
respectively. Several other proteins, which are tyrosine-phosphorylated
in activated T cells, including the chain(23) ,
Lck(24) , HS-1(25) , CD5(26) , and cbl (27) , were not detected in anti-
-tubulin
immunoprecipitates (data not shown). Syk (6) was also
undetectable in anti-
tubulin immunoprecipitates from Jurkat T
cells. However, this was probably due to its low expression levels in
these cells as Syk was the major tubulin-associated Tyr(P) band in WEHI
231 B cells (data not shown). In reciprocal experiments, both
-
and
-tubulin co-immunoprecipitated with ZAP-70 and Vav (Fig. 1b). Competition with the immunizing ZAP-4 and
Vav-1 peptides in the cell lysates inhibited the co-immunoprecipitation
of both
- and
-tubulin by anti-ZAP and anti-Vav antibodies,
respectively (Fig. 1b). Thus the detected associations
of tubulin with ZAP-70 and Vav were specific. ZAP-70-tubulin and
Vav-tubulin interactions were constitutive and did not alter following
CD3 stimulation.
To test whether ZAP-70 and Vav required each other,
or distinct hematopoietic-specific proteins, to interact with tubulin,
COS-7 cells, which do not express endogenous ZAP-70 or Vav, were
transiently transfected with plasmids containing ZAP-70 or Vav cDNA.
Both ZAP-70 and Vav were detected in anti--tubulin
immunoprecipitates (Fig. 1c). Co-transfection of ZAP-70
and Vav did not alter the level of interaction of these proteins with
tubulin (data not shown). Thus the interactions of ZAP-70 and Vav with
tubulin were independent and did not require other proteins which were
hematopoietic cell-specific.
A 100-kDa Tyr(P) band, with the same mobility as Vav, co-immunoprecipitated with ZAP-70 in CD3-stimulated Jurkat T cells (Fig. 2a, top panel). Immunoblotting with anti-Vav antibody demonstrated that Vav was constitutively co-purified in anti-ZAP-70 immunoprecipitates and probably corresponded to the 100-kDa Tyr(P) protein (Fig. 2a, middle panel). In some experiments, it was also possible to detect very low levels of ZAP-70 specifically co-purifying in anti-Vav immunoprecipitates, also in a constitutive fashion (data not shown). Although co-immunoprecipitation of Vav with ZAP-70 was constitutive, a GST fusion protein of the Vav-SH2 domain precipitated ZAP-70 only after TCR stimulation (Fig. 2b), as has been reported previously by Katzav et al.(28) . Our data suggest that a constitutive interaction between Vav and ZAP-70 existed which was not mediated via the Vav SH2 domain. One possible explanation for this is that TCR stimulation induces a conformational change allowing the SH2 domain of Vav, which is already complexed with ZAP-70, to bind to its tyrosine-phoshorylated target sequence on ZAP-70. However, it cannot be excluded that a small fraction of total Vav may be induced to associate with ZAP-70 after TCR stimulation via its SH2 domain, but this increase may be below the detection limit of the assay. The possibility that tubulin mediated the interaction between Vav and ZAP-70 is considered below.
Figure 2: Constitutive co-immunoprecipitation of ZAP-70 and Vav. Extracts were prepared from unstimulated(-) or OKT3-stimulated (+) Jurkat T cells. In a, cell lysates were immunoprecipitated with anti-ZAP-70 (ZAP) or anti-Vav (Vav) antisera in absence or presence (+P) of immunizing peptide. Purified proteins were Western blotted and then probed sequentially for Tyr(P) (PTyr), Vav, and ZAP-70, as indicated on the left-hand side of the panel. In b, lysates were precipitated with GST or GST-Vav SH2 fusion protein. Bound proteins and total cell lysates (lysate) were Western blotted and probed for ZAP-70.
In contrast to the present study, Katzav et al.(28) found that the interaction between ZAP-70 and Vav was induced by CD3 stimulation using an anti-Vav antibody for immunoprecipitation. This may reflect the use of an anti-Vav antibody which cannot recognize Vav that is constitutively associated with ZAP-70. Katzav et al.(28) also did not detect Vav in immunoprecipitates using the 1222 anti-ZAP-70 antibody. A direct comparison of 1222 anti-ZAP-70 antibody with ZAP-4 anti-ZAP-70 antibody, which is used in the present study, has demonstrated that only ZAP-4 antibody co-immunoprecipitated Vav (data not shown). These results suggest that there are qualitative differences between the two anti-ZAP-70 antibodies, such that Vav associated with ZAP-70 is only immunoprecipitated by ZAP-4 antibody and not by 1222 antibody in detectable quantities.
Microtubule-associated proteins can be
operationally defined as proteins which co-purify with tubulin
polymerized in vitro(29) . The presence of ZAP-70 and
Vav in anti- tubulin immunoprecipitates suggested that these
proteins might also co-purify with polymerized tubulin. To test this
hypothesis, taxol and GTP were added to Jurkat cytosolic extracts to
promote in vitro tubulin polymerization(30) .
Taxol/GTP treatment removed over 80-90% of tubulin from cytosolic
extracts, and large amounts of tubulin were detected in the pellet, as
expected (Fig. 3a). The pellet fraction contained only
trace amounts of actin, and actin was not depleted from the cytosol
taxol/GTP treatment, confirming the specificity of this method for
polymerization of tubulin (Fig. 3a). Immunoblotting
demonstrated that 35% (±5% S.E.; n = 4) of
ZAP-70 and 6% (±2% S.E.; n = 4) of Vav were
depleted from cytosolic extracts by taxol/GTP treatment, and both of
these molecules were detected in the pellet fraction (Fig. 3a). Two other cytosolic Tyr(P) proteins, HS-1
and cbl, were not depleted from the cytosol or detected in the pellet
fraction after taxol/GTP treatment (data not shown), suggesting that
the co-purification of ZAP-70 and Vav with polymerized tubulin was
specific. The
chain, Lck, and CD5 were not present in the
cytosolic fraction, as they are membrane-associated. These data
indicated that both ZAP-70 and Vav could interact with polymerized
tubulin in vitro. Extraction of Jurkat T cells with PM2G
buffer, which maintains the microtubule cytoskeleton
intact(21, 31) , indicated that both ZAP-70 and Vav
were associated with soluble and polymerized tubulin in vivo (data not shown).
Figure 3:
Association of ZAP-70 and Vav with tubulin
polymerized in vitro. Cytosolic extracts were prepared from
unstimulated(-) or OKT3-stimulated (+) Jurkat T cells. In a, tubulin was polymerized in vitro from cytosol by
addition of taxol and GTP (Tax) and the centrifuged to
generate the ``pellet'' fraction. No taxol and GTP was added
to the control cytosol (cont.). Cytosol and pellet fractions
were Western blotted and probed sequentially for Vav, ZAP-70,
-tubulin, and actin, as indicated on the left-hand side of the panels. The amount of each fraction loaded was adjusted to
give the same number of cell equivalents. In b, Vav (Vav
I.P.) and ZAP-70 (ZAP-70 I.P.) were immunoprecipitated
from cytosolic extracts prepared as in a, with and without
taxol/GTP treatment. Immunopurified proteins were Western blotted and
probed for the antigens indicated beside the panels. In c,
control and taxol-treated cytosol were immunoprecipitated with
anti-ZAP-70 antiserum. The immunoprecipitated proteins were Western
blotted and probed for ZAP-70 and Vav (left-hand panels). In
the right-hand panel, total cytosol (control and
taxol-treated) were Western blotted and probed for
-tubulin
content.
Similar amounts of ZAP-70 were immunoprecipitated from the control and taxol-treated cytosolic extracts under conditions where ZAP-70 antibody was limiting (Fig. 3b). Under these conditions, taxol/GTP reduced the relative abundance of tyrosine-phosphorylated ZAP-70 in the cytoplasm by 70% (±5%; n = 4), as judged by anti-Tyr(P) immunoblotting (Fig. 3b). As expected, a tyrosine-phosphorylated band with identical mobility to ZAP-70 preferentially accumulated in the microtubule pellet from activated cell extracts (data not shown). These data indicate that ZAP-70 that was tyrosine-phosphorylated after TCR stimulation was preferentially associated with tubulin polymerized in vitro. This further suggests that the interaction of ZAP-70 with tubulin may be important for the activation of this kinase. In contrast, cytosolic tubulin depletion had little effect on the amount of tyrosine-phosphorylated Vav (Fig. 3b) detected in anti-Vav immunoprecipitates (reduction of 13% ± 5% S.E.; n = 4).
The association of both ZAP-70 and Vav with tubulin (Fig. 2a) suggested that the presence of Vav in ZAP-70 immunoprecipitates might be mediated via tubulin. However, taxol/GTP depletion of the majority of cytosolic tubulin only slightly reduced the level of Vav in ZAP-70 immunoprecipitates (Fig. 3c). These data indicated that Vav interaction with ZAP-70 was largely independent of tubulin.
The interactions of
ZAP-70 and Vav with components of the microtubule cytoskeleton and with
each other raised the possibility that these proteins might be
functionally interlinked in TCR signal transduction. As Vav is
tyrosine-phosphorylated after TCR
stimulation(32, 33) , this suggested that Vav might be
a downstream substrate of ZAP-70 PTK. To investigate this, the kinetics
of phosphorylation of ZAP-70 and Vav were analyzed after CD3
stimulation in Jurkat T cells. Tyrosine phosphorylation of Vav was
extremely rapid, reaching a maximum at 0.5 to 1 min and then falling to
base-line levels by 10 min (Fig. 4). In contrast the tyrosine
phosphorylation of ZAP-70 was slower, peaking at 5-10 min and
falling to base-line levels by 30 min. The kinetics of tyrosine
phosphorylation of ZAP-70 and Vav that co-immunoprecipitated with
-tubulin were very similar to that detected in anti-ZAP-70 and
anti-Vav immunoprecipitates (Fig. 4, bottom panel).
ZAP-70 PTK is activated by tyrosine phosphorylation(10) . These
data (Fig. 4) suggested that Vav was tyrosine-phosphorylated
before ZAP-70 was activated and, therefore, that Vav was unlikely to be
a downstream target of ZAP-70.
Figure 4:
Kinetics of tyrosine phosphorylation of
ZAP-70 and Vav. Jurkat T cells were stimulated with OKT3 antibody for
the times indicated and then rapidly lysed with 2 IPB. Vav,
ZAP-70, and
-tubulin (as indicated) were immunoprecipitated from
cell extracts and Western blotted and probed for Tyr(P) (PTyr). The positions of the tyrosine-phosphorylated ZAP-70
and Vav in the anti-
-tubulin immunoprecipitates are indicated with arrows. The kinetics of tyrosine phosphorylation of Vav and
ZAP-70 are also shown graphically at the top of the figure.
These data were determined by laser densitometric scanning of Vav and
ZAP-70 immunoprecipitates probed for Tyr(P) and are the mean of three
experiments (±S.E.). For comparison, the density of Tyr(P) blots
was adjusted to peak value of 1 for both ZAP-70 and
Vav.
In conclusion, these data demonstrate that ZAP-70 and Vav interact with tubulin and each other in T lymphocytes. A functional link, therefore, is likely to exist between these signaling molecules and the tubulin cytoskeleton. The possibility that the intracellular targeting of ZAP-70 and Vav is dependent on interactions with tubulin is currently being investigated.