Activation and Association of the Tec Tyrosine Kinase with the Human Prolactin Receptor: Mapping of a Tec/Vav1-Receptor Binding Site
J. Bradford Kline,
Daniel J. Moore and
Charles V. Clevenger
Department of Pathology and Laboratory Medicine University of
Pennsylvania Medical Center Philadelphia, Pennsylvania 19104
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ABSTRACT
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Stimulation of the PRL receptor (PRLr) results in
the activation of the guanine nucleotide exchange factor (GEF)
p95Vav1 with corresponding alterations in
cytoarchitecture and cell motility. To better understand the mechanisms
involved in the regulation of Vav1 activity, the role of the tyrosine
kinase p70Tec was examined.
Coimmunoprecipitation and in vitro kinase assays revealed
that ligand stimulation of the PRLr resulted in the rapid activation of
Tec and its concomitant association with the PRLr. When coexpressed in
COS-1 cells, both Vav1 and Tec were found to associate with the PRLr in
the presence of ligand. In the absence of receptor, a constitutive
complex between Vav1 and Tec was noted. Both Vav1 and Tec, however,
were capable of independent engagement of a bipartite intracellular
domain of the PRLr. Deletion mapping studies confined this interaction
to residues 323 to 527 of the intracellular domain of the PRLr.
Furthermore, Tec enhanced the GEF activity of Vav1 as evidenced by an
increase in GTP-bound Rac1. These data would suggest a pivotal function
for the formation of a Tec/Vav1/PRLr complex during PRL-driven signal
transduction, given the role of Vav1 in the control of cell
proliferation and the regulation of Rho family-mediated cytoskeletal
alterations.
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INTRODUCTION
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PRL is necessary for the terminal differentiation and maturation
of breast tissues during pregnancy; in its absence lobulo-alveolar
differentiation and milk production fail to occur (1). While the
province of PRL within normal mammary tissues is widely acknowledged,
an increasing body of evidence indicates a significant role for this
hormone in the pathogenesis and progression of rodent mammary carcinoma
(2, 3) and human breast cancer (4, 5, 6). Indeed, recent epidemiological
studies confirm a positive correlation between serum PRL levels and the
development and evolution of human breast cancer (7). Precedent data
would suggest that PRL can serve as mitogen for human breast cancer (8, 9) and as a survival factor (10). Recent data from our laboratory,
however, also indicate that PRL stimulates the progression of human
breast cancer by its actions as a chemoattractant (11). These studies
have revealed that PRL dramatically increases the motility of human
breast cancer by its rapid up-regulation of the filamentous actin
cytoskeleton, an event found to be dependent upon the generation of
phosphoinositide metabolites.
The effects of PRL are mediated by the PRL receptor (PRLr), a member of
the cytokine receptor superfamily of type I transmembrane receptors
(12). Lacking intrinsic kinase activity, the PRLr is dependent upon
associated protein kinases for its functional activity (13). These
include Jak2 (14, 15), Fyn (16), phosphoinositol-3-kinase (PI3K) (17, 18), and the Raf-mitogen-activated protein kinase (MAPK) cascade (19, 20). In addition, the guanine nucleotide exchange factor (GEF) activity
of the 95-kDa protooncogene Vav1 is triggered by PRL stimulation (21).
The stimulation of this activity is associated with the rapid
phosphorylation and binding of Vav1 to the PRLr. Activation of Vav1 GEF
activity results in the exchange of GDP for GTP on GTP binding proteins
of the Rho family namely, Rac1 and RhoA (22, 23, 24, 25). In turn, GTP-bound
Rac1 and RhoA serve to modulate the activity of several kinases in
intimate association with the cytoskeleton (26, 27, 28). The activation of
the Vav1/Rac/Rhoassociated signaling network ultimately results in
the formation of cellular structures, such as stress fibers and
lamellipodia, that directly contribute to cellular motility. Thus,
Vav1, or related family members such as Vav2 or Vav3 (29, 30), could
integrate early signals emerging from the ligand-dimerized receptor
complex, and thereby mediate the PRL-induced alteration of
cytoarchitecture and motility of human breast cancer.
Our laboratory has sought to study how Vav1 associates with the PRLr
complex and is activated by the kinases associated with this receptor.
Our examination of this complex was the first to reveal an association
and potential role for the Src-family kinase, Fyn (16, 31, 32), a
finding subsequently confirmed by others (33, 34). Indeed,
phosphorylation of Vav1 by Src-family kinases contributes to the
activation of its GEF (35). Additional analysis of the PRLr complex has
led us to question whether the 70-kDa protein-tyrosine kinase Tec also
could contribute to the activation of PRLr-associated Vav1. Tec is a
member of the Tec family of tyrosine kinases comprised of Tec, Btk,
Itk, and Bmx. Tec demonstrates considerable homology to the Src-family
of protein-tyrosine kinases. Both families of protein-tyrosine kinases
contain C-terminal SH2, SH3, and kinase domains. Unlike the Src-family
kinases, Tec also contains a phosphoinositide-binding pleckstrin
homology (PH) domain, a Tec homology domain, and a proline-rich region
within its N terminus (36, 37). Tec has also been reported to associate
with Jak- and Src-family tyrosine kinases (38, 39, 40). Thus, Tec would be
centrally located to mediate early signals from tyrosine and
phosphoinositide kinases associated with the PRLr complex. Confirming
such a hypothesis, the data presented here demonstrate that PRL induces
the activation and phosphorylation of Tec, and the association of a
Vav1/Tec complex with the PRLr. Furthermore, the tyrosine kinase
activity of Tec was found to enhance the GEF function of Vav1.
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RESULTS
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Tec Kinase Associates with the PRLr and Is Activated upon Ligand
Binding
Previous work in our laboratory sought to study how the
protooncogene product Vav1 associates with the PRLr (41), leading us to
question whether the protein tyrosine kinase Tec could also associate
with Vav1 and contribute to PRLr-mediated signal transduction. The
human breast carcinoma cell line T47D expresses both the PRLr and Tec
kinase and was therefore used to analyze the activation of Tec through
PRL stimulation. As shown in Fig. 1A
, the
stimulation of rested T47D cells with PRL resulted in the formation of
a receptor-associated signaling complex that contains both the PRLr and
Tec. To determine whether Tec could be activated by PRLr association,
in vitro kinase assays were performed in Chinese hamster
ovary (CHO) cell transfectants that lack endogenous Vav1 (Fig. 1B
).
After 10 min of PRL stimulation, cells coexpressing the PRLr and Tec
exhibited an 18-fold induction of Tec autokinase activity not observed
in cells expressing Tec with vector alone.

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Figure 1. Association and Activation of Tec Kinase with the
PRLr after Ligand-Induced Receptor Homodimerization
A, In vivo association and activation of the PRLr with
Tec kinase. T47D cells (3 x 105) were rested
overnight in DMEM/ITS+ and then stimulated with 250 ng/ml human PRL for
10 min. Tec and PRLr were immunoprecipitated with corresponding
antisera. After washing, precipitates were separated by 10% SDS-PAGE,
transferred to nitrocellulose, and immunoblotted with anti-PRLr or
anti-Tec antibodies. B, In vitro kinase activity of Tec
kinase upon PRL stimulation. CHO cells expressing the human long PRLr
in conjunction with Tec kinase were stimulated with 250 ng/ml human PRL
for the indicated times. Cell lysates were immunoprecipitated with
anti-Tec antiserum and incubated with [ 32-P]ATP
without exogenous substrates. After washing, the precipitates were
separated by 10% SDS-PAGE and visualized by autoradiography. The same
amounts of the samples used in the in vitro kinase assay
were immunoprecipitated and immunoblotted to show equal Tec expression
between samples (lower panel). Representative of one
of two experiments.
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The PRLr Interacts Directly with Tec Kinase and proto-onco
Vav1
Based upon the observations that both Vav1 and Tec were found to
associate with the PRLr upon PRL stimulation, we tested whether this
complex could be reconstituted in a cell line nonresponsive to PRL.
Therefore, transient transfections of either Tec or Vav1 in conjunction
with the PRLr were performed in COS-1 cells. As shown in Fig. 2
, after the addition of PRL the PRLr was
found to coimmunoprecipitate with Tec. This protein-protein interaction
was specific, as coexpression of the PRLr with a control vector
expressing ß-galactosidase showed no interaction. Additionally, the
interaction of Tec with the PRLr in the COS-1 transfectants did not
require the presence of Vav1, as these cells lack this protein.

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Figure 2. In Vivo Association of the PRLr with
Tec Kinase
Lysates from PRL-stimulated (250 ng/ml) COS-1 cells transiently
transfected with the PRLr and Tec (or a LacZ control vector) were
immunoprecipitated with corresponding antisera. After washing,
precipitates were separated by 10% SDS-PAGE, transferred to
nitrocellulose, and blotted with anti-PRLr monoclonal antibody or
anti-Tec antisera. Representative of one of three experiments.
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Interactions between Tec and Vav1 have been shown in other
signaling systems. However, this association was shown to be cytokine
dependent in some systems (42), and constitutive in another (43). To
determine whether the association of Vav1 and Tec could occur directly
in the absence of the PRLr, immunoprecipitations were performed on
COS-1 cotransfectants expressing Tec and Vav1, but lacking the PRLr.
This analysis revealed the direct interaction between these signaling
proteins in vivo (Fig. 3
).
This experiment also revealed that the association of Vav1 with Tec is
constitutive, and does not require the PRLr.

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Figure 3. Constitutive Association of Tec Kinase with Vav1
Lysates from COS-1 cells transiently transfected with Tec and Vav1 or
LacZ control vector and Vav1 were immunoprecipitated with anti-Tec
antiserum. After washing, precipitates were separated by 10% SDS-PAGE,
transferred to nitrocellulose, and blotted with anti-Vav1 antiserum.
Representative of one of two experiments.
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Since both Tec and Vav1 were observed to associate with the PRLr and to
constitutively associate with each other, we examined whether both
signaling molecules complexed with the PRLr in the presence of PRL.
COS-1 cells transiently expressing the PRLr, Tec, and Vav1 were
stimulated with PRL, and cell lysates were immunoprecipitated with
antibodies to determine whether a complex between the PRLr and Vav1/Tec
was formed (Fig. 4
). Once again, Vav1 was
observed to constitutively associate with Tec, while Tec associated
with the PRLr only in the presence of PRL. Taken together, these
experiments suggest the formation of a complex of Vav1/Tec/PRLr upon
the addition of ligand.

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Figure 4. Immunoprecipitation of a PRLr/Vav1/Tec Complex upon
PRL Stimulation Transiently transfected COS-1 cells were rested
overnight and then stimulated with 250 ng/ml hPRL for 10 min. Lysates
were immunoprecipitated with anti-Tec antiserum, separated by 10%
SDS-PAGE, and electrotransferred to nitrocellulose. Blots were probed
with anti-PRLr, -Tec, -Vav1, or -ß-gal antibodies to look for
signaling complex formation. Representative of one of four experiments.
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Vav1 and Tec Bind to a 204-Amino Acid Segment of the Intracellular
Domain of the PRLr
The results obtained above and previous results indicated that the
presence of PRL was sufficient for Vav1 and Tec association with the
PRLr. To further examine the interaction of the PRLr with Tec and Vav1,
glutathione-S-transferase (GST) chimeras containing regions
of the intracellular domain of the PRLr were used in pull-down
experiments. Recent data have convincingly demonstrated that GST exists
in its native state as a dimer (44). Indeed, enzymes requiring
dimerization can be activated when recombinantly expressed as a
GST-chimera (45). Therefore, we reasoned that a dimerized GST-PRLr
chimera could induce the necessary structural changes within the PRLr
complex necessary for the association of Tec and Vav1. Using lysates
from PRL-stimulated Nb2 cells as a source for Vav1 and Tec, pull-down
experiments with GST-PRLr chimera revealed that a 204-amino acid domain
(aa 323 to 527) was capable of binding both Tec and Vav1 (Fig. 5
). This region is C-terminal to the
conserved box 1/variable box/box 2 motif in the human PRLr. Bisection
of the 204-amino acid fragment of intracellular domain of the PRLr
revealed that both halves of this region (aa 323425 and 425527)
were capable of a direct engagement of Vav1 and Tec. This suggests that
the 204-residue fragment contains a bipartite binding motif, as both
halves were capable of binding both signaling molecules.

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Figure 5. In Vitro Deletion Mapping of the
PRLr Binding Motifs of Tec Kinase and Vav1 GEF
A, Pull-down of Tec and Vav1 binding to GST-PRLr chimeras. Five
micrograms of GST-PRLr chimeras (expressed in E. coli)
were bound to glutathione sepharose and incubated with Nb2 cell lysates
for 2 h at room temperature. Precipitates were washed, separated
by 10% SDS-PAGE, and blotted with anti-Tec or anti-Vav1 antisera.
Representative of one of five experiments. B, Deletion map of PRLr
domains involved in Vav1 and Tec association. ECD, Extracellular
domain; TM, transmembrane domain; 1, box 1 motif; V, variable box
motif; 2, box 2 motif. Positions of tyrosine residues are indicated
(Y).
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To confirm the in vitro pull-down data in
vivo, immunoprecipitations were performed (Fig. 6
). PRLr constructs were generated
consisting of the full-length PRLr extracellular domain and
transmembrane region coupled to regions aa 323425 and aa 425527 of
the intracellular domain. These constructs were transiently transfected
with Tec kinase or Vav1 into NIH 3T3 cells and analyzed for their
ability to associate independently with Tec or Vav1 upon the addition
of ligand. PRLr constructs expressing both of these fragments of the
intracellular domain associated with Tec or Vav1 in a PRLdependent
manner. In contrast, the recently characterized human intermediate PRLr
(46), which does not encode for these amino acid sequences but contains
the conserved box 1/variable box/box 2 motif, was incapable of
associating with either Tec or Vav1. These in vivo results
therefore confirm the direct Tec/PRLr and Vav1/PRLr associations
observed with the GST chimera. In addition, these results reveal that
either Tec or Vav1 is capable of PRL-induced association with the PRLr
independently of the other partner.

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Figure 6. In Vivo Deletion Mapping of the PRLr
Binding Motifs of the Tec Kinase and Vav1 GEF
A, Immunoprecipitation of PRLr/Tec or PRLr/Vav1 complexes from
transiently transfected NIH 3T3 cells. Transfectants expressing Tec and
PRLr (upper panel) or Vav1 and PRLr (lower
panel) were rested overnight and then stimulated for 10 min
with 250 ng/ml hPRL. Cell lysates were immunoprecipitated with
anti-PRLr antiserum and blotted with anti-Tec (upper
panel) or -Vav1 (lower panel) antisera. B, PRLr
constructs used in mapping the in vivo Vav1 and Tec
association. Int. PRLr, Human intermediate PRLr; ECD, extracellular
domain; TM, transmembrane domain; 1, box 1 motif; V, variable box
motif; 2, box 2 motif. Positions of tyrosine residues are indicated
(Y).
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Tec Kinase Enhances the Activation of Rac1 by Vav1
To address the functional significance of the association of a
Vav1/Tec complex with the PRLr, we sought to test whether Tec kinase
activity modulated the downstream effects of Vav1-mediated signaling.
To this end, Vav1 GEF activity in the presence of Tec was measured
indirectly by the activation of the small GTP-binding protein and Vav1
substrate Rac1. Activated Vav1, via its GEF activity, induces GDP/GTP
exchange on Rac1, thereby activating it. Activated (GTP-bound) Rac1 is
capable of binding its downstream effectors such as paxillin (47),
IQGAP, and PIP5 kinase (48). Thus, to measure GTP-Rac1 and thereby Vav1
activity, lysates from cells transiently transfected with PRLr, Vav1,
Tec, or a kinase-inactive form of Tec (Tec-K397E) were incubated with
GST-PAK and analyzed by anti-Rac1 immunoblot analysis (Fig. 7
). As PAK kinase associates only with
GTP-bound Rac1, GST-PAK/Rac1 precipitates can be used to assess the
levels of activated Rac1 (47). This analysis revealed a PRL-induced
increase in activated Rac1 (manifest by increased binding to GST-PAK)
in transfectants expressing both the PRLr and Vav1. This activation was
greatly enhanced by the simultaneous cotransfection of wild-type Tec.
Conversely, the PRL-induced activation of Rac1 by Vav1 was not enhanced
by the introduction of the kinase-dead form of Tec. These data indicate
that the tyrosine kinase activity of Tec significantly contributes to
the GEF activity of Vav1, resulting in the consequent GDP/GTP exchange
and activation of Rac1.

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Figure 7. Activation of the Cytoskeletal Regulator Rac-1 Is
Enhanced by Tec Kinase
GST-PAK (25 µg) was incubated with lysates of resting or
hPRL-stimulated (250 ng/ml, 20 min) NIH 3T3 cell transfectants
expressing PRLr in conjunction with Tec, Vav1, or kinase-inactive Tec
(Tec-K397E). Activated (GTP-bound) Rac1 bound to the GST-PAK was
detected by subsequent immunoblotting with an anti-Rac1 monoclonal
antibody. The blot was then stained with amido black to show equal
loading of GST-PAK precipitated (lower panel).
Representative of one of two experiments.
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DISCUSSION
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Like the PRLr, members of the superfamily of cytokine receptors
undergo ligand-induced hetero- or homodimerization and are dependent
upon associated intracellular signaling factors for the activation of
transduction networks. The tyrosine kinase p70Tec
is activated when the appropriate ligand is applied to members of this
superfamily including the receptors for interleukin-3 (IL-3) (40, 42),
interleukin-6 (IL-6) (38), granulocyte-macrophage colony stimulating
factor (GM-CSF) (39), erythropoietin (42), and granulocyte-colony
stimulating factor (G-CSF) (43), and other receptors found in cells of
the immune system including the T cell receptor and CD28 (49). Although
widely expressed in cells of the immune system, Tec can also be found
within tissues or cells from the liver, kidney, ovary, heart, and
breast (37) (C. V. Clevenger and J. B. Kline, unpublished
results). Given our prior observation of the activation and association
of the GEF p95Vav1 with the PRLr, we questioned
whether Tec was associated and activated by this complex. This
hypothesis was confirmed by the demonstration of the rapid association
and activation of Tec by the PRL ligand in the PRL-responsive cell line
T47D and PRLr-transfected CHO and COS-1 cells. These findings are
consistent with prior studies with other cytokine receptors that have
demonstrated the activation of Tec within 510 min of ligand
engagement (39, 42).
Precedent studies have indicated an interaction between Tec and Vav1
(38, 42, 43). A level of discord between these studies exists, as some
groups have found the Tec-Vav1 interaction to be IL-3- or
erythropoietin-induced (42), while others have noted a constitutive
interaction (43) in G-CSF-dependent systems. Both direct
coimmunoprecipitation and pull-down approaches with cell lysates were
used in these disparate studies to confirm the formation of Vav1-Tec
complexes. These approaches, however, could not assess whether the
interaction between Tec/Vav1 was direct or mediated indirectly by an
associated protein. To address whether a constitutive and/or direct
interaction between Tec and Vav1 could occur in a milieu that enabled
appropriate posttranslational protein folding, both of these proteins
were transiently overexpressed in COS-1 cells and tested for
interaction. Our findings indicate that appreciable quantities of Tec
and Vav1 can associate in the absence of any cytokine receptor or
associated ligand, and do so directly in COS-1 cells. These findings
are consistent with prior yeast two-hybrid analysis demonstrating that
a mutant form of Tec lacking tyrosine kinase activity was capable of
interacting with Vav1 (38). Thus, these data would suggest that the
interaction between Tec and Vav1 is unlikely to be mediated by
ligand-induced tyrosine phosphorylation of Tec and Vav1,
i.e. via SH2 domain-phosphotyrosine interaction. Instead,
these data would indicate that such an interaction might be mediated by
intrinsic structures found in Tec and Vav1 such as the SH3-proline-rich
regions (50). Further mutagenesis studies of Vav1 and Tec, currently
underway in our laboratory, should shed further light on this
interaction.
Only a single precedent study has examined the interaction of Tec with
an activating cell surface receptor, namely CD28 (51). Like the PRLr,
the association of Tec with CD28 was ligand dependent and paralleled
the stimulation of Tec kinase activity. Pull-down approaches, using
cell lysates and mutant GST-Tec chimera, revealed that this interaction
was dependent upon the SH3 domain of Tec and proline-rich sequences
within CD28. However, whether the interaction between Tec and any
receptor was direct or mediated through its interaction with other
signaling factors with which it interacted, such as Jak2, PI3K, or
Vav1, was not established. To address this question here, Tec was
independently cotransfected with the hPRLr into COS-1 cells. As seen in
Fig. 2
, the findings from these experiments clearly establish that the
PRLr in the presence of ligand is capable of an independent association
with Tec. The structural basis for the interaction of the PRLr with Tec
and Vav1 was further examined through the use of dimerized GST-chimeras
containing portions of the intracellular domain of the hPRLr.
Interaction studies with these chimeras in pull-down experiments
revealed that both Vav1 and Tec interact with a central region (aa
323527) within the hPRLr C-terminal to the box 1/variable box/box 2.
Further analysis with these chimeras mapped the binding site of Vav1
and Tec to each half of this domain (aa 323425 and 425527). This
was also confirmed in vivo, as both fragments were found to
bind Tec and Vav1 upon PRL stimulation of 3T3 cells expressing
truncated intracellular domains consisting entirely of these two
regions. We speculate that a motif found in each of these domains,
namely a conserved [W(L/P)LPQ], may play a role in such interactions
and may contribute to the formation of a ternary complex between the
PRLr and Vav1 and Tec. Other motifs within residues 323527 of the
human PRLr, however, may also contribute to this interaction. This
speculation is based on precedent data from our laboratory (21), which
indicates that the rat intermediate PRLr also inducibly binds and
activates Vav1. While the rat intermediate PRLr intracytoplasmic domain
is similar to the GST-PRLr 235/322528/599 construct, it is not
identical in the C terminus. Such differences could contribute to the
rat intermediate PRLr association with Vav1. Studies are currently
underway to test the respective roles of these human and rat PRLr
motifs toward Vav1 binding.
Several disparate signaling pathways appear to interact with and
modulate the activity of Tec early during receptor-mediated signaling.
Members of both the Jak and Src kinase family are known to bind,
phosphorylate, and up-regulate the kinase activity of Tec family
members (39, 40, 52). Indeed, cotransfection studies with a
kinase-inactive form of Jak2 suppressed the IL3-induced, Tec-mediated
activation of the c-fos locus (39), indicating a downstream
relationship for Tec with respect to Jak2. These interactions are
mediated via the interactions between phosphotyrosine residues and the
SH2 domain of Tec. Other tyrosine kinases, such as Bmx, that are
activated by heterotrimeric G proteins, may also up-regulate Tec
activity (53). The activity of Tec is also regulated by
phosphoinositide metabolites, specifically
phosphatidyl-3,4,5-triphosphate (54), a phosphoinositide metabolite
known to also activate Vav1 (55). Not surprisingly, Tec also interacts
with both the p85 and p55 subunits of PI3K. Finally, negative
regulation of Tec is in part mediated by the suppressors of cytokine
signaling (SOCS) proteins, as SOCS-1 binds to Tec and down-regulates
its kinase activity (56). Each of these regulators of Tec activity has
also been found in association with and activated by the PRLr,
e.g. Jak2, Fyn, PI3K, and SOCS-1. Such cross-talk may enable
some degree of integration between the cytokine receptors and other
cell surface receptor superfamilies. Whether the PRL-induced activation
of Jak2 and Fyn is sufficient for the tyrosine phosphorylation of Vav1,
or requires the intermediate phosphorylation and activation of Tec,
remains an area of active investigation in our laboratory. Indeed, one
recent study (53) has suggested that the activity of Src and Tec may
synergize in the activation of the Vav1 effector Rho. Taken together,
however, these data would suggest that a rapid engagement and
activation of a sizable complex of signaling factors including Tec and
Vav1 occur on the PRLr intracellular domain as a consequence of
ligand-induced receptor dimerization.
Downstream effectors of the activated Tec-Vav1 complex include the Rho
family of small GTP-binding proteins (22, 23, 53), specifically Rac-1,
RhoA, and Cdc42. Activation of the Rho family has been linked to
cytoskeletal reorganization and cellular movement (26, 55, 57). Our
laboratory has recently demonstrated that the directional motility of
human breast cancer is stimulated by a PRL gradient. Assessment of the
filamentous actin cytoskeleton also revealed a rapid induction of
stress fibers and lamellipodia after PRL stimulation, an event
dependent upon the generation of phosphoinositide metabolites (11). A
central regulator of cytoskeletal structure is the GTP-bound form of
Rac1. GTP-bound Rac1 directly activates effector proteins within the
cytoskeleton including PAK and PIP5 kinase (48). To determine whether
Tec kinase activity might increase GTP-bound Rac1 levels via the GEF
Vav1, Tec and Vav1 were coexpressed with the PRLr in 3T3 cells. A
significant increase in the level of GTP-bound Rac1 was observed when
both Tec and Vav1 were present, while the introduction of a
kinase-deficient Tec with Vav1 inhibited the potentiation of Vav1
activity. Taken together, this suggests a role for Tec in the process
of cytoskeletal reorganization via Vav1 activation. As both Tec and
Vav1 family members are widely expressed in human breast cancer
(our unpublished observations), our findings would suggest a
proximal role for this complex in the mediation of PRL-induced
motility. Thus, further study of the Tec-Vav1 complex, in the context
of the PRLr and the multihormonal environment of human breast cancer,
may provide further insights into in the pathophysiology of breast
cancer motility and metastasis.
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MATERIALS AND METHODS
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Cell Culture
Human PRL was a gift from the National Hormone and Pituitary
Program, National Institute of Diabetes and Digestive and Kidney
Diseases. T47D and COS-1 cells were maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10%
FBS, 50 U/ml penicillin, and 50 µg/ml streptomycin. NIH 3T3 cells
were maintained in DMEM supplemented with 10% calf serum, 10% horse
serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. Nb2 cells were
maintained in Fishers medium (Life Technologies, Inc.)
supplemented with 10% FBS (containing at least 20 ng PRL/ml), 50 U/ml
penicillin, and 50 µg/ml streptomycin. CHO-K1 cells were maintained
in Hams F-12 medium (Life Technologies, Inc.)
supplemented with 10% FBS, 50 U/ml penicillin, and 50 µg/ml
streptomycin. When appropriate, cells were allowed to remain in
medium consisting of DMEM (Life Technologies, Inc.)
supplemented with sodium selenide, linoleic acid, insulin, and
transferrin (ITS+; Calbiochem, La Jolla, CA).
Generation of cDNA Constructs
Recombinant forms of the human PRLr, Tec kinase, and Vav1
were generated by PCR using primers homologous to the 5'- and 3'-ends
of their respective open reading frames (ORFs) containing
EcoRI and XhoI restriction sites (PRLr and Vav1)
or EcoRI and EcoRV sites (Tec). For the PRLr,
these primers were PRLR-Kl (5'-CGAATTCCACCATGAAGGAAAATGTGGCA-3') and
PRLR-LONG' (5'-CGCTCGAGGTGAAAGGAGTGTGTAAA-3'); Tec, TEC-ERI
(5'-CGAATTCACCATGAATTTTAACACT-3') and TEC-ERV
(5'-CGATATCTTCCAAAAGTTTC-3'); Vav1, PVAV1-KPRO
(5'-CGCGAATTCACCATGGAGCTGTGGCGCCAA-3') and PVAV1-CRYS'
(5'-GCGCTCGAGGCAGTATTCAGAATA-3'). Primary cycling reactions consisted
of 94 C for 2 min, which was followed by 30 cycles of 94 C for 30 sec,
50 C for 30 sec, and 72 C for 2 min. It was then extended at 72 C for 3
min. The 3'-primers did not contain endogenous stop codons, allowing
the C-terminal addition of V5/poly-histidine epitope tags. PCR
fragments were ligated into the eukaryotic expression vector
pEF1-V5/HisA (Invitrogen, San Diego, CA). DNA fragments
encoding regions of the intracellular domain of the human long PRLr
were amplified in a manner similar to that described above with primers
encoding EcoRI and XhoI restriction sites. The
digested fragments were ligated into the corresponding restriction
sites of pGEX-4T (Pharmacia Biotech, Piscataway, NJ) for
prokaryotic protein expression. All clones were subsequently checked
for amplification errors by dideoxynucleotide sequencing. A
kinase-inactive form of Tec kinase (Tec-K397E) (51) was generated by
amplification of 5'- and 3'-halves using primers TEC-ERI and TECK397E'
(5'-CCGAATAGCTTCGATTGCGAC-3') or TEC-ERV and TECK397E
(5'-GTCGCAATCGAAGCTATTCGG-3'). Purified amplification products were
mixed in a second round of amplification using primers TEC-ERI and
TEC-ERV. The PCR product was ligated into pEF1-V5/HisA as described
above and sequenced to ensure codon 398 was changed to glutamic acid.
To generate eukaryotically expressed PRLr truncation mutants, a similar
approach was undertaken. Primers PRLR-Kl and PRLRTM'
(5'-CAAAGCCACTGCCCAGAC-3') were used to amplify the extracellular and
transmembrane domains (ECD/TM) of the PRLr. In parallel, intracellular
regions coding for residues 323425 and 425527 of the PRLr were
amplified with PRLR-425' (5'-GCGCTCGAGTCAAGTGGCCGGTGCACCTGC-3') and
TM323 (5'-GTCTGGGCAGTGGCTTTGACTGACTCAGGCCGGGGG-3') or PRLR-527'
(GCGCTCGAGTCACCCGGACACCTTGGCATA-3') and TM425
(5'-GTCTGGGCAGTGGCTTTGACTCTGTTGAATGAAGCA-3'). Amplified fragments
were reamplified with the product coding for the ECD/TM region of the
PRLr using primers PRLR-Kl and PRLR-425' or PRLR-Kl and PRLR-527' to
generate the PRLr deletion constructs. Amplified products were ligated
into pEF1-V5/HisA and sequenced.
GTPase Assays
NIH 3T3 cells transiently transfected with PRLr and either Vav1
and Tec, or inactive Tec-K397E, were rested overnight in DMEM with
0.5% BSA. Cells were stimulated with 250 ng/ml PRL for 20 min and then
lysed in buffer containing 0.5% NP-40, 50 mM Tris-HCl, pH
7.4, 150 mM NaCl, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride (PMSF), protease inhibitors, and 25 µg
recombinant GST-PAK (47). Lysates were then incubated with
glutathione-agarose beads (Pharmacia Biotech) for 1 h
at 4 C, washed with lysis buffer, and boiled with SDS sample buffer.
Bound Rac-1 was analyzed by Western blotting using a monoclonal
anti-Rac-1 antibody (Upstate Biotechnology, Inc., Lake
Placid, NY). To confirm equal precipitation of GST-PAK in samples, the
blot was then stained for 1 min with amido black, followed by
destaining with 25% isopropanol/10% acetic acid for 30 min.
Transient Transfections
COS-1 cells were transiently cotransfected with pEF1-V5/HisA
constructs using a CaCl2 transfection kit
(Invitrogen) for 5 h as instructed. Cells were washed
twice and incubated an additional 48 h in 10% complete DMEM.
Transient transfection of CHO and 3T3 cells with pEF1-V5/HisA
constructs was performed using Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN) as instructed. Briefly, 1 µg
pEF1-V5/HisA:PRLr was mixed with 1 µg pEF1-V5/HisA or 1 µg
pEF1-V5/HisA:Tec. DMEM (100 µl) containing 6 µl Fugene 6 was then
added to the DNA and incubated for 20 min at room temperature. The
mixture was added dropwise to 2 x 105 CHO
cells in 2 ml 10% complete DMEM and incubated an additional 48 h
before use.
Immunoprecipitation and in Vitro Kinase Assays
T47D cells (3 x 105) remained
overnight in DMEM/ITS+ and then were stimulated with 250 ng/ml human
PRL for 10 min. Cells were lysed and immunoprecipiated overnight as
previously described (58) using 3 µl anti-PRLr (59) or anti-Tec
antisera (Upstate Biotechnology, Inc.). Antigen-antibody
complexes were isolated by the addition of 50 µl protein-A beads.
After three washes with lysis buffer, immunoprecipitates were boiled in
30 µl Laemmli buffer with 2-mercaptoethanol, and the reaction
products were separated by 10% SDS-PAGE. COS-1 transfectants were
lysed and immunoprecipitated overnight in a similar manner using 3 µl
of the appropriate anti-Tec or anti-Vav1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibodies. For CHO
in vitro kinase assays, 2 x 105
transfectants were rested overnight in 2 ml DMEM/ITS+, stimulated with
250 ng/ml human PRL for 030 min, and immunoprecipitated with anti-Tec
antiserum. Antigen-antibody complexes were isolated by the addition of
50 µl protein-A beads. After three washes with lysis buffer,
immunoprecipitates were washed once with low-salt buffer [10
mM Tris-HCl (pH 7.0), 100
mM NaCl, and 100 µM
Na3VO4]. The
immunoprecipitates were then suspended in 30 µl autokinase buffer
[25 mM Tris-HCl (pH 7.0), 10
mM MgCl2, and 10 µCi
[
-32P]ATP]. After 20 min at 30 C, the
reactions were stopped by the addition of 2x Laemmli buffer with
2mercaptoethanol, and the reaction products were analyzed by 10%
SDS-PAGE followed by autoradiography. Tec activation was
quantitated by scanning densitometry using ImageQuaNT software
(Molecular Dynamics, Inc., Sunnyvale, CA).
GST Pull-Down Assays
For in vitro pull-downs, GST-PRLr chimeras were
expressed using the GST expression system (Pharmacia Biotech) as instructed. Briefly, 100 ml cultures of
Escherichia. coli transformants were grown to midlog phase
and induced with 0.1 mM isopropyl
ß-D-thiogalactoside (IPTG) for 4 h. Pelleted
cells were suspended in lysis buffer, sonicated, and cleared of debris
by centrifugation. Five micrograms of constructs were conjugated to
glutathione beads (Pharmacia Biotech) for 20 min and
washed three times with lysis buffer. One milliliter of Nb2 cell lysate
(107 cells/ml) was incubated with the beads for
2 h at room temperature, washed three times with lysis buffer,
resuspended in 30 µl Laemmli buffer, and boiled. Protein complexes
were separated by SDS-PAGE.
Immunoblot Analysis
immunoprecipitates separated by SDS-PAGE were transferred to
nitrocellulose, and nonspecific binding was blocked with 5% milk in
PBS/Tween 20. Antigens were labeled with a 1:1,000 dilution of
appropriate antibodies, followed by a 1:2,500 dilution of the
appropriate horseradish peroxidase (HRP)-conjugated secondary
antibodies (Sigma, St. Louis, MO). Antigen-antibody
complexes were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL).
 |
ACKNOWLEDGMENTS
|
---|
We wish to thank Dr. James Ihle for providing a Tec cDNA
construct, and Dr. Margaret Chou for her gift of GST-PAK cDNA.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Charles V. Clevenger, Department of Pathology & Laboratory Medicine, University of Pennsylvania Medical Center, 513 Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104. E-mail:
clevengc{at}mail.med.upenn.edu
This study was supported in part by the NIH Grants 2R01CA-69294 and
1R01DK-50771 (to C.V.C.) and 1F32DK-09727 (to J.B.K.).
Received for publication May 5, 2000.
Revision received January 9, 2001.
Accepted for publication January 11, 2001.
 |
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