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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo, 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. 1BGo). 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 [{gamma}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.

 
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. 2Go, 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.

 
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. 3Go). 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.

 
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. 4Go). 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.

 
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. 5Go). 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 323–425 and 425–527) 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).

 
To confirm the in vitro pull-down data in vivo, immunoprecipitations were performed (Fig. 6Go). PRLr constructs were generated consisting of the full-length PRLr extracellular domain and transmembrane region coupled to regions aa 323–425 and aa 425–527 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).

 
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. 7Go). 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 5–10 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. 2Go, 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 323–527) 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 323–425 and 425–527). 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 323–527 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 235/322–528/599 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 Fisher’s 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 Ham’s 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 323–425 and 425–527 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 0–30 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 [{gamma}-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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