Novel Association of Vav2 and Nek3 Modulates Signaling through the Human Prolactin Receptor
Sommer L. Miller,
Jamie E. DeMaria,
David O. Freier,
Angela M. Riegel and
Charles V. Clevenger
Department of Pathology and Laboratory Medicine (S.L.M., C.V.C.), University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104; Guilford Pharmaceuticals (J.E.D.), Baltimore, Maryland 21224; Lynchburg College (D.O.F.), Lynchburg, Virginia 24501; and Philadelphia College of Osteopathic Medicine (A.M.R.), Philadelphia, Pennsylvania 19131
Address all correspondence and requests for reprints to: Charles V. Clevenger, University of Pennsylvania Medical Center, 513 Stellar Chance Laboratories, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104. E-mail: clevengc{at}mail.med.upenn.edu.
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ABSTRACT
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Prolactin (PRL) receptor activation contributes to the progression and motility of human breast cancer. This event activates multimeric signaling pathways, including the activation of the Vav family of guanine nucleotide exchange factors. To detect novel proteins interacting with Vav, yeast two-hybrid analysis was performed and demonstrated an interaction between the serine/threonine NIMA (never in mitosis A)-related family kinase p56Nek3 and Vav1. The PRL-dependent interaction of Nek3 with Vav1 and Vav2 was confirmed by coimmunoprecipitation analysis. PRL stimulation of T47D cells induced Nek3 kinase activity and the interaction of Vav2/Nek3 with the PRL receptor. Increased Nek3 levels up-regulated Vav2 serine and tyrosine phosphorylation, whereas knockdown of Nek3 resulted in a reduction of Vav2 phosphorylation. Activation of guanosine triphosphatase Rac-1 in Chinese hamster ovary transfectants required both Nek3 and Vav2 and was inhibited by the overexpression of a kinase inactivating Nek3 mutant. However, overexpression of either Nek3 or kinase-inactive Nek3 had no effect on Vav2-potentiated signal transducer and activator of transcription 5-mediated gene expression. Overexpression of kinase inactive Nek3 in T47D cells led to a 50% increase in apoptosis vs. controls. These data suggest that the PRL-mediated activation of Nek3 contributes differentially to Vav2 signaling pathways involving Rac1 and signal transducer and activator of transcription 5 and implicates Nek3 during PRL-mediated actions in breast cancer.
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INTRODUCTION
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PROLACTIN (PRL) IS a pleiotropic hormone implicated in the regulation of lactogenesis, reproduction, immune function, and the progression and motility of human breast cancer (1, 2). PRL may alter the growth and differentiation of mammary tissue by both endocrine and autocrine/paracrine mechanisms (3, 4) and significantly increase proliferation of mammary carcinoma cells (5). PRL has recently been shown to stimulate cyclin D1 expression (5), an activity that contributes to mammary tumorigenesis in transgenic mice (6). In addition, overexpression of PRL in transgenic mice, resulting in lines with increased levels of PRL at either the autocrine/paracrine or endocrine levels, is sufficient to induce the formation of mammary cancers (7, 8). Furthermore, haploinsufficiency of the human (h) PRL receptor (PRLr), accomplished by germ-line disruption, reduces the number and progression of polyoma middle T antigen-induced mammary cancers (9, 10).
The actions of PRL in the mammary gland are mediated by PRL binding to its receptor, which initiates multimeric signaling pathways, including, but not limited to, Jak (Janus family of tyrosine kinases)/Stat (signal transducer and activator of transcription) (11, 12), MAPK (13), Grb2/Shc/Raf (14, 15), and phosphatidylinositol 3-kinase (14, 15, 16). In addition to these pathways, our laboratory has shown a constitutive complex of Tec tyrosine kinase and Vav1 guanine nucleotide exchange factor associating with the hPRLr in ligand-stimulated lymphoma cells (17). Activation of this pathway permits exchange of GDP for GTP on Rho family members, including Rac1 and RhoA (18, 19), which ultimately results in formation of stress fibers and lamellipodia, an observed response to PRL in several mammary tumor cell lines (16).
The Vav family of guanine nucleotide exchange factors (GEFs) currently numbers three. Vav1 is found exclusively in hematopoietic tissues (20), whereas Vav2 and Vav3 demonstrate broader expression patterns (21, 22). Similar to other enzymes that drive the activation of small guanosine triphosphatases (GTPases) (i.e. Rho/Rac), Vav2 has both Dbl and pleckstrin homology (PH) domains (20). The Dbl domain of Vav2 is necessary for the GDP to GTP exchange (23), whereas the PH domain may facilitate membrane association, in addition to being required for biological and catalytic activity in vivo (24). The PH domain also serves as a target for phosphatidylinositol 3-kinase products that enhance the transforming and signaling activities of Vav2 (24). Vav2 also contains two Src homology 3 and one Src homology 2 (SH3-SH2-SH3) domains. These C-terminal motifs contribute to the interaction between Vav2 and tyrosine-phosphorylated proteins (20). In addition, the calponin homology (CH) and acidic domains at the amino terminus serve negative regulatory roles in Vav function, via presumed alteration of protein structure (20). Phosphorylation of tyrosine-174 within the acidic domain of Vav relieves this negative regulatory function (25, 26). Due to this mechanism, Vav proteins appear to be dependent on tyrosine phosphorylation for the activation of their catalytic and biological activities (25, 26). Vav proteins are phosphorylated by associated kinases, including, but not limited to, Tec (27), Src family proteins (28, 29), Jak (30), and a heretofore unidentified serine/threonine kinase (31). After phosphorylation (19, 32, 33, 34) and binding of phosphoinositides (35, 36), Vav2 has been shown to activate Rac1 and Cdc42 (18, 19) in addition to RhoA and RhoG (33). Activation of Rho proteins leads to activation of several downstream signaling targets implicated in tumor progression and motility, including, but not limited to, the serine/threonine p21-associated kinase (PAK) (37).
The involvement of Vav1 in hPRLr signaling in cell lines of hematopoietic lineage has been previously reported (31). In the present study, we have set out to detect novel proteins interacting with Vav, to identify possible mechanisms by which hPRLr may regulate the activity of these proteins in breast cancer epithelial cells. In these experiments we identify a unique PRL-dependent interaction between the serine/threonine kinase Nek3 and Vav1/Vav2 that appears to differentially contribute to downstream hPRLr function.
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RESULTS
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Nek3 Kinase Associates with Vav1 and Vav2
Previous studies have shown stimulation of the human hPRLr results in the activation of the guanine nucleotide exchange factor activity of Vav1 in PRL-dependent Nb2 rat T cell lymphoma lysates (31). To better understand the activation mechanisms of Vav proteins, yeast two-hybrid analysis was conducted and an interaction was found between Vav1 and Nek3 (data not shown). To confirm the interaction between these proteins, coimmunoprecipitation studies were conducted with Nek3 antibodies using Nb2 whole-cell lysates, treated with PRL. The stimulation of rested Nb2 cells with PRL resulted in 3.4-fold increase in the association of Vav1 and Nek3 by 7.5 min (Fig. 1A
).

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Fig. 1. PRL-Induced Association of Nek3 with Vav1 and Vav2
A, Association with the Nek3 serine/threonine kinase and Vav1. Total cell lysates from Nb2 cells (12.0 x 106) stimulated or unstimulated with PRL were immunoprecipitated with anti-Nek3 antibody and were subjected to sequential immunoblot analysis with anti-Vav1 and anti-Nek3. Upon PRL stimulation, there was a 3.4 ± 0.57 (X ± SEM fold increase in Vav1 co-precipitating with Nek3 at 7.5 min. B, Association with Nek3 and Vav2. Total cell lysates from T47D cells (5.0 x 106) were treated as in panel A. Data are representative of one of three experiments.
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Given the broader expression profile of Vav2 in human epithelium, an association between Vav2 and Nek3 in T47D cells was tested. Like Vav1, an inducible interaction between Vav2 and Nek3 was noted after 7.5 min of PRL stimulation (Fig. 1B
). The interaction between Vav2 and Nek3 was further confirmed in a transfection model system. Chinese hamster ovary (CHO) cells were transiently transfected with V5-tagged hPRLr, wt (wild type) Vav2, and wt Nek3 expression constructs, rested for 24 h, and stimulated with PRL. Immunoprecipitation of whole-cell lysates with Nek3-specific antibodies and subsequent immunoblotting for Vav2 revealed a 2-fold increase in the association between Vav2 and Nek3 (Fig. 2A
). These findings temporally paralleled results in T47D and Nb2 cells.
Having demonstrated an in vivo interaction between Vav2 and Nek3, the site of interaction between these proteins was mapped. CHO cells were transfected with the indicated constructs (Fig. 2A
).
1144 Vav2 lacks the CH domain, whereas
1183 Vav2 lacks both the CH and acidic domains. PRL-stimulated transfectant lysates were immunoprecipitated with Nek3 antibody and immunoblotted for Vav2 proteins. Nek3 was found to require the acidic domain of Vav2, as no interaction was observed with
1183 Vav2 in contrast to
1144 Vav2 (Fig. 2A
). In contrast to wt Vav2, the kinetics of PRL-mediated Nek3 and
1144 Vav2 association appeared to be biphasic with increases in coprecipitating
1144 Vav2 at 7.5 and 30 min. To determine the site of Vav2 interaction with Nek3, immunoprecipitation studies were conducted with in vitro translated 35S-labeled, wt Vav2, 1285 Nek3, and 286510 Nek3 proteins spanning the N- and C-terminal domains of wt Nek3, respectively. As shown in Fig. 2B
, 1285 Nek3, but not 286510 Nek3, coprecipitated with wt Vav2, indicating that Vav2 interacts with Nek3 at the N-terminal region containing the serine/threonine kinase domain. The utilization of recombinant proteins demonstrated that the Vav-Nek3 interaction is direct and occurred without other interacting and/or adaptor proteins.
Nek3 and Vav2 Associate with the hPRLr and Are Activated upon Ligand Binding
To investigate the PRL-dependent association between Nek3 and Vav proteins, coimmunoprecipitation analysis was conducted to determine whether there was an association between Nek3 and Vav2 with the long isoform of the hPRLr. As shown in Fig. 3
, A and B, stimulation of rested T47D cells with PRL resulted in an approximate 3.5-fold increase in the association of both Vav2 and Nek3 with the hPRLr by 15 min. To determine whether Nek3 kinase activity could be activated by PRL, in vitro kinase assays were performed using T47D cell lysates and casein as an exogenous substrate as previously described (38). After PRL stimulation, there was a significant increase in Nek3 kinase activity (Fig. 3C
).

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Fig. 3. PRL-Induced Association of Vav2/Nek3 with the hPRLr and Activation of Nek3 Kinase
PRL-induced association of wt Vav2 (A) and wt Nek3 (B) with hPRLr. A, T47D (2.0 x 106) cell lysates that were stimulated or unstimulated with PRL were incubated with Vav2, and precipitates were subjected to sequential immunoblot analysis with anti-hPRLr and Vav2 antibodies. B, T47D cells were treated as in panel A, and cell lysates were immunoprecipitated with anti-Nek3 and immunoblotted with anti-hPRLr and anti-Nek3 antibodies. Upon PRL stimulation, there was a 3.1 ± 0.54 (X ± SEM) fold increase in Vav2 (A) and a 3.7 ± 0.82 (X ± SEM) fold increase in Nek3 (B) coprecipitating with hPRLr at 7.5 min. C, PRL-mediated Nek3 in vitro kinase activity. T47D cells (2.0 x 106), stimulated or unstimulated with PRL, were immunoprecipitated with Nek3 or rabbit IgG (control) antibodies and incubated with [ 32-P]ATP in the presence of dephosphorylated casein as an exogenous substrate. Precipitates were resolved by SDS-PAGE and visualized by autoradiography. Equal amounts of cell lysates treated as above in the in vitro kinase assay were immunoprecipitated and immunoblotted to show equal Nek3 expression between samples (lower panel). Data are representative of one of three experiments.
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Upon cell stimulation, Vav proteins become transiently phosphorylated on tyrosine residues, leading to the activation of their GDP-GTP exchange activity toward Rho/Rac proteins (39, 40, 41, 42, 43). To investigate whether PRL induces Vav2 phosphorylation, untransfected T47D cells were stimulated with PRL, and anti-Vav2 immunoprecipitates were examined for tyrosine as well as serine phosphorylation. Antiphosphotyrosine and -serine immunoblots revealed a 2-fold increase in phosphorylation, on both tyrosine and serine residues at 15 min (Fig. 4
, B and C). To our knowledge these data represent the first demonstration of a ligand-inducible phosphorylation of Vav2 on both serine and tyrosine residues. To determine whether Nek3 kinase activity contributed to PRL-mediated Vav2 phosphorylation, the levels of Nek3 were reduced by knockdown with Nek3 short interfering RNA (siRNA) or appropriate siRNA control. Cells treated with an 80 nM final concentration of Nek3 siRNA showed a significant reduction in total Nek3 protein levels (Fig. 4A
), as compared with mock and control transfectants. Anti-Vav2 immunoprecipitates from T47D cells transfected with Nek3 siRNA showed a loss of PRL-inducible serine as well as tyrosine phosphorylation, when compared with control transfectants (Fig. 4
, B and C). To complement these siRNA knock-down experiments, overexpression studies with wt Nek3 or control vector expression constructs were conducted in CHO cells (Fig. 4
, D and E). In contrast to vector control, expression of wt Nek3 enabled Vav2 tyrosine and serine phosphorylation, after PRL stimulation (Fig. 4
, D and E). These results were similar to that noted in T47D (Fig. 4
, B and C). Taken together, the data within Figs. 3
and 4
demonstrated that PRL stimulation induced Nek3 kinase activity and significantly contributed to both serine and tyrosine phosphorylation of Vav2.

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Fig. 4. Knockdown and Overexpression of Nek3 Affects PRL-Mediated Vav2 Phosphorylation
A, Western blot analysis 72 h after transfection of total Nek3 (top) and actin (bottom) in T47D cells mock treated or transfected with control and Nek3 siRNAs at 40 and 80 nM final concentrations. The ratio of total Nek3 to actin was determined by densitometry, and the levels were normalized to mock transfected cells. B and C, 1.5 x 106 T47D cells were transfected with an 80 nM final concentration of control and Nek3 siRNAs. Cells were stimulated 72 h after transfection with PRL, for the indicated times. Lysates were immunoprecipitated with anti-Vav2 antibody, and prepared samples were split and analyzed for phosphotyrosine and phosphoserine content. Membranes were stripped and reprobed with anti-Vav2 antibody. D and E, CHO cells (1.0 x 106) were transfected with 2 µg of vector, hPRLr, wt Vav2, wt Nek3, or vector control, and were stimulated with PRL 48 h after transfection. Immunoprecipitations were conducted as stated above. The ratio of phospho-Vav2 to immunoprecipitated Vav2 was determined by densitometry and normalized to the 15-min (panels B and D) and 7.5-min (panels D and E) time points. Data are representative of one of three experiments.
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Nek3 and Tec Kinases Enhance the Activation of Rac1 by Vav2
To address the functional significance of the association of Vav2 and Nek3 with the hPRLr, the effect of Nek3 and Tec expression on downstream effects of Vav2-mediated signaling was evaluated. One downstream target of Vav2 activation is the GTPase, Rac1 (18). To test the role of Nek3 and Tec during PRL-induced activation of Rac1, CHO cells were transiently transfected with the indicated constructs (Fig. 5
). Whole-cell lysates stimulated with PRL were incubated with GST (glutathione S-transferase)-PAK and analyzed by anti-Rac1 immunoblot analysis as previously described (17).

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Fig. 5. Nek3 and Tec Kinase Expression Enhance Vav2-Mediated Rac-1 Activity
GST-PAK was incubated with lysates of resting or PRL-stimulated CHO cell transfectants (2.5 x 105) expressing hPRLr only (A); hPRLr/wt Vav2, hPRLr/wt Vav2/wt Nek3, hPRLr/wt Vav2/D143A Nek3 (B); hPRLr/wtVav2/wt Tec/wt Nek3, hPRLr/wt Vav2/wt Tec (C); hPRLr/ 1183 Vav2/wt Nek3, hPRLr/ 1183 Vav2/D143A Nek3 (D). GTP-bound Rac-1 bound to the GST-PAK was resolved by 12% SDS-PAGE and immunoblotted with anti-Rac1 antibody. The membrane was subsequently stained with amido black to show equal loading. Data are representative of one of three experiments.
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We have previously demonstrated that the tyrosine kinase, Tec, will enhance the Rac1 activation of Vav1 (17), and its inclusion in this study was to test its efficacy, along with Nek3, in enhancing Vav2-mediated Rac1 activation. Expression of hPRLr alone, hPRLr/wt Nek3, hPRLr/wt Tec, or hPRLr/Vav2 was not sufficient to activate Rac1 after PRL stimulation (data not shown; Fig. 5
, A and B). The addition, however, of wt Nek3 or wt Tec and PRLr/Vav2 led to robust activation of Rac1 after PRL stimulation within 7.5 min (Fig. 5
, B and C). However, addition of both Nek3 and Tec, although leading to increased activation of Rac, was 50% less (Fig. 5C
) than when the kinases were expressed individually (Fig. 5
, B and C). In contrast, CHO cells cotransfected with hPRLr/wt Vav2/D143A Nek3, although showing somewhat elevated levels of basal Rac activity, demonstrated no PRL-driven Rac-1 activation, illustrating the need for functional Nek3 kinase in this system (Fig. 5B
, right panel). Interestingly, cotransfection of the oncogenic
1183 Vav2 (21, 33), with either wt Nek3 or D143A Nek3, led to constitutive activation of Rac1, which was not increased in the presence of PRL (Fig. 5D
). These data indicate that the kinase activity of both Tec and Nek3 significantly contributes to the PRL-mediated GEF activity of wt Vav2, whereas
1183 Vav2 was independent of Nek3 kinase function for its activity.
Vav2 Enhances PRL-Induced ß-Casein Gene Transcription
Upon ligand binding and dimerization of the hPRLr, the Jak/Stat pathway is activated (44). This results in the transactivation of numerous PRL-specific genes, including ß-casein. Recently, the expression of constitutively active
1187 Vav1, or activated forms of Ras or Rho family members, has been shown to lead to Stat3-specific activation (45).
To determine the effects of Nek3 and Vav2 on Stat5-mediated gene transcription, Vav2 and Nek3 constructs were transiently overexpressed in T47D cells. A LH response element (LHRE)-luciferase construct containing multiple Stat5 DNA-binding sites was cotransfected with an internal control Renilla reporter construct, and cells were rested and then stimulated with PRL for 24 h. The relative luciferase activity of unstimulated cells overexpressing wt Vav2 was 4-fold higher than PRL-stimulated controls. A 10-fold increase was observed upon PRL stimulation of Vav2-transfected cells, and expression of
1183 Vav2 enabled a significant increase in both basal and PRL-induced luciferase activity (Fig. 6
, lanes 3 and 4). Coexpression of a GEF-inactive form of Vav2 (L212A) (36, 46) with wt Vav2 attenuated the affects of wt Vav2, reducing luciferase activity to basal levels (Fig. 6
, lane 6). However, the increase in Stat5-mediated gene transcription seen with the expression of
1183 Vav2 was not significantly altered by the coexpression of L212A Vav2 (Fig. 6
, lane 7).

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Fig. 6. Vav2 Enhances Stat5-Mediated Gene Expression
T47D cells (6.0 x 105) were transfected with 12 µg of empty vector control, wt Vav2, 1183 Vav2, L212A Vav2, and D143A Nek3, as well as 0.5 µg of ß-casein and Renilla luciferase reporter constructs. Stimulated or unstimulated total cell lysates were analyzed for luciferase activity. Error bars represent SEM; as compared with vector controls, * denotes P < 0.01 and ** denotes P < 0.005. Data are representative one three of experiments conducted with triplicate transfections.
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Given the importance of Nek3 in Vav2-mediated Rac activity (Fig. 5
), we sought to determine the role, if any, of Nek3 in Vav2-mediated Stat5 activity. Interestingly, the expression of wt Nek3 (data not shown) or D143A Nek3 (Fig. 6
) did not affect either basal or PRL-induced LHRE-driven luciferase activity in this system. Taken together, these data suggested that the PRL-mediated activation of Nek3 contributes differentially to Vav2-regulated pathways involving Rac1 and Stat5.
Kinase-Inactive Nek3 Induces Apoptosis in Breast Cancer Cells
The activation of Rho family GTPases, such as Rac1, results in the induction of several intracellular signaling cascades, and both Rho and Stat proteins have been shown to impact diverse cellular responses, including cell survival and proliferation (47, 48). In addition, PRL also actively inhibits apoptosis of mammary tumor cells, as measured by DNA cleavage (49) and caspase activation (50, 51). These observations led us to assess the survival function of Nek3 in human breast cancer cell lines. To that end, T47D cells were transfected with fGFP (farnesylated green fluorescent protein) and either empty vector, wt Nek3, or D143A Nek3. After transfection (24 h), there was minimal (<5%) pyknosis in cells cotransfected with fGFP and vector only or wt Nek3 (Fig. 7A
). However, more than 50% of the T47D cells cotransfected with fGFP and D143A Nek3 exhibited characteristic nuclear shrinkage and cellular detachment. A fluorescent TdT labeling assay was used to confirm that these cellular changes represented death by apoptosis. Indeed, expression of D143A Nek3 resulted in a 2-fold increase in the percentage of apoptotic cells over empty vector controls (Fig. 7B
).

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Fig. 7. Kinase-Inactive Nek3 Increases Pyknosis and Apoptosis in T47D Cells
T47D cells (5.0 x 104) transfected with fGFP and vector control, wt Nek3, or D143A Nek3 were analyzed for nuclear fragmentation and cellular detachment indicative of pyknosis, or subjected to in situ TdT fluorescent labeling. The percentage of pyknotic (A) and apoptotic (B) GFP-positive cells was calculated for each transfection group. Error bars represent SEM; *, P < 0.01 as compared with vector controls. Data are representative of one of three experiments.
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DISCUSSION
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In the present study, we identified a novel interaction between Vav1 and the serine/threonine kinase Nek3 in a yeast two-hybrid system. Subsequent coimmunoprecipitation analysis in mammalian cells confirmed the interaction between Vav1 and Vav2 isoforms with Nek3. Our data indicate that this interaction is PRL dependent and that Vav2 and Nek3 also inducibly associate with the hPRLr. Upon ligand stimulation, Nek3 is activated and Vav2 becomes phosphorylated on serine and tyrosine residues.
Previously, our laboratory has shown the importance of Vav1 in PRL-mediated signaling in immune function (31). The novel interaction between Vav2 and the hPRLr described here supports this and other reports of Vav family proteins binding to diverse receptors, including the erythropoietin receptor (52) and growth factor receptors, epidermal growth factor and platelet-derived growth factor (34). Vav proteins have also been shown to be associated with and/or activated by several protein tyrosine kinases, including Zap70, Tec, Lck, and Fyn (17, 53). In the current study, we identify a novel Vav serine-threonine-associated kinase, Nek3, by yeast two-hybrid studies, and coimmunoprecipitation analysis of T47D lysates demonstrated a PRL-inducible interaction between Nek3 and the hPRLr (Fig. 3B
). However, the association of Nek3 with the hPRLr appears to be more transient than Vav2, as the amount of coprecipitated protein returns to basal levels by 30 min (Fig. 3B
). These data implicate both Vav2 and Nek3 in hPRLr signaling in breast cancer.
Nek3 is a novel mammalian gene product structurally related to the cell cycle regulatory kinase NIMA (never in mitosis A) of Aspergillus nidulans (38). Currently, there is no evidence supporting a role for Nek3 in cell cycle control, and no other putative functions have been assigned to this protein. Nek3 displays a typical serine/threonine protein kinase domain within its N-terminal half (38), and our studies revealed that Vav2 could directly interact with the N-terminal portion (Fig. 2B
), in the absence of additional adaptor proteins. In addition, the acidic domain within the N-terminus of Vav2 is required for Nek3 binding (Fig. 2A
). Future studies will determine the molecular mechanisms of the Vav2 and Nek3 interaction.
Tyrosine phosphorylation of Vav family members is thought to stimulate their intrinsic GEF activity (33). Interestingly, it has been shown that when stimulated with PRL, Vav1 is also inducibly phosphorylated on serine-threonine residues in Nb2 cells (31). Given this, the phosphorylation status of Vav2 during PRL stimulation was tested and revealed inducible tyrosine and serine phosphorylation of Vav2 (Fig. 4
). Paralleling these data, Nek3 immunoprecipitates from T47D cells showed a significant increase in kinase activity after PRL stimulation (Fig. 3C
). Complementary knockdown and overexpression studies were then conducted to determine the effects of Nek3 on Vav2 phosphorylation. Knockdown of endogenous Nek3 in PRL-stimulated T47D cells transfected with Nek3 siRNA led to approximately 60% reduction in serine and tyrosine phosphorylation of Vav2 (Fig. 4
, B and C). Conversely, overexpression of exogenous wt Nek3 in CHO cells (incapable of Vav2 activation in the untransfected state), resulted in PRL-induced phosphorylation of Vav2 comparable to that observed in untransfected (data not shown) and control siRNA transfected (Fig. 4
, B and C) T47D cells. These data suggest that Nek3 contributes to PRL-mediated Vav2 serine phosphorylation and facilitates Vav2 tyrosine phosphorylation, by both direct and indirect mechanisms.
Downstream effectors of activated Vav2 include members of the Rho family of small GTP-binding proteins, such as Rac1 RhoA and Cdc42 (18, 19, 33). Activation of the Rho family has been linked to cytoskeletal reorganization and cellular movement (54, 55) after GDP-GTP exchange on Rac1 (56). After PRL stimulation, activation of Vav1 by the Tec tyrosine kinase also resulted in increased GTP-bound Rac1 (17). To determine whether Nek3 kinase activity might increase the level of GTP-bound Rac1 mediated by Vav2, coexpression studies with hPRLr, Vav2, Nek3, and Tec were performed. Upon ligand stimulation, a significant increase in the level of GTP-bound Rac1 was observed when Nek3 and Tec kinases were transfected individually (Fig. 5
, B and C). When both kinases were coexpressed, the increase in GTP-bound Rac1 was not as robust as when expressed individually (Fig. 5C
, left panel), suggesting that the effect of Nek3 and Tec on Vav2 activation is not synergistic. The introduction of D143A Nek3 led to a significant decrease in GTP-bound Rac1 when coexpressed with wt Vav2 in contrast to the overexpression of
1183 Vav2, which resulted in high basal levels of Rac1 (Fig. 5C
). These results are in line with other studies that show increased Rac1 activity in the presence of similar constitutive Vav2 constructs (18, 19). Taken together, these results suggest a role for Nek3 and Tec kinases in Rac-mediated actions such as cytoskeletal reorganization.
Recent studies have suggested a link between the activity of Stats and Rho family GTPases (45, 57), as both RhoA and Rac1 have been shown to modulate Stat1 and Stat3 activation. These reports, along with our data suggesting a role for Nek3 in Vav2-mediated Rac1 activation, prompted the examination of the effects of Nek3 and Vav2 overexpression on Stat5-mediated gene transcription. The expression of wt Vav2 led to an inducible increase in Stat5-mediated gene transcription whereas
1183 Vav2 expression furthered these effects, even under basal conditions (Fig. 6
), and represents the first report of a functional relationship between Vav2 and Stat5. Unexpectedly, coexpression of either wt (data not shown) or D143A Nek3 (Fig. 6
) had no significant effect in this system on wt or
1183 Vav2 potentiation of Stat5-mediated gene transcription. This is in direct contrast to the effect of Nek3 on Vav2-mediated Rac1 activity, suggesting that the PRL-mediated activation of Nek3 contributes differentially to Vav2 signaling pathways involving Rac1 and Stat5.
Both the activation of Stats and Rac1 have been implicated in cell survival; therefore we examined the effects of wt and D143A Nek3 on breast cancer survival. In contrast to controls, overexpression of D143A Nek3 in T47D cells resulted in more than 50% of cells exhibiting hallmarks of pyknosis and apoptosis. Evidence of a role for PRL in promoting mammary epithelial cell survival is now beginning to emerge, as recent studies have reported that PRL antagonists (49, 58) induce cell death in T47D cells. Coupled with the chemoattractant effects of PRL (16), these data suggest a role for the hPRLr-Vav2-Nek3 complex in the PRL-mediated effects on human breast cancer survival and progression.
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MATERIALS AND METHODS
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Yeast Two-Hybrid Analysis
Full-length cDNA for wt Vav1 was generated by PCR using specific primers containing EcoRI/XhoI restriction sites: forward (5'-CGAATTCGCCATGGAGCTGTGGC-3') and reverse (5'-CTCGAGTCAGCAGTATTC-3'), cloned into the yeast vector pJK202 and cotransfected into the yeast strain EGY48 with a HeLa cDNA library inserted into the pJG45 vector (courtesy of Dr. Erica Golemis). Selection of positive interacting clones was performed as previously described (59).
Generation of cDNA Constructs
Using the partial Nek3 cDNA fragment obtained above, a full-length Nek3 cDNA was isolated and cloned into pEF1-V5/His (Invitrogen, San Diego, CA) using the EcoRI/Not I restriction sites, with the following primers: forward, 5'-CGAATTCACCATGGACAACTACACAGTC-3'; and reverse, 5'-AGCGGCCGCGGCACGTTCTCCGAGGAGTTG-3'. Mutation of aspartic acid 143 to alanine (D143A Nek3) using forward primer (5'-GTGAAATTGGGAGCTTTTGGCTCTGCC-3') and reverse (5'-GGCAGAGCCAAAAGCTCCCCAATTTCAC-3') rendered wt Nek3 kinase inactive (38). The 1285 Nek3 construct encompassing the amino-terminal region of wt Nek3 containing the serine/threonine kinase domain (38) was cloned into pcDNA3.1-V5/His (Invitrogen) using the EcoRI/XhoI sites and the following primers: forward, 5'-GTGGAATTCCACCATGATGGACAACTACACAGTC-3'; and reverse, 5'-CTCGAGCGGATTCTTAGGCGTGGATATTTTGATTTG-3'. 286510 Nek3 containing the remaining carboxy-terminal region of wt Nek3 was cloned into pcDNA3.1-V5/His (Invitrogen) using the EcoRI/XhoI sites and the following primers: forward, 5'-GTGGAATTCCACCATGATGAAAAAACAGGACTCCAACAG-3'; and reverse, 5'-CTCGAGCGGGGCACGT TCTCCGAGGAGTTGTCC-3'.
wt Vav2 cDNA and
1183Vav2 were also cloned into pEF1-V5/His with the following primers, respectively: forward, 5'-GCAAATTCCCACCATGGAGCAGTGGCGACAGTGCGGC-3'; and reverse, 5'-CGCTCGAGCTGGATGCCCTCCTCTTC-3'; forward, 5'-CGAATTCCGCGCCGCCATGATTAGATACATGCAG-3'; and reverse, 5'-CGCTCGAGCTGGATGCCCTCCTCTTC-3'. L212A Vav2 was generated using the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) with the following primers: forward, 5'-GTACTACCGCACCGCGGAGGACATTGAG-3'; and reverse, 5'-CTCAATGTCCTCCGCGGTGCGGTAGTAC-3'.
PCR amplification of all constructs were performed as previously described (17). Endogenous stop codons were removed to permit addition of the V5/His tag, and all clones were checked for the absence of amplification errors by dideoxynucleotide sequencing. The pEF-hPRLr full-length isoform of the human receptor used in the CHO transfection model has been previously described (17).
Cell Culture and Transient Transfections
Nb2, CHO, and T47D cells were maintained as previously described (60). Before stimulation with NIDDK recombinant PRL, cells were rested in defined media supplemented with ITS+ (sodium selenide, linoleic acid, insulin, and transferrin) (Calbiochem, La Jolla, CA) or 0.1% BSA (Sigma Chemical Co., St. Louis, MO) for 2448 h. CHO and T47D cells were transfected following manufacturers instructions with Fugene (Roche Clinical Laboratories, Indianapolis, IN) and Lipofectamine Plus (Invitrogen), respectively. T47D human breast cancer epithelial cells express PRLr, estrogen, and epidermal growth factor receptors and were used to study the mechanisms of endogenously expressed proteins. CHO cells were used in transient overexpression studies as they express undetectable levels of Vav2 and Nek3. For siRNA knockdown experiments, siGENOME SMARTpool reagent consisting of four individual siRNAs targeting the Nek3 gene was purchased from Dharmacon (Lafayette, CO), along with a nontargeting siCONTROL pool. T47D siRNA transfections were conducted using Lipofectamine 2000 in serum-free media at 40 nM and 80 nM final concentrations.
In Vitro Interaction Assays, Immunoprecipitations, and Immunoblot Analysis
To assess the in vitro interaction between Nek3 and Vav2, 35S-labeled wt Nek3, 1285 Nek3, 286510 Nek3, and wt Vav2 were generated by the TnT Quick Coupled Transcription/Translation system (Promega Corp., Madison, WI), following manufacturers instructions. Labeled wt Vav2 (15 µl) was admixed in 250 µl Nonidet P-40 (NP-40) lysis buffer (17) with 5 µl Vav2 antibody (Zymed Laboratories, South San Francisco, CA) at room temperature for 1 h. Protein A agarose beads (Invitrogen) were subsequently added for 1 h, followed by the addition of 15 µl of labeled Nek3 TnT reaction mix for a final 1-h incubation. Immunoprecipitates were subjected to SDS-PAGE and autoradiography.
To assess in vivo interactions, rested Nb2 cells and T47D and CHO transfectants were stimulated with 10 ng/ml and 100 ng/ml of PRL, respectively. Nek3 and Vav2 immunoprecipitations were conducted as previously described (17). Immunoblot analysis was conducted with the following antibodies: anti-Actin (Sigma), anti-V5 HRP (Invitrogen), anti-Nek3, anti-Vav2 (Zymed Laboratories), anti-Vav1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-hPRLr (17), anti-pY-plus (Zymed), and anti-pSer (Biomeda, Foster City, CA). Immunoblotting of whole-cell lysates verified equal expression of transfected proteins (data not shown), and quantification of resolved proteins was conducted using a FUJI LAS-3000 imaging apparatus.
In Vitro Kinase Assay
Rested T47D cells were stimulated with 100 ng/ml hPRL for indicated times. Cells were washed with ice-cold PBS with 1 mM Na3VO4 and harvested with NP-40 lysis buffer, on ice, into 1.5-ml microcentrifuge tubes. After centrifugation, lysate supernatants were incubated with 2 µg Nek3 antibody (Santa Cruz) for 60 min at 4 C. Immune complexes were isolated with protein G-agarose beads and washed three times with NP-40 lysis buffer. Samples were then resuspended in 20 µl protein kinase buffer (50 mM HEPES, pH 7.1; 0.1 mM EDTA; 0.01% Brij 35; 0.1 mg/ml BSA; 0.1% 2-mercaptoethanol; and 0.15 M NaCl). Twenty microliters of ATP mix (50 mM ATP; 2 M MgCl2; 10 mCi/ml [
32P]ATP; diluted in protein kinase buffer) were then added in the presence of 1 mg/ml dephosphorylated casein (Sigma) (38) as a substrate, and the kinase reaction was performed at 30 C for 20 min. Kinase reactions were stopped by the addition of 0.5 vol of Laemmli buffer containing sodium dodecyl sulfate and 2-mercaptoethanol, boiled for 5 min and subjected to SDS-PAGE, and autoradiography.
Rac-1 Activation Assay
Transiently transfected, rested CHO cells were stimulated for indicated times with 100 ng/ml of hPRL and lysed in GST-PAK lysis buffer (20 mM Tris-Base, pH 7.4; 150 mM NaCl; 5 mM MgCl2; 0.5% Nonidet P-40; 5 mM ß-glycerophosphate; 1 mM dithiothreitol; 1 mM phenylmethylsulfonylfluoride; 10 µg/ml leupeptin; and 1 µM pepstatin) plus 25 µg GST-PAK, obtained as previously described (61). Lysates were incubated with 50 µl glutathione sepharose beads (Amersham Pharmacia Biotech, Arlington Heights, IL) for 30 min at 4 C. Beads were washed three times in GST-PAK lysis buffer, and samples were subjected to SDS-PAGE and autoradiography. Rac-1 was labeled with a 1:1000 dilution of the Rac-1 antibody (Upstate Biotechnology, Inc., Lake Placid, NY) followed by 1:2500 dilution of antimouse horseradish peroxidase-conjugated secondary antibody (Sigma). After autoradiography, membranes were then stained with amido black (Sigma) for 1 min and destained with 25% isopropanol/10% acetic acid for 30 min to verify equal loading of GST-PAK samples.
Reporter Gene Assays
The reporter construct LHRE-TK-Luc containing the Stat5 DNA-binding sites from the promoter region of ß-casein 5' to a luciferase reporter was a kind gift of R. J. M. Ross (Sheffield University, Sheffield, UK). T47D cells were transfected with 12 µg of empty vector, wt Vav2,
1183 Vav2, or L212A Vav2, as indicated, and 1.0 µg of LHRE-TK-Luc and 0.5 µg of a control Renilla luciferase reporter construct. After transfection (24 h), cells were rested for 24 h and then treated with 100 ng/ml of PRL for 24 h. Luciferase assays were conducted by standard methods using the Dual Luciferase Assay System (Promega) and the Luminoskan Ascent Type 392 (Thermo Labsystems, Inc., Franklin, MA). Statistical analysis was generated by standard t test, and data are representative of one of three experiments conducted with triplicate transfections.
Apoptosis Assays
T47D cells were seeded (5 x 104 cells per well) on four-chamber CC2 Lab-Tel II glass chamber slides (Nalge Nunc International, Rochester, NY). Cells were cotransfected with 1 µg of fGFP (BD Biosciences, Palo Alto, CA) and empty vector, wt Nek3, or D143A Nek3. After transfection (24 h), fGFP-positive cells were analyzed for microscopic evidence of pyknosis or subjected to in situ labeling of free 3'-OH DNA termini, following the manufacturers protocol for the ApopTag Red Apoptosis Detection Kit (Intergen, Purchase, NY). Pyknotic and apoptotic fGFP-positive cells were counted for each transfection group (
200 cells per treatment), and the means of three separate experiments were analyzed by one-way ANOVA.
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ACKNOWLEDGMENTS
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We thank the National Hormone and Pituitary Program, NIDDK for recombinant PRL.
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FOOTNOTES
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This work was supported, in part, by National Institutes of Health (NIH) Grants RO1CA69294 and RO1CA92265 (to C.V.C.) and a NIH supplement to RO1CA92265 (to S.M.) from the National Cancer Institute, Comprehensive Minority Biomedical Branch.
First Published Online December 23, 2004
Abbreviations: CH, Calponin homology; CHO, Chinese hamster ovary; fGFP, farnesylated green fluorescent protein; GEF, guanine nucleotide exchange factor; GST, glutathione-S-transferase; GTPase, guanosine triphosphatase; Jak, Janus family of tyrosine kinases; LHRE, LH response element; NP-40, Nonidet P-40; PAK, p21-associated kinase; PH, pleckstrin homology; PRL, prolactin; PRLr, PRL receptor; siRNA, short interfering RNA; Stat, signal transducer and activator of transcription; wt, wild type.
Received for publication November 2, 2004.
Accepted for publication December 15, 2004.
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REFERENCES
|
---|
- Clevenger CV, Furth PA, Hankinson SE, Schuler LA 2003 The role of prolactin in mammary carcinoma. Endocr Rev 24:127[Abstract/Free Full Text]
- Goffin V, Binart N, Touraine P, Kelly PA 2002 Prolactin: the new biology of an old hormone. Annu Rev Physiol 64:4767[CrossRef][Medline]
- Clevenger CV, Chang WP, Ngo W, Pasha TL, Montone KT, Tomaszewski JE 1995 Expression of prolactin and prolactin receptor in human breast carcinoma. Evidence for an autocrine/paracrine loop. Am J Pathol 146:695705[Abstract]
- Ginsburg E, Vonderhaar BK 1995 Prolactin synthesis and secretion by human breast cancer cells. Cancer Res 55:25912595[Abstract]
- Brockman JL, Schroeder MD, Schuler LA 2002 PRL activates the cyclin d1 promoter via the jak2/stat pathway. Mol Endocrinol 16:774784[Abstract/Free Full Text]
- Bartkova J, Lukas J, Muller H, Lutzhoft D, Strauss M, Bartek J 1994 Cyclin D1 protein expression and function in human breast cancer. Int J Cancer 57:353361[Medline]
- Wennbo H, Gebre-Medhin M, Griti-Linde A, Ohlsson C, Isaksson OGP, Tornell J 1997 Activation of the prolactin receptor but not the growth hormone receptor is important for induction of mammary tumors in transgenic mice. J Clin Invest 100:27442751[Abstract/Free Full Text]
- Wennbo H, Tornell J 2000 The role of prolactin and growth hormone in breast cancer. Oncogene 19:10721076[CrossRef][Medline]
- Horseman ND, Zhao W, Montecino-Rodriguiez E, Tanaka M, Nakashima K, Engle SJ, Smith F, Markoff E, Dorshkind K 1997 Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene. EMBO J 16:69266935[Abstract/Free Full Text]
- Vomachka AJ, Pratt SL, Lockefeer JA, Horseman ND 2000 Prolactin gene-disruption arrests mammary gland development and retards T-antigen-induced tumor growth. Oncogene 19:10771084[CrossRef][Medline]
- Lebrun JJ, Ali S, Sofer L, Ullrich A, Kelly PA 1994 Prolactin-induced proliferation Nb2 cells involves tyrosine phosphorylation of the prolactin receptor and its associated tyrosine kinase JAK2. J Biol Chem 269:1402114026[Abstract/Free Full Text]
- Rui H, Lebrun J-J, Kirken RA, Kelly PA, Farrar WL 1994 JAK2 activation and cell proliferation induced by antibody-mediated prolactin receptor dimerization. Endocrinology 135:12991306[Abstract]
- Das R, Vonderhaar BK 1996 Activation of raf-1, MEK, and MAP kinase in prolactin responsive mammary cells. Breast Cancer Res Treat 40:141149[Medline]
- Clevenger CV, Torigoe T, Reed JC 1994 Prolactin induces rapid phosphorylation and activation of prolactin receptor associated Raf-1 kinase in a T-cell line. J Biol Chem 269:55595565[Abstract/Free Full Text]
- Das R, Vonderhaar BK 1996 Involvement of Shc, Grb2, Sos, and Ras in prolactin signal transduction in mammary epithelial cells. Oncogene 13:11391145[Medline]
- Maus MV, Reilly SC, Clevenger CV 1999 Prolactin as a chemoattractant for human breast carcinoma. Endocrinology 140:54475450[Abstract/Free Full Text]
- Kline JB, Moore DJ, Clevenger CV 2001 Activation and association of the Tec tyrosine kinase with the human prolactin receptor: mapping of a Tec/Vav1-receptor binding site. Mol Endocrinol 15:832841[Abstract/Free Full Text]
- Abe K, Rossman KL, Liu B, Ritola KD, Chiang D, Campbell SL, Burridge K, Der CJ 2000 Vav2 is an activator of Cdc42, Rac1, and RhoA. J Biol Chem 275:1014110149[Abstract/Free Full Text]
- Liu BP, Burridge K 2000 Vav2 activates Rac1, Cdc42, and RhoA downstream from growth factor receptors but not ß1 integrins. Mol Cell Biol 20:71607169[Abstract/Free Full Text]
- Bustelo XR 2000 Regulatory and signaling properties of the Vav family. Mol Cell Biol 20:14611477[Free Full Text]
- Schuebel KE, Bustelo XR, Nielsen DA, Song B-J, Barbacid M, Goldman D, Lee IJ 1996 Isolation and characterization of murine vav2, a member of the vav family of protooncogenes. Oncogene 13:363371[Medline]
- Movilla N, Bustelo XR 1999 Biological and regulatory properties of Vav-3, an new member of the Vav family of oncoproteins. Mol Cell Biol 19:78707885[Abstract/Free Full Text]
- Adams JM, Houston H, Allen J, Lints T, Harvey R 1992 The hematopoietically expressed vav proto-oncogene shares homology with the dbl GDP-GTP exchange factor, the bcr gene and a yeast gene (CDC24) involved in cytoskeletal organization. Oncogene 7:611618[Medline]
- Booden MA, Campbell SL, Der CJ 2002 Critical but distinct roles for the pleckstrin homology and cysteine-rich domains as positive modulators of Vav2 signaling and transformation. Mol Cell Biol 22:24872497[Abstract/Free Full Text]
- Aghazadeh B, Lowry WE, Huang XY, Rosen MK 2000 Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation. Cell 102:625633[Medline]
- Bustelo XR 2002 Regulation of Vav proteins by intramolecular events. Front Biosci 7:d24d30
- Machide M, Mano H, Todokoro K 1995 Interleukin 3 and erythropoietin induce association of Vav with Tec kinase through Tec homology domain. Oncogene 11:619625[Medline]
- Servitja JM, Marinissen MJ, Sodhi A, Bustelo XR, Gutkind JS 2003 Rac1 function is required for Src-induced transformation. Evidence of a role for Tiam1 and Vav2 in Rac activation by Src. J Biol Chem 278:3433934346[Abstract/Free Full Text]
- Tartare-Deckert S, Monthouel MN, Charvet C, Foucault I, Van Obberghen E, Bernard A, Altman A, Deckert M 2001 Vav2 activates c-fos serum response element and CD69 expression but negatively regulates nuclear factor of activated T cells and interleukin-2 gene activation in T lymphocyte. J Biol Chem 276:2084920857[Abstract/Free Full Text]
- Matsuguchi T, Inhorn RC, Carlesso N, Xu G, Druker B, Griffin JD 1995 Tyrosine phosphorylation of p95Vav in myeloid cells is regulated by GM-CSF, IL-3, and Steel factor and is constitutively increased by p210BCR/ABL. EMBO J 14:257265[Abstract]
- Clevenger CV, Ngo W, Sokol DL, Luger SM, Gewirtz AM 1995 Vav is necessary for prolactin-stimulated proliferation and is translocated into the nucleus of a T-cell line. J Biol Chem 270:1324613253[Abstract/Free Full Text]
- Tamas P, Solti Z, Buday L 2001 Membrane-targeting is critical for the phosphorylation of Vav2 by activated EGF receptor. Cell Signal 13:475481[CrossRef][Medline]
- Schuebel KE, Movilla N, Rosa JL, Bustelo XR 1998 Phosphorylation-dependent and constitutive activation of Rho proteins by wild-type and oncogenic Vav-2. EMBO J 17:66086621[Abstract/Free Full Text]
- Moores SL, Selfors LM, Fredericks J, Breit T, Fujikawa K, Alt F, Grugge JS, Swat W 2000 Vav family proteins couple to diverse cell surface receptors. Mol Cell Biol 20:63646373[Abstract/Free Full Text]
- Tamas P, Solti Z, Bauer P, Illes A, Sipeki S, Bauer A, Farago A, Downward J, Buday L 2003 Mechanism of epidermal growth factor regulation of Vav2, a guanine nucleotide exchange factor for Rac. J Biol Chem 278:51635171[Abstract/Free Full Text]
- Marignani PA, Carpenter CL 2001 Vav2 is required for cell spreading. J Cell Biol 154:177186[Abstract/Free Full Text]
- Manser E, Loo R-H, Koh C-G, Zhao Z-S, Chen X-Q, Tan L, Tan I, Leung T, Lim L 1998 PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. Mol Cell 1:183192[CrossRef][Medline]
- Tanaka K, Nigg EA 1999 Cloning and characterization of the murine Nek3 protein kinase, a novel member of the NIMA family of putative cell cycle regulators. J Biol Chem 274:1349113497[Abstract/Free Full Text]
- Crespo P, Schuebel KE, Ostrom AA, Gutkind JS, Bustelo XR 1997 Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product. Nature 385:169172[CrossRef][Medline]
- Han J, Das B, Wei W, Van Aelst L, Mosteller RD, Khosravi-Far R, Westwick JK, Der CJ, Broek D 1997 Lck regulates Vav activation of members of the Rho family of GTPases. Mol Cell Biol 17:13461353[Abstract]
- Miranti CK, Leng L, Maschberger P, Brugge JS, Shattil SJ 1998 Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1. Curr Biol 8:12891299[Medline]
- Salojin KV, Zhang J, Delovitch TL 1999 TCR and CD28 are coupled via ZAP-70 to the activation fo the Vav/Rac-1/PAK-1/p38 MAPK signaling pathway. J Immunol 163:844853[Abstract/Free Full Text]
- Lopez-Lago M, Lee H, Cruz C, Movilla N, Bustelo XR 2000 Tyrosine phosphorylation mediates both activation and downmodulation of the biological activity of Vav. Mol Cell Biol 20:16781691[Abstract/Free Full Text]
- Grimley PM, Dong F, Rui H 1999 Stat5a and Stat5b: fraternal twins of signal transduction and transcriptional activation. Cytokine Growth Factor Rev 10:131157[CrossRef][Medline]
- Faruqi TR, Gomez D, Bustelo XR, Bar-Sagi D, Reich NC 2001 Rac1 mediates STAT3 activation by autocrine IL-6. Proc Natl Acad Sci USA 98:90149019[Abstract/Free Full Text]
- Kodama A, Matozaki T, Fukuhara A, Kikyo M, Ichihashi M, Takai Y 2000 Involvement of an SHP-2-Rho small G protein pathway in hepatocyte growth factor/scatter factor-induced cell scattering. Mol Biol Cell 11:25652575[Abstract/Free Full Text]
- Aznar S, Lacal JC 2001 Rho signals to cell growth and apoptosis. Cancer Lett 165:110[CrossRef][Medline]
- Socolovsky M, Fallon AE, Wang S, Brugnara C, Lodish HF 1999 Fetal anemia and apoptosis of red cell progenitors in Stat5a/-5b/ mice: a direct role for Stat5 in Bcl-X(L) induction. Cell 98:181191[Medline]
- Chen WY, Ramamoorthy P, Chen N, Sticca R, Wagner TE 1999 A human prolactin antagonist, hPRL-G129R, inhibits breast cancer cell proliferation through induction of apoptosis. Clin Cancer Res 5:35833593[Abstract/Free Full Text]
- Beck MT, Peirce SK, Chen WY 2002 Regulation of bcl-2 gene expression in human breast cancer cells by prolactin and its antagonist, hPRL-G129R. Oncogene 21:50475055[CrossRef][Medline]
- Ramamoorthy P, Sticca R, Wagner TE, Chen WY 2001 In vitro studies of a prolactin antagonist, hPRL-G129R in human breast cancer cells. Int J Oncol 18:2532[Medline]
- Shigematsu H, Iwasaki H, Otsuka T, Ohno Y, Arima F, Niho Y 1997 Role of the vav proto-oncogene product (Vav) in erythropoietin-mediated cell proliferation and phosphatidylinositol 3-kinase activity. J Biol Chem 272:1433414340[Abstract/Free Full Text]
- Michel F, Grimaud L, Tuosto L, Acuto O 1998 Fyn and ZAP-70 are required for Vav phosphorylation in T cells stimulated by antigen-presenting cells. J Biol Chem 273:3193231938[Abstract/Free Full Text]
- Hall A 1998 Rho GTPases and the actin cytoskeleton. Science 279:509514[Abstract/Free Full Text]
- Han J, Luby-Phelps K, Das B, Shiu X, Mosteller RD, Krishna UM, Falck JR, White MA, Broek D 1998 Role of substrates and products of PI 3-kinase in regulating activation of rac-related guanosine triphosphatases by Vav. Science 279:558560[Abstract/Free Full Text]
- Aspenstrom P 1999 Effectors for the Rho GTPases. Curr Opin Cell Biol 11:95102[CrossRef][Medline]
- Schuringa JJ, Jonk LJ, Dokter WH, Vellenga E, Kruijer W 2000 Interleukin-6-induced STAT3 transactivation and Ser727 phosphorylation involves Vav, Rac-1 and the kinase SEK-1/MKK-4 as signal transduction components. Biochem J 347: 8996
- Fuh G, Wells JA 1995 Prolactin receptor antagonists that inhibit the growth of breast cancer cell lines. J Biol Chem 270:1313313137[Abstract/Free Full Text]
- Golemis EA, Serebriiskii I, Gyuris J, Brent R 1997 Interaction trap/two-hybrid system to identify interacting proteins. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, eds. Current protocols in molecular biology. New York: John Wiley & Sons; 135
- Rycyzyn MA, Clevenger CV 2002 The intranuclear prolactin/cyclophilin B complex as a transcriptional inducer. Proc Natl Acad Sci USA 99:67906795[Abstract/Free Full Text]
- del Pozo MA, Price LS, Alderson NB, Ren X-D, Schwartz MA 2000 Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. EMBO J 19:20082014[Abstract/Free Full Text]