©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Vav Is Necessary for Prolactin-stimulated Proliferation and Is Translocated into the Nucleus of a T-cell Line (*)

Charles V. Clevenger (1)(§), Winnie Ngo (1), Deborah L. Sokol (1), Selina M. Luger (2), , Alan M. Gewirtz (1) (2)

From the (1) Departments of Pathology and Laboratory Medicine and (2) Internal Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Stimulation of the prolactin receptor (PRLr) with ligand activates multiple kinase cascades. The proximal mediators involved in the activation of the PRL-activated Raf-1 cascade in T-cells, however, remain poorly characterized. The role of one proximal signaling protein, namely p95, during PRLr signal transduction was examined in the Nb2 T-cell line. The novel results obtained here indicate that Vav is transiently associated with the PRLr and is necessary for PRL-stimulated proliferation. During PRL stimulation, a rapid and dramatic increase in guanine nucleotide exchange factor (GEF) activity was found to be associated with Vav immunoprecipitates. Concomitantly, an increase in Vav phosphorylation on serine-threonine residues was observed. The Vav-associated GEF activation could be inhibited by staurosporine and calphostin, but not herbimycin, suggesting a modulatory role for phosphorylation at serine-threonine residues. Treatment of Nb2 cells with antisense Vav oligonucleotide ablated Vav expression and blocked PRL-driven proliferation, but failed to inhibit PRL-induced GEF activation within Nb2 lysates. These data indicate that GEF activity may not be intrinsic to Vav as has been previously suggested, but either resides in or is complemented by an associated GEF. Subsequent to the transient activation of associated GEF activity, Vav was found to translocate into the Nb2 cell nucleus. Thus, Vav may utilize two independent mechanisms in T-cells, namely the activation of an associated GEF and direct nuclear internalization, to mediate PRLr signaling.


INTRODUCTION

The pleiotropic actions of prolactin (PRL)() are mediated by its specific receptor (PRLr). Signals mediated by the PRLr induce the expression of specific genes that include cell cycle associated proteins (1, 2, 3) , receptors (4) , milk proteins (5, 6) , and proteins of uncertain function (7, 8). The PRLr is a member of the gene superfamily of growth factor receptors that include the receptors for growth hormone, erythropoietin, granulocyte-macrophage colony-stimulating factor, and the interleukins 2-7 (9) . Unlike the tyrosine-kinase family of receptors (i.e. receptors for epidermal growth factor and platelet-derived growth factor), the growth factor receptor family lacks intrinsic enzymatic activity, and the mechanisms of PRLr signal transduction have remained uncertain. Members of the growth factor receptor family, including the PRLr, demonstrate conserved cysteine disulfide linkages and a tryptophan-serine-X-tryptophan-serine motif in the extracellular domain. Both of these structures are believed to be necessary for the formation of the ligand binding pocket (10, 11) . The intracytoplasmic structure of the growth factor receptor superfamily is heterogeneous except for the so-called ``Box 1'' and ``Box 2'' motifs. The Box 1 subdomain represents a conserved hydrophobic proline-rich region that strongly resembles a SH3-binding domain (12) and may contribute to growth factor receptor-mediated gene transcription (13, 14) . The Box 2 domain is hydrophobic and acidic, and its signaling function is largely uncharacterized. Interactions between these PRLr domains and their associated signal transduction complexes are largely uncharacterized. Several studies have alternatively suggested that the activation of protein kinase C, G-proteins, and the Na-H antiport (15, 16, 17) occurs as a function of PRLr stimulation. Recent data have demonstrated that the serine-threonine kinase p72-74 is involved in PRLr signaling (18) . Other kinases activated and associated with the PRLr during signal transduction in T-lymphocytes include the tyrosine kinases p59 and p130(19, 20, 21) . These data suggest that multiple kinase cascades are activated as a function of PRLr stimulation.

Raf occupies a central position in the activation of downstream kinases, namely MAPK and MEK (Mitogen Activated Protein Kinase and MAPK or ERK Kinase, respectively) that induce the phosphorylation of numerous transcription factors (22, 23, 24, 25, 26) . A critical upstream activator of Raf is the GTP-binding protein p21. Ras participates in the recruitment of Raf to the cell membrane (27, 28) and contributes to its activation (29, 30) . Ras is activated, in turn, when bound GDP is exchanged for GTP, a process mediated by the so-called guanine nucleotide exchange factors (GEF (31) ). Recently isolated mammalian GEFs include Sos (32, 33, 34, 35) , Ras-GRF (36) , C3G (37) , and the p95 protooncogene, Vav (38, 39, 40) .

The p95 product of the Vav proto-oncogene is expressed selectively in hematopoietic cells (41, 42, 43, 44) . One interpretation of this phenomenon, and other functional data, has been that Vav may contribute to the differentiation (45) , or growth factor-mediated signaling (46) in cells of the hematopoietic system. How critical a role this is and whether Vav is absolutely required in these processes has been debated (45, 46) . A GEF activity associated with Vav has recently been demonstrated during stimulation of the T cell receptor and membrane immunoglobulin in T and B lymphocytes, respectively (38, 47) . This activity appears to be regulated by protein tyrosine kinases (38) or by phorbol esters and diacylglycerides (39) . The nature of the GEF activity associated with Vav has also been the subject of controversy. Initial studies indicated that this GEF activity was intrinsic to Vav (38) ; however, additional reports have disputed this finding (37, 48) . In addition to its associated GEF activity, Vav contains multiple protein-protein interaction motifs namely, SH2, SH3, leucine zipper, helix-loop-helix-like, acidic rich, pleckstrin-homology, and nuclear translocation domains (49, 50) . Loss or alteration of the amino terminus of Vav which contains the helix-loop-helix-like and leucine zipper motifs activates the transforming potential of Vav (50) . Thus, in addition to its associated GEF activity, Vav may also contribute to the formation of multienzyme complexes associated with receptor signaling (51, 52) .

The experiments presented here examine the role of Vav during stimulation of the PRLr in the PRL-dependent T-cell line Nb2. These data demonstrate that while Vav is necessary for PRL-driven proliferation, the GEF activity found within anti-Vav immunoprecipitates is associated with, but not necessarily intrinsic to, Vav. Data are presented that indicate that two separate, possibly phosphorylation-dependent, mechanisms may be utilized by Vav in its transmittal of the proliferative signal initiated by PRLr stimulation. These data confirm previous functional interpretations of the amino acid sequence of Vav (43, 50, 53) that have indicated multifunctional roles for this signal transduction factor.


MATERIALS AND METHODS

Cell Culture and Labeling

The rat T-cell lymphoma line Nb2/11c was maintained in Fisher's medium containing 10% bovine fetal calf serum, 10% gelding serum, 10 -mercaptoethanol, and penicillin/streptomycin (54) at 37 °C. This subline was directly obtained from Dr. P. Gout and found to express only the intermediate isoform (other sublines had been previously found to demonstrate heterogeneous PRLr isoform expression (18, 19) ). For experimental purposes, the cells were rested for 48 h before PRL stimulation in chemically defined medium lacking PRL, consisting of Dulbecco's modified Eagle's medium supplemented with sodium selenide, linoleic acid, insulin, and transferrin (ITS+ supplement, Becton Dickinson, Sunnyvale, CA). PRL used in these studies was purified rat PRL obtained from the NIDDK. For stimulation of the Nb2 cells, 10 ng PRL/ml was used; this dose had been determined in previous studies (18, 55) to induce the maximal proliferation of Nb2 cells. Nuclei and cytosol were isolated using hypotonic swelling and Dounce homogenization, as described previously (56) . For P labeling, 1 10 Nb2 cells were placed in 5 ml of phosphate-free Dulbecco's medium for 1 h prior to the addition 0.1 mCi of PO/ml for 4 h prior to PRL stimulation. To examine the effect of kinase inhibitors, Nb2 cells were pretreated for 12 h with 10 µM herbimycin A, for 20 min with 0.5 or 1 µM staurosporine, or for 20 min with 1 µM calphostin prior to PRL stimulation.

Immunoprecipitation and Immunoblotting

After PRL stimulation, 1 10 cells were promptly washed with ice-cold PBS and lysed in 500 µl of lysis buffer consisting of 10 mM Tris, pH 7.6, 50 mM NaCl, 12 mM MgCl, 30 mM sodium pyrophosphate, 50 mM NaF, 2.5 mM EDTA, 1 mM NaVO, 0.23 unit aprotonin/ml, 10 µg leupeptin/ml, and 2% Nonidet P-40 at 4 °C. After removal of debris by centrifugation, the lysate was precleared with 60 µl of a 1:1 mixture of protein A + G-conjugated Sepharose beads (Life Technologies, Inc.). Immunoprecipitations were then performed on precleared lysates (100 µl) using anti-Vav (3 µl; Santa Cruz Biotechnology) or anti-PRLr (5 µl of U6, gift of Dr. Paul Kelly). Control immunoprecipitations utilized an irrelevant isotype matched antibody (MOPC-21, Sigma) Antigen-antibody complexes were isolated by the addition of 50 µl of protein-A/G beads. After three washes with lysis buffer, the immunoprecipitates were prepared for electrophoresis or GDP exchange assay. For preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), washed beads were resuspended in 2-fold concentrated Laemmli buffer with mercaptoethanol and boiled prior to analysis on a 10% gel. Hydrolysis of phosphate groups from serine and threonine residues was accomplished by incubation of the SDS-PAGE in 1 M KOH at 55 °C for 1 h.

Immunoblot analysis was performed as described previously (19) using the above immunoprecipitates or 5 10 washed cells in 2 Laemmli buffer. After transfer to nitrocellulose, antigen was labeled with a 1:2,000 dilution of anti-Vav, 1:1,000 anti-phosphotyrosine (4G10, UBI) or a 1:1,000 dilution of anti-PRLr or isotype matched control antibody. Antigen-antibody complexes were then detected using enhanced chemiluminescense (ECL kit, Amersham Corp.).

GDP-GTP Exchange Assay

The solid-phase GDP-GTP exchange assay previously described (38) was modified slightly. Briefly, 0.9 µM recombinant Harvey Ras was loaded with 10 µCi of [H]GDP for 90 min at 30 °C in a buffer consisting of 25 mM Tris, pH 7.5, 100 mM KCl, 0.01% bovine serum albumin, 0.2 mM dithiothreitol, and 12 mM EDTA. The [H]GDPRas complexes were stabilized by the addition of MgCl to 22 mM and further incubation at 30 °C for 30 min. These complexes were applied with gentle vacuum to a slot-blot apparatus containing nitrocellulose (Amersham). The complexes were then washed with three times with wash buffer (25 mM Tris, pH 7.5, 100 mM KCl, 0.2 mM dithiothreitol, and 12 mM MgCl) at 4 °C. After careful and exacting excision of the filter strip, the solid phase GDP-Ras was incubated with 150 µl of cell lysates or immunoprecipitates. Blunt handling of the filter strip during excision or during incubation with lysates was noted to induce an undesirable release of radiolabel in control specimens; this could be avoided by meticulous handling of the specimens. At various (but precisely timed) intervals, 30 µl of fluid was removed for scintillography to monitor the release of [H]GDP into the aqueous phase.

Oligodeoxynucleotides and Treatment of Cells

Unmodified phosphodiester oligomers were obtained and used as described previously (57-59). Lyophilized oligomers were dissolved in tissue culture medium before use at a concentration of 1 µg/µl. Oligomer sequences based on the published human proto-vav cDNA sequence (43, 50) were as follows: (5`-AAG GCA CAG GAA CTG GGA-3`), antisense ODN and (5`-AGC TCG AAA GAC AGG GGA-3`), scrambled ODN. 1 10 Nb2 cells/ml were exposed to oligomers in defined medium containing 10 ng PRL/ml. ODN (20-100 µg/ml) were added at time 0, and 50% of the initial dose was added again 24 h later (final concentration 5-25 µM). Before the addition of the second dose of oligomer, cells from some experiments were removed for analysis of Vav content. Twenty-four h after the addition of the second dose of oligomer, additional cells were removed for Vav content analysis or were labeled for 4 h with 0.5 µCi [H]thymidine/ml prior to harvest and scintillography.

Immunofluorescence Microscopy

Cells were prepared for immunofluorescence microscopy using standard technique (60) . Briefly, washed Nb2 cells were immobilized on glass slides with a Cytospin apparatus (Shandon). The cells were then fixed for 10 min with 1% paraformaldehyde (Polysciences) in PBS, followed by permeabilization with 0.1% Triton X-100 in PBS for 3 min, both at 4° C. The cells were then incubated with a 1:100 dilution of anti-Vav antibody for 45 min. After washing antigen-antibody complexes were then detected by incubating with a fluorescein isothiocyanate conjugated goat anti-rabbit antibody (Tago, 1:40).


RESULTS

To examine if GEF activity was associated with PRLr signal transduction, the rat T-cell lymphoma line Nb2 was utilized. This line has served as an excellent model for studying PRLr signaling as it can be propagated under defined conditions with PRL as the sole growth factor (61) . PRL-induced GEF activation was initially assessed in resting and PRL-stimulated Nb2 lysates by a solid-phase [H]GDP release assay (GEF assay). This had been used previously to demonstrate the activation of Vav-associated GEF activity during antigen-stimulated T-cell receptor and membrane immunoglobulin signal transduction (38, 47) . The induction of GEF activity was examined in Nb2 cells stimulated with 10 ng PRL/ml (a concentration previously shown to induce the optimal proliferation of Nb2 cells (19, 61) ), for 5 min. PRL stimulation induced a 4-5-fold increase in GEF activity of Nb2 lysates, in comparison to lysates obtained from resting cells (Fig. 1). These data also demonstrate the linearity of the GEF assay for the Nb2 lysates. For all subsequent studies a 2-min incubation was used for the GEF assay.


Figure 1: PRL stimulation of Nb2 cells induces GEF activity. Nb2 cells were stimulated for 5 min in the presence or absence of 10 ng/ml PRL. Cleared cellular lysates or buffer alone (Background) were temporally examined for exchange activity through the use of a filter [H]GDP release assay (GEF assay). Approximately 150,000 counts/min of [H]GDP-Ras was bound to each filter at the initiation of these experiments. The exchange assay was initiated by co-incubation of lysates or buffer with the [H]GDP-Ras filter. At indicated time intervals, 30-µl aliquots were removed for scintillography. Presented data were multiplied by 5 to correct for the total reaction volume (150 µl). For each data point, n = 3 ± S.E.



The basis for the PRL-induced GEF activity in Nb2 cells was examined. Immunoblot analysis revealed abundant Vav within Nb2 cells (Fig. 3B), but little if any Sos (data not shown). To assess if the GEF activity in PRL-stimulated Nb2 lysates was associated with Vav, GEF assay of anti-Vav immunoprecipitates and immunodepleted lysates was performed (Fig. 2). At least 70% of the PRL-induced GEF activity in Nb2 cells was specifically recovered in the anti-Vav immunoprecipitates. These data indicate that most of the GEF activity within Nb2 cells is associated with anti-Vav immunoprecipitates, but do not distinguish whether such activity was intrinsic to the immunoprecipitated Vav or extrinsic to the immunocomplex in the form of an associated GEF.


Figure 3: Temporal induction of GEF-specific activity by PRL stimulation. Lysates were obtained from Nb2 cells stimulated for various intervals with 10 ng PRL/ml. A, anti-Vav immunoprecipitates obtained from these lysates were subject to GEF assay as per Fig. 2. For each data point, n = 3 ± S.E. B, immunoblot analysis of these stimulated lysates using anti-Vav antibody reveals constant levels of Vav protein. Lower molecular mass species (at 50 and 85 kDa) may represent proteolytic products and are not seen in anti-Vav immunoprecipitates.




Figure 2: PRL-induced GEF activity in Nb2 cells is associated with anti-Vav immunoprecipitates. Lysates from Nb2 cells stimulated for 5 min with 10 ng PRL/ml (Nb2 lysate) were immunoprecipitated with anti-Vav, anti-Vav blocked overnight with a specific Vav peptide (a-Vav + Vav), or preimmune serum. GEF assay was performed on these immunoprecipitates and cell lysates, both pre- (Nb2 lysate) and post- (a-Vav depleted lysate) immunoprecipitation. Immunoprecipitates and immunodepleted lysates were obtained from the same lysate; all data presented in this figure utilized the same volume of cell lysate initially (i.e. each data point represents the GEF activity of approximately 2 10 Nb2 cells). Exchange assay was performed as in Fig. 1, after 2 min of co-incubation between the lysate/immunoprecipitate and the solid phase [H]GDP-Ras. For each data point, n = 3 ± S.E.



The temporal activation of the Vav-associated GEF activity was studied in Nb2 cell lysates stimulated with PRL for varying time intervals. The PRL-induced activation of Vav occurred rapidly after PRL stimulation, peaking at 5-min post-stimulation (Fig. 3A). This activation was transient and returned to base-line levels within 10 min of initial PRL stimulation. The transient PRL-induced activation of Vav-associated GEF is consistent with observations in other signaling systems (38, 47) . Immunoblot analysis of PRL-stimulated Nb2 cell lysates with an anti-Vav antibody (Fig. 3B) revealed a doublet migrating at approximately at 95 kDa. These molecular weight differences may be due to differential protein processing (i.e. phosphorylation, glycosylation) and have been observed in other T-cell systems (52) . Quantitation of these bands revealed little change as a function of PRL stimulation. Taken with the data presented in Fig. 3A, these observations indicate that the PRL-induced exchange activity in Nb2 cells was not correlated with an overall increase in intracellular Vav levels.

The association of Vav with the PRLr present within the Nb2 cells was examined. Immunoblot analysis of anti-Vav and anti-PRLr immunoprecipitates (Fig. 4) revealed a PRL-induced association of Vav with a protein species migrating at approximately 65 kDa, consistent with the intermediate isoform of the PRLr. The inducible association occurred rapidly within 5 min and persisted, albeit to a lesser degree, after 20 min of PRL stimulation. Thus, although the PRL induction of Vav-associated GEF activity and the association of Vav with the PRLr are temporally associated, the inactivation of GEF activity and the disassociation of Vav from the PRLr are not (compare Fig. 4 with Fig. 3A). The interaction of other factors (i.e. phosphatases, ancillary kinases) may lead to an inactivation of Vav-associated GEF activity, without a concomitant release of Vav from the PRLr. Preliminary data (not shown) indicate that Ras and Raf are also associated with anti-Vav immunoprecipitates, suggesting the formation of a multienzyme complex during PRLr signaling.


Figure 4: PRL stimulation induces the association of Vav with the PRLr. Lysates from fed (F), resting (0 min), or 10 ng/ml PRL-stimulated Nb2 cells (5 or 20 min) were immunoprecipitated (Ippt) with anti-Vav or anti-PRLr antibody. The immunoprecipitated proteins were subjected to immunoblot (Iblt) analysis with these or control antibodies. Prior to immunoblotting, antigens were released from the immunoprecipitates with 1% SDS and heat, and the denatured antibodies removed with protein A/G beads, as described previously (18, 80). The anti-PRLr antibody used in these studies (U6) recognizes a extracellular epitope common to the long, intermediate, and short PRLr isoforms. Molecular masses on right are in kDa.



Two mechanisms have been identified that may regulate Vav GEF activity subsequent to the interaction of ligand with receptor. Tyrosine phosphorylation (38, 41, 62, 63, 64) , diacylglycerol (DAG) and phorbol ester (39, 40) , and ceramide (40) have been implicated in the activation of Vav. Such pathways could contribute to the PRL-induced activation of Vav in Nb2 cells. Thus, the role of kinase and phorbol ester stimulation was examined in Nb2 cells. Anti-Vav immunoprecipitates from PO-labeled Nb2 cells demonstrated a rapid 5-fold increase in phosphorylation upon PRL stimulation (Fig. 5). Multiple immunoblots of anti-Vav immunoprecipitates probed with anti-phosphotyrosine antibodies, however, revealed an absence of significant phosphorylation of tyrosine residues (data not shown). That PRL-induced Vav phosphorylation might be occurring on serine and threonine residues was further confirmed by the observation that virtually all of the radiolabel in anti-Vav immunoprecipitates could be removed by KOH treatment. These results were consistent with previous findings in our laboratory (18) suggesting that the activation of the Vav/Ras/Raf in Nb2 cells occurred via serine and threonine phosphorylation.


Figure 5: PRL- induced phosphorylation of Vav. P-Labeled Nb2 cells stimulated with 10 ng/ml PRL and harvested at various time intervals. Lysates from these cells were immunoprecipitated with anti-Vav or pre-immune control antibodies and subject to SDS-PAGE analysis. Molecular masses on left are in kDa. Immunoblot analysis of similar unlabeled lysates with anti-phosphotyrosine antibodies did not reveal significant phosphorylation on tyrosine residues (data not shown).



The relative role of DAG stimulation and protein kinases during PRL-induced Vav-associated GEF activation was assessed through the use of the pharmacologic inhibitors herbimycin A (a specific protein tyrosine kinase inhibitor), staurosporine (a nonspecific kinase inhibitor that demonstrates the highest IC for protein kinase C), and calphostin (a diglyceride antagonist). Nb2 cells were pretreated with these pharmacologic agents (at doses used conventionally to inhibit signal transduction) prior to PRL stimulation and subsequent GEF assay. The data presented in Fig. 6 indicate that the inhibition of DAG/protein kinase C pathways by staurosporine and calphostin significantly reduces the PRL-induced activation of Vav-associated GEF. In contrast, inhibition of protein tyrosine kinase action by herbimycin A did not affect the activation of Vav-associated GEF activity and is supportive of the data presented in Fig. 5that suggests a minimal role for protein tyrosine kinases in PRL-induced Vav signal transduction.


Figure 6: Pharmacologic inhibition of PRL-induced Vav activation indicates that Vav activation is independent of protein tyrosine kinase activation. Resting Nb2 cells were pretreated with either herbimycin A (10 µM, 12 h, herb), staurosporine (0.5 or 1 µM, 20 min, staurosp), or calphostin (1 µM, 20 min) prior to stimulation for 5 min with 10 ng PRL/ml. Lysates from these cells were immunoprecipitated with anti-Vav antiserum and subject to GEF assay as per Fig. 2. Approximately 100,000 cpm of [H]GDP-Ras was bound to each filter at the initiation of these experiments. Each data point represents the mean ± S.E., n = 3.



If Vav was significantly contributing to the PRL-driven proliferation, down-regulation of its expression should inhibit cell proliferation. To test this hypothesis, PRL-stimulated Nb2 cells were incubated with antisense or scrambled Vav phosphodiester oligodeoxynucleotides (ODN). After 2 days of incubation with the maximal dose of antisense Vav ODN tested, the expression of Vav protein in Nb2 cell lysates was not detectable by immunoblot analysis (Fig. 7A), while incubation with the scrambled ODN controls had little effect on Vav expression. The effect of Vav antisense on cell proliferation was extensively examined with respect to dose and duration; after 3 days of co-incubation with the maximal dose of antisense Vav ODN most of the Nb2 cells failed to exclude trypan blue, while control ODN had little effect on cell viability assessed in this manner (data not shown). A similar phenomenon has been noted to occur when Nb2 cells are deprived of PRL (61, 65) . This was further confirmed by examining the effect of varying doses of Vav ODN on the [H]thymidine incorporation in PRL-stimulated Nb2 cells after 2 days of co-incubation (Fig. 7B), a time point when cell viability in all cultures was >90%. These data demonstrate that Vav antisense inhibited PRL-dependent proliferation in a dose-dependent manner, while the scrambled control ODN had little effect on PRL-driven proliferation. PAGE analysis of culture supernatants obtained throughout the culture period revealed no significant degradation of the ODN (data not shown). These results support the hypothesis that the observed inhibition of cell growth was due to antisense-mediated loss of Vav protein and not to suppression of cell growth by ODN degradation products.


Figure 7: Effects of antisense ODN ablation of Vav expression in Nb2 cells. A, Vav is down-regulated by antisense ODN. Nb2 cells (1 10/ml) were incubated in defined medium containing 10 ng PRL/ml and antisense or scrambled Vav ODN. 100 µg of ODN was added at the initiation of the experiment (Day 0); on the following day (Day 1) cultures were either harvested, or 50 µg/ml of additional ODN was added to the remaining cultures prior to their harvest on Day 2. Lysates from these cells were obtained after 1 or 2 days of incubation and subject to immunoblot analysis (10 µg of total protein/lane) with anti-Vav antibody. Densitometry of the bands was performed and values are reported in arbitrary units (au). B, antisense Vav inhibits PRL-driven cell proliferation. Nb2 cells were incubated in defined medium in the absence of PRL (-PRL) or in the presence of 10 ng PRL/ml with varying concentrations of antisense or scrambled Vav ODN. Values present on the x axis represent dosage of oligomer added to the cells within the first dose (to calculate total dosage of oligomer received by the cells add 50%). After 2 days of incubation, proliferation was assessed by the addition of 0.5 µCi of [H]thymidine for 4 h prior to cell harvesting and scintillography. Each data point represents the mean ± S.E., n = 4. C, ablation of Vav expression fails to inhibit PRL-induced GEF activity in Nb2 cell lysates. Nb2 cells were incubated for 2 days as per B in the absence of PRL with 100 µg/ml (at initiation) of antisense or scrambled Vav ODN (or PBS control) prior to stimulation for 5 min with 10 ng/ml PRL. After harvest of the stimulated cells, cell lysates and anti-Vav immunoprecipitates were prepared and analyzed for GEF activity. These data reveal that the antisense ablation of Vav dramatically reduces GEF activity in anti-Vav immunoprecipitates, but does not significantly effect overall PRL-induced GEF activity in PRL-stimulated Nb2 cell lysates. These data suggest that PRL-induced GEF may not be intrinsic to Vav, but either resides in or is complemented by an associated GEF. Each data point represents the mean ± S.E., n = 3.



Ablation of Vav expression by antisense treatment of Nb2 cells was also used to examine the nature of the GEF activity associated with Vav. If GEF activity was intrinsic to Vav, treatment with Vav antisense should largely abolish the PRL-induced exchange activity in both Nb2 cell lysates and anti-Vav immunoprecipitates. However, if the GEF activity was extrinsic to Vav, in the form of an associated, and perhaps novel, protein with exchange activity, a reduction of PRL-induced GEF activity within the Nb2 lysates would not be expected to occur. To test these alternative hypothesis, Nb2 cells were cultured, as above, in the presence of Vav antisense or scrambled ODN for 2 days. After stimulation with PRL, the cultures were harvested and both cell lysates and anti-Vav immunoprecipitates tested for GEF activity (Fig. 7C). Not suprisingly, the anti-Vav immunoprecipitates from antisense-treated cell lysate lacked GEF activity, secondary to the lack of immunoprecipitatable Vav within these lysates. However, analysis of the GEF activity in the antisense-treated cell lysates revealed no significant reduction of the PRL-induced exchange activity. These data indicate that the PRL-induced GEF activity within Nb2 cells can be dissociated from Vav expression and indicate that the exchange activity observed in anti-Vav immunoprecipitates either resides in, or is complemented by, an associated GEF.

Although Vav is thought to interact with receptor signaling complexes at the cell surface (41, 49) , it has been noted that Vav contains two nuclear translocation motifs. Indeed, it has been suggested (but not formally demonstrated) that stimulation of the IgE receptor in mast cells induces the nuclear translocation of Vav (64) . Moreover, a recent report has indicated that Vav can interact with the heterogenous ribonucleoprotein K, which is located in both the nucleus and cytoplasm (66). To examine if a nuclear translocation of Vav occurred during PRLr signaling, PRL-stimulated Nb2 cells were harvested at intervals and prepared for examination by indirect immunofluorescence with anti-Vav antibody. These data (Fig. 8A) indicate that Vav is transiently internalized into the nucleus of Nb2 cells 10-30 min after PRL stimulation. This observation was further confirmed (Fig. 8B) at the biochemical level by immunoblot analysis with anti-Vav antibodies of lysates obtained from isolated nuclei and cytosol of PRL-stimulated Nb2 cells. These data indicate that a significant, but transient, increase in intranuclear Vav occurs approximately 10 min after PRL stimulation; interestingly, this translocation correlates with the return of Vav-associated GEF activity to base-line levels (as seen in Fig. 3A).


Figure 8: Vav is translocated to the nucleus during PRL stimulation. A, Nb2 cells cultured in defined medium were stimulated with 10 ng PRL/ml for 0, 5, 10, 30, or 60 min (panels A-E, respectively) prior to harvest, fixation with 1% paraformaldehyde, 0.1% Triton X-100, and indirect immunofluorescence labeling with 1 µg anti-Vav/ml (panels A-E) or preimmune serum (panel F, 30 min of incubation with PRL). Preincubation of anti-Vav antibody with Vav peptide resulted in labeling similar to that seen in panel F. B, Nb2 cells were stimulated at varied intervals with 10 ng PRL/ml, prior to harvesting and separation into cytoplasmic (cyto) or nuclear (nuc) fractions, prior to immunoblotting with anti-Vav. Densitometric quantitation of the bands revealed >10-fold increase in intranuclear Vav transiently occurred during PRL stimulation.




DISCUSSION

The results presented here demonstrate that PRL stimulation of Nb2 cells induced the activation of Vav-associated GEF (Fig. 1-3) and its association with the PRLr (Fig. 4). Thus, the PRLr joins a growing list of receptors associated with hematopoietic differentiation and immune response, such as the T-cell receptor (38) , membrane immunoglobulin (47), Fc receptor (64) , interferon receptor (67) , c-kit (68), and the receptors for interleukin-1 (40) and -2 (63) , that either phosphorylate and/or activate Vav during signal transduction. This study, however, represents the first report of an association between Vav and a member of the superfamily of growth factor receptors. Such an interaction would bring Vav into close proximity to other kinases known to associate with the PRLr, namely, Raf (18) , Fyn (19) , and JAK2 (20, 21) . Like Vav, these kinases are transiently activated. However, the rapid activation of Vav-associated GEF activity (Fig. 3, peak activity within 5 min of stimulation) appears to precede that of Fyn (19) and Raf (18) and parallel that of JAK2 (20) . This observation may suggest that Vav serves a proximal role in PRLr signaling. Unlike Vav, the kinases Fyn, Raf, and JAK2 are constitutively associated with the PRLr, while the association of Vav with PRLr is induced by ligand binding and presumed receptor dimerization (69) .

When stimulated by PRL, Vav was inducibly phosphorylated (Fig. 5) in Nb2 cells. Unlike the T-cell receptor (38) , membrane immunoglobulin (47), Fc receptor (64) , interferon receptor (67) , c-kit (68), and interleukin-2 receptor (63) , however, this PRL-induced phosphorylation appeared not to occur on tyrosine residues. This observation was further supported by the lack of herbimycin A inhibition of PRL-stimulated Vav activation. These data suggest that phosphorylation of Vav in Nb2 cells occurs on serine-threonine residues and that protein tyrosine kinases are not directly involved in the activation of Vav during PRLr signaling. Several alternative pathways, therefore, may activate Vav-associated GEF during PRLr signal transduction. One possibility is that an unknown or known (i.e. protein kinase C, or a relative) serine-threonine kinase phosphorylates and activates Vav. This theory is supported by the inhibition of PRL-induced Vav activation by the ATP analogue, and preferential inhibitor of protein kinase C, staurosporine (Fig. 6). Protein kinase C has been previously implicated in PRLr signal transduction (15, 70) . The kinetics of PRL-induced protein kinase C activation, however, lag behind those of Vav. Thus, protein kinase C is an unlikely candidate as a proximal mediator of Vav activation. The possibility of a novel protein kinase C-like, serine-threonine kinase, proximal to Vav, that is involved in PRLr signaling is therefore under examination in our laboratory. Another pathway that may contribute to Vav activation is signaling by DAG. Similar to the PRLr, the interleukin-1 stimulation of Vav occurs through a non-protein tyrosine kinase mediated mechanism in EL4 cells (40, 71, 72) . Indeed, these studies have demonstrated that stimulation by DAG analogues or phorbol ester treatment can induce Vav GEF activity. Vav activation by interleukin-1 was inhibited by calphostin, a diglyceride antagonist. As was seen in Fig. 6 , calphostin also inhibited the activation of Vav by PRL. These data would suggest that DAG contributes to the PRL-induced activation of Vav-associated GEF. Vav is known to contain the cysteine-rich motif Cys-X-Cys-X-Cys-X-Cys-X-His-X-Cys-X-Cys that mediates DAG binding (44) . Thus, it is possible that a PRL-induced elaboration of DAG directly stimulates Vav. Alternatively, DAG may serve to stimulate a proximal kinase that is necessary for the PRL-induced activation of Vav.

The data presented in Fig. 4 reveal that the association of Vav with the 65-kDa intermediate PRLr isoform present in the Nb2 line used in these studies was induced by PRL stimulation. Whether Vav interacts with the long (85 kDa) or short (45 kDa) PRLr isoforms is not currently known in vivo, although in vitro binding studies performed in our laboratory suggest that Vav can interact with the recombinantly expressed long isoform (data not shown). These data would suggest that the 198 amino acids deleted from the intracytoplasmic domain of intermediate Nb2 PRLr isoform are not necessary for Vav's interaction with the PRLr. There are numerous domains within Vav that could mediate this interaction with the PRLr. In addition to its SH3-SH2-SH3 motif, Vav also contains a leucine zipper motif and an acidic rich domain (43, 44, 53) . The contribution of these domains to PRLr-Vav interactions is currently under study in our laboratory.

The role of Vav in hematopoietic cell development is somewhat controversial. A study by Wulf et al.(45) reported that when mouse embryonic stem cells were transfected with a construct expressing Vav antisense RNA, hematopoietic cell differentiation was markedly inhibited. These results suggested that Vav was indispensable for early hematopoietic cell development. In contrast, a report by Zhang et al.(46) has recently reported that Vav null embryonic stem cells created by homologous recombination retain their ability to differentiate in hematopoietic cells. This report also demonstrated that Vav cells contributed to hematopoiesis in Vav/wild-type mouse chimeras. Whether such mice respond normally to an imposed hematopoietic stress was not commented upon. Accordingly, analogous to the more subtle myelopoietic defect in kit defective white spotting mice (73) , these results do not negate the potential importance of Vav in hematopoietic cell development. Rather, they do suggest that embryonic cells retain the ability to complement Vav function with other signaling proteins. Whether this can occur in adult hematopoietic cells is uncertain and may vary with cell lineage (74) . Of interest as well, the Vav lymphocytes have impaired receptor-mediated signal transduction (46) . Thus, while Vav may not be necessary for basal myelopoiesis, a critical role in immune cell proliferation and function is strongly suggested by this work (46) . Since co-stimulation with PRL is necessary during the clonal expansion of T-cells (75, 76, 77) , the necessity of Vav expression during PRL-driven Nb2 T cell proliferation was examined using antisense Vav DNA. These studies (Fig. 7, A and B) show that antisense DNA can reduce Vav protein expression to non-detectable levels within 2 days and profoundly inhibit the PRL-driven proliferation of Nb2 cells. Thus, the expression of Vav appears necessary for the PRL-driven proliferation of the Nb2 line.

The Vav-associated Ras GEF activity demonstrated here has been described in other signaling systems (38, 39, 40, 47) . GEFs exchange GDP for GTP bound to p21, activating this molecule and the Raf-1 kinase cascade. The molecular basis for this Vav-associated Ras GEF activity, however, has produced considerable debate. Sequence analysis of Vav has demonstrated homology to the dbl oncogene (53, 78) , a GEF for CDC42. CDC42 is a member of the Rho/Rac family of small GTP-binding proteins. A glutathione S-transferase fusion protein of Dbl failed, however, to demonstrate GEF activity for Ras (78). Other attempts at demonstrating a Ras-GEF activity in Vav expressed in Escherichia coli or in baculovirus-infected Sf9 cells have not been successful (48) . If Vav was acting directly as a Ras-GEF, transformation and over-expression in a responsive cell line, such as NIH3T3, should induce morphologic and biochemical changes similar to those seen with bona fide Ras exchange factors, such as CDC25. This, however, was not observed in a recent study (79) . Indeed, no increase in the levels of Ras-GTP were noted in NIH3T3 cells transformed with oncogenic Vav or Dbl, although activation of mitogen-activated protein kinase was noted in Dbl transformed cells. Thus, taken together, these results (48, 78, 79) contradict previous data demonstrating a Ras-GEF activity within anti-Vav immunoprecipitates from stimulated T and B cell lysates (38, 47) and suggest that Vav and Dbl may function through alternative pathways during receptor-mediated signaling. To test this hypothesis during PRLr-mediated signal transduction, anti-Vav immunoprecipitates and cell lysates from PRL-stimulated Nb2 cells pretreated with Vav ODN were analyzed for GEF activity. These analyses revealed that the PRL-induced activation of GEF activity did not require the expression of Vav (Fig. 7C), even though most of this activity was co-immunoprecipitated with anti-Vav antibodies (Fig. 2). A plausible interpretation of these data is that the GEF activity previously associated with Vav in immunoprecipitates (38, 47) is not intrinsic to this signaling factor. Rather, an associated, but as yet unidentified, protein may serve as the Vav-associated GEF. Thus, like GRB2, Vav may serve as an adaptor protein, and link this unidentified GEF, and perhaps other signaling factors, with Ras during receptor-mediated signaling. Nevertheless, it is not entirely possible to exclude from the data presented here that Vav does have intrinsic GEF activity, which in the absence of Vav is complemented by a currently unknown GEF.

Another mechanism through which Vav may mediate PRL-driven T-cell proliferation is through its translocation into the cell nucleus. Although various reports, without presenting data, have suggested (64) and discounted (49) the possibility of the nuclear internalization of Vav, the data presented here (Fig. 8) represent the first documentation of a growth factor-induced translocation of Vav. The internalization of Vav in the nucleus appears to be transient and interestingly occurs after its associated GEF activity returns to base-line levels, i.e. after approximately 10 min of PRL stimulation. Preliminary data from our laboratory indicate that the decrease in GEF activity is not due to its translocation into the nucleus with Vav (not shown). Thus, the PRL-induced activation of Vav-associated GEF activity appears to precede the internalization of this signaling factor. There are multiple mechanisms through which Vav could translocate into the nucleus. Vav has two putative nuclear translocation signal sequences (43), and these may be sufficient to mediate its internalization. Alternatively, Vav has recently been found to interact with heterogeneous ribonucleoprotein K (66) , a protein that is found within both the nucleus and cytoplasm of cells. Within the nucleus, Vav could interact with numerous proteins via its leucine-rich, acidic-rich, SH2, and SH3 domains. It is possible that through these domains, and/or its interaction with heterogeneous ribonucleoprotein K, Vav could influence RNA transcription or maturation. Given its multifunctional domains and proximal and potentially distal signaling functions, Vav may therefore serve to coordinate multiple events during PRLr signal transduction necessary for cell proliferation and the clonal expansion of T-cells during an immune response.


FOOTNOTES

*
This work was supported in part by Grants AI-33510 (to C. V. C.) and CA-54384 and CA-36896 (to A. M. G.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 509 BRB1, Dept. of Pathology and Laboratory Medicine, 422 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-0734; Fax: 215-349-5910.

The abbreviations used are: PRL, prolactin; PRLr, prolactin receptor; ODN, oligodeoxynucleotide; PBS, phosphate-buffered saline; GEF, guanine nucleotide exchange factor; DAG, diacylglycerol; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Drs. Erich Gulbins and Amnon Altman for their advice in establishing the solid-phase GEF assay and Dr. G. Dreyfuss for his helpful comments regarding heterogenous ribonucleoprotein K.

Note Added in Proof-Zmuidzinas et al, (81) have also reported that vav embryonic stem cells are capable of generating erythroid, myeloid, and mast cell lineages.


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