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From the
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
The pleiotropic actions of prolactin (PRL)
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
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.
Immunoblot analysis was performed as described previously
(19) using the above immunoprecipitates or 5
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
[
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.
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).
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
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
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
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
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.
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
Volume 270,
Number 22,
Issue of June 2, pp. 13246-13253, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, 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.
()
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.
. 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) .
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 Na
VO,
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.
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).
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.
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.
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.
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) .
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.
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.
, 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.
embryonic stem cells are capable of generating erythroid,
myeloid, and mast cell lineages.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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