Phosphorylation of cbl after Stimulation of Nb2 Cells with Prolactin and Its Association with Phosphatidylinositol 3-Kinase
Seija Hunter,
Becky L. Koch and
Steven M. Anderson
University of Colorado Health Sciences Center Department of
Pathology Denver, Colorado 80262
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ABSTRACT
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Stimulation of Nb2 cells with PRL results in the
rapid phosphorylation of a 120-kDa protein identified as the adapter
protein cbl on tyrosine residues. Maximal phosphorylation
of cbl occurs at 20 min after PRL stimulation and declines
thereafter. Stimulation with as little as 5 nM
PRL resulted in the phosphorylation of cbl;
increasing the concentration of PRL to 100 nM
had only a minimal effect upon the phosphorylation of cbl.
The cbl protein appears to be constitutively associated
with grb2 and the p85 subunit of phosphatidylinositol 3-kinase (PI
3-kinase). The constitutive association of cbl with the p85
subunit of PI 3-kinase was observed in Nb2 cells as well as in 32Dcl3
cells transfected with either the rat Nb2 (intermediate) form of the
PRL receptor or the long form of the human PRL receptor. A glutathione
S-transferase fusion protein encoding the SH3 domain of the
p85 subunit of PI 3-kinase bound to cbl in lysates of both
unstimulated and PRL-stimulated Nb2 cells; however, neither of the SH2
domains of p85 bound to cbl under the same conditions. PRL
stimulation increased the cbl-associated PI kinase
activity. The majority of PI kinase activity appeared to be
cbl-associated after PRL stimulation. These results suggest
that cbl may function as an adapter protein in PRL-mediated
signaling events and regulate activation of PI 3-kinase. Our model
suggests that the p85 subunit of PI 3-kinase is constitutively
associated with cbl through binding of the p85 SH3 domain
to a proline-rich sequence in cbl. After the tyrosine
phosphorylation of cbl, an SH2 domain(s) of p85 binds to a
specific phosphorylation site(s) in cbl, leading to the
activation of PI 3-kinase.
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INTRODUCTION
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Cbl is the cellular homolog of the oncogene present in
the Cas-NS-1 retrovirus, which induces B cell lymphomas (1, 2).
Sequence analysis of the cbl cDNA revealed that the protein
contains 913 amino acids, a putative nuclear localization sequence in
its N-terminal region, a RING finger motif typical of DNA-binding
proteins, and several proline-rich sequences in its C-terminal half
that may serve as SH3-binding sites (1, 3). There is no evidence,
however, that cbl is present in the nucleus or that it binds
to DNA. Amino acid sequence analysis of cbl does not predict
the presence of any catalytic domain of a known signaling molecule or
enzyme. The fact that cbl becomes tyrosine-phosphorylated
after activation of a variety of receptors, however, does suggest that
it may serve as an adapter molecule in a manner analogous to that of
insulin-regulated substrate-1 in signal transduction mediated by the
insulin receptor.
Numerous recent studies have demonstrated that cbl becomes
tyrosine-phosphorylated after stimulation of a wide variety of
receptors including: the T cell receptor (4, 5, 6), the B cell antigen
receptor (7, 8), the Fc receptor (9, 10), the epidermal growth factor
receptor (11, 12, 13, 14), the colony-stimulating factor-1 receptor (15), the
erythropoietin receptor (16), the receptor for granulocyte-macrophage
colony-stimulating factor (16), and the receptor for interleukin-3
(17). The cbl protein is also phosphorylated in cells
expressing activated oncogenes such as v-abl or BCR-ABL
(18, 19, 20). In these studies, cbl was observed to be
associated with a variety of proteins by either coimmunoprecipitation
studies or binding to bacterial fusion proteins. Association of the p85
subunit of phosphatidylinositol 3-kinase (PI 3-kinase) with
tyrosine-phosphorylated cbl has been described to occur in a
phosphotyrosine-dependent manner via the SH2 domains of p85, and in a
phosphotyrosine-independent manner involving the SH3 domain of p85 (4, 6, 11, 19). It is likely, however, that both domains are important in
the interaction of cbl with p85 leading to activation of PI 3-kinase.
cbl has also been observed to interact with the SH3 domain
of lyn (9, 10), with the SH2 and SH3 domains of
fyn (6, 9), with the SH2 domain of crk (19), and
with the SH3 domain of grb2 in a constitutive manner (5, 14, 16).
Interactions involving the SH2 domains are largely thought to occur in
a phosphotyrosine-dependent manner, although one study has suggested
that this may not be the case (17). The association of cbl
with the p85 subunit of PI 3-kinase suggests that phosphorylation of
cbl may regulate activation of PI 3-kinase. The constitutive
association of the p85 subunit of PI 3-kinase with cbl,
mediated by the binding of the p85 SH3 domain to a proline-rich
sequence in cbl, would allow the rapid activation of PI
3-kinase after the binding of the SH2 domain(s) of p85 bind to a
specific phosphorylation site(s) in cbl. Supporting this
hypothesis is the observation that activated PI 3-kinase activity is
associated with cbl in stimulated T- and B lymphocytes (4, 6, 7) as well as in interleukin-3-stimulated myeloid cells (17).
The PRL receptor is a member of the cytokine receptor superfamily
(21, 22, 23, 24, 25). Stimulation of the receptors for erythropoietin,
granulocyte-macrophage colony-stimulating factor, and
interleukin-3, three other cytokine receptor family members, results in
tyrosine phosphorylation of cbl, suggesting that PRL
stimulation might also result in the phosphorylation of cbl.
In this manuscript we demonstrate that cbl is phosphorylated
after PRL stimulation of Nb2 cells as well as in 32Dcl3 cells
transfected with the long form of the human PRLR or the Nb2
(intermediate) form of the rat PRLR. Cbl is constitutively
associated with the SH2-containing adapter protein grb2 and the p85
subunit of PI 3-kinase. Quantitative studies suggest that the majority
of PI kinase activity is associated with cbl after PRL
stimulation of Nb2 cells. This suggests that cbl may
function as an adapter or docking molecule in a manner similar to that
observed with insulin-regulated substrate-1 in insulin receptor
signaling. After the ligand-induced tyrosine phosphorylation of
cbl, multiple signaling molecules could bind to specific
phosphorylation sites in cbl, presumably through the binding
of their SH2 domains to specific phosphorylation sites, leading to the
activation of these signaling molecules.
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RESULTS
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Cbl Is Rapidly Phosphorylated on Tyrosine Residues
after PRL Stimulation
To determine whether cbl was phosphorylated on tyrosine
residues after PRL stimulation, Nb2 cells were stimulated with 10
nM PRL for 10 min and immunoprecipitated with either
antiphosphotyrosine or anti-cbl antibodies. The results
indicate that cbl becomes tyrosine phosphorylated after
stimulation of Nb2 cells with PRL (Fig. 1
). The major
tyrosine-phosphorylated protein present in antiphosphotyrosine
immunoprecipitates of PRL-stimulated Nb2 cells has a molecular mass of
130,000 and corresponds to JAK2 (26, 27, 28). By densitometry there is a
6-fold increase in the phosphorylation of JAK2 after PRL stimulation
(Fig. 1
, first and second lanes). Immunoblotting with
anti-cbl antibody indicates that PRL stimulation did not
change the amount of cbl present in the cell (Fig. 1
, seventh and eighth lanes). The anti-cbl immunoblot also
indicates that only a small amount of the cbl protein
appears in the antiphosphotyrosine immunoprecipitate of PRL-stimulated
Nb2 cells (Fig. 1
, sixth and eighth lanes). Densitometric measurements
indicate that only about 5% of the cbl in the
anti-cbl immunoprecipitate could be detected in the
anti-phosphotyrosine immunoprecipitate of PRL-stimulated Nb2 cells
(Fig. 1
, lane 6 vs. lane 8). Only 25% of the
anti-cbl immunoprecipitate was loaded in lanes 7 and 8 of
Fig. 1
to allow for a more accurate quantification of the amounts of
cbl in the anti-cbl and anti-phosphotyrosine
immunoprecipitates; however, the figure of 5% takes into account this
difference in loading. This result was consistent in four different
studies with Nb2 cells. From the study shown in Fig. 1
, it is clear
that cbl does not comigrate with any of the major
tyrosine-phosphorylated proteins present in the antiphosphotyrosine
immunoprecipitate (Fig. 1
, lanes 14). Therefore, the phosphorylation
of cbl in PRL-stimulated Nb2 cells would not have been
anticipated by an examination of the sizes of tyrosine-phosphorylated
proteins in anti-phosphotyrosine immunoblots prepared from these cells.
Furthermore, there are no detectable tyrosine-phosphorylated proteins
that coprecipitate with cbl (Fig. 1
, lane 4). Three major
tyrosine-phosphorylated proteins are detected in the 4G10
immunoprecipitate with mol wts of 130,000, 66,000, and 42,000 (Fig. 1
).
Based upon immunoblot studies with anti-JAK2 antiserum, we believe that
130 kDa protein to be JAK2 (data not shown). The 66 and 42 kDa proteins
may correspond to shc and mitogen-activated protein kinase,
respectively.

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Figure 1. Cbl Is Phosphorylated on Tyrosine
Residues after Stimulation with PRL
Nb2 cells were cultured overnight in RPMI 1640 with 5%
charcoal-stripped serum, then stimulated with 10 nM rat PRL
for 0 (lanes marked -) or 10 min (lanes marked +). Cells were lysed
with modified RIPA and immunoprecipitated with either
anti-phosphotyrosine monoclonal antibody 4G10 (lanes 1, 2, 5, and 6) or
anti-cbl antibody (lanes 3, 4, 7, and 8). Each
immunoprecipitate contained lysate prepared from 2 x
107 cells. After display of the immunoprecipitated proteins
on a 7% SDS polyacrylamide gel, the proteins were transferred to
Immobilon and immunoblotted with either antiphosphotyrosine antibody
4G10 (lanes 14) or anti-cbl antibody (lanes 58). The
position of prestained molecular mass markers are indicated on the
left side of the figure, and the position of
cbl is indicated in the center of the
figure.
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The kinetics of cbl phosphorylation were also examined by
stimulating Nb2 cells with 10 nM PRL for 0120 min.
Maximal phosphorylation was detected approximately 20 min after PRL
stimulation and decreased after this time (Fig. 2A
, top panel). The phosphorylation of cbl was
significantly decreased by 1 h, and no increased phosphorylation
could be detected by 2 h (Fig. 2
). No coimmunoprecipitating
tyrosine-phosphorylated proteins were noted in the anti-cbl
immunoprecipitates at any of the times examined (data not shown). The
time course of cbl phosphorylation described in Fig. 2
was
consistently observed in four different studies with Nb2 cells.
Immunoblotting with anti-cbl antiserum indicated that the
amount of cbl protein did not change over the time period
examined (Fig. 2A
, bottom panel).

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Figure 2. Kinetics and Dose-Response of cbl
Phosphorylation
A, Nb2 cells were cultured overnight as described in Fig. 1 , then
stimulated for 0120 min with 10 nM rat PRL. Cells were
lysed in EB and immunoprecipitated with anti-cbl
antibody. The time of PRL stimulation, in minutes, is indicated at the
top of each lane. Immunoblotting was with
antiphosphotyrosine antibody 4G10 (top panel). The
immunoblot was then stripped as described (17 ) and reprobed with
anti-cbl antibody to indicate the amount of
cbl protein in each immunoprecipitate. B, Nb2 cells were
cultured overnight in RPMI 1640 with 5% charcoal-stripped serum, then
stimulated with 0200 ng/ml rat PRL for 10 min. Cells were lysed in EB
and immunoprecipitated with anti-cbl antibody, after
which extent of phosphorylation was examined by antiphosphotyrosine
immunoblotting. The immunoblot was reprobed with
anti-cbl antibody to indicate the amount of
cbl in each immunoprecipitate. The concentration of rat
PRL used to stimulate the Nb2 cells, in nanograms/ml is indicated at
the top of each lane.
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The effect of increasing concentrations of PRL upon the phosphorylation
of cbl was also examined. Nb2 cells were stimulated with
0200 ng/ml PRL for 15 min before lysis and analysis of cbl
phosphorylation by antiphosphotyrosine immunoblotting (Fig. 2B
).
Although a low level of cbl phosphorylation could be
observed in unstimulated cells (Fig. 2B
, lane 1), PRL stimulated the
tyrosine-phosphorylation of cbl (Fig. 2B
, lanes 56). The
amount of tyrosine phosphorylation remained fairly constant over a
range of 10100 ng/ml, and in some studies there appears to be a
slight decrease in the extent of cbl phosphorylation in
cells stimulated with 200 ng/ml PRL. There was no significant change in
the level of cbl protein after PRL stimulation over this
dose range (Fig. 2B
, bottom panel). This result was
consistently observed in four independent studies of Nb2 cells.
Association of cbl with the SH2/SH3-Containing Adapter
Protein grb2
As noted in the introduction above, cbl has been noted
to associate with numerous proteins, including src-like
tyrosine kinases, the SH2-containing adapter protein grb2, and the p85
subunit of PI 3-kinase (4, 5, 6, 9, 10, 11, 16, 19). The ability of
cbl to coimmunoprecipitate with some of these proteins was
examined in lysates of both unstimulated and PRL-stimulated Nb2
cells.
The adapter protein grb2 couples the GTP exchange protein SOS to
activated growth factor receptors, leading to the activation of SOS and
subsequent activation of ras (29, 30, 31, 32, 33). We observed the
constitutive association of grb2 with cbl (Fig. 3
). Unstimulated and stimulated Nb2 cells were
immunoprecipitated with either anti-grb2 or anti-cbl
antibodies, and the immunoprecipitates were subjected to immunoblotting
with either anti-phosphotyrosine, anti-cbl, or anti-grb2
antibodies. Antiphosphotyrosine immunoblotting confirmed the
PRL-stimulated phosphorylation of cbl (Fig. 3
, lanes 3 and
4), although there was a detectable amount of phosphorylated
cbl in the unstimulated Nb2 cells. No cbl protein
was detected in anti-grb2 immunoprecipitates immunoblotted with
anti-cbl antibody (Fig. 3
, lanes 5 and 6); however, grb2
protein was present in anti-cbl immunoprecipitates from both
unstimulated and stimulated cells (Fig. 3
, lanes 11 and 12). Note that
the gels used for analysis of grb2 were 12% gels, whereas those probed
with anti-phosphotyrosine or anti-cbl were 7% gels. There was no
difference in the amount of grb2 protein present in anti-cbl
immunoprecipitates from unstimulated vs. PRL-stimulated Nb2
cells. By densitometry, the amount of grb2 protein detected in the
anti-cbl immunoprecipitates represented only 12% (range of
914% was observed in three different studies of Nb2 cells) of the
amount observed in anti-grb2 immunoprecipitates (Fig. 3
, compare lanes
912). When lanes 912 of Fig. 3
were immunoblotted with the
anti-cbl antibody, no cross-reaction of the antiserum with
grb2 was detected. This suggests that the anti-cbl antibody
does not nonspecifically react with grb2. These results suggest that a
small portion of cbl is constitutively associated with grb2,
and the amount of this complex does not change after PRL stimulation.
We do not understand why we were unable to detect cbl in the
anti-grb2 immunoprecipitates by immunoblotting; however, this may
reflect the small amount of cbl associated with grb2. There
was no evidence that grb2 became tyrosine-phosphorylated after PRL
stimulation (data not shown). Anti-grb2 immunoprecipitates also
contained a phosphotyrosine-containing protein larger than
cbl, with a mol wt of approximately 130,000140,000 (Fig. 3
, lanes 1 and 2). This protein remains to be identified.

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Figure 3. Constitutive Association of cbl with
grb2
Nb2 cells were cultured as described in Fig. 1 , then stimulated for 0
(lanes marked -) or 10 (lanes marked +) minutes with 10 nM
PRL. The cells were lysed with EB and immunoprecipitated with either
anti-grb2 (lanes 1, 2, 5, 6, 9, and 10) or anti-cbl
antibody (lanes 3, 4, 7, 8, 11, and 12). The immunoprecipitates were
resolved on 7% (left and center panels)
or 12% (right panel) SDS polyacrylamide gels and
transferred to Immobilon, and the immunoblots were probed with
antiphosphotyrosine antibody 4G10 (lanes 14), anti-cbl
antibody (lanes 58), or anti-grb2 antibody (lanes 912). The
positions of prestained molecular mass markers are indicated. The
position of cbl is indicated on the left,
and the position of grb2 is indicated on the right.
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Constitutive Association of the p85 Subunit of PI 3-Kinase with
cbl
To further examine the role of cbl as an adapter
protein, the association of cbl with PI 3-kinase was also
examined. Unstimulated and stimulated cells were immunoprecipitated
with antiphosphotyrosine, anti-cbl, or an anti-p85 subunit
antibody that immunoprecipitates both the
- and ß-isoforms of p85.
Immunoblotting with an anti-p85 subunit antibody detected p85 in both
the anti-cbl and anti-p85 immunoprecipitates of both
unstimulated and PRL-stimulated Nb2 cells (Fig. 4A
, lanes 4 and 6). The p85 protein was also detected in the
anti-phosphotyrosine immunoprecipitate of unstimulated cells; however,
there was a 4-fold increase in the amount of p85 in the
antiphosphotyrosine immunoprecipitate after PRL stimulation of Nb2
cells (Fig. 4
, lanes 1 and 2). Densitometric analysis of the p85 bands
in Fig. 4
indicate that the anti-cbl immunoprecipitates
contain 1020% of the protein present in the anti-p85
immunoprecipitate obtained with the monoclonal anti-p85 antibody used
in this study. The anti-cbl immunoprecipitates were also
observed to contain the 110-kDa catalytic subunit of PI 3-kinase as
determined by immunoblotting with a monoclonal antibody directed
against this protein (data not shown). The antiphosphotyrosine
immunoprecipitate of PRL-stimulated cells contained 2030% of the p85
subunit present in the anti-p85 immunoprecipitate. These numbers
reflect the range of values observed in four different studies with Nb2
cells. Although p85 was present in the antiphosphotyrosine
immunoprecipitate, we could not detect the tyrosine phosphorylation of
p85 (Fig. 1
and data not shown). Reprobing the immunoblot with
anti-cbl antibody revealed the present of cbl
protein in the anti-cbl immunoprecipitates but not the
anti-p85 immunoprecipitates (Fig. 4A
, lanes 3 and 4, bottom
panel). Longer exposures of the immunoblot, which revealed the presence
of cbl in the antiphosphotyrosine immunoprecipitate, failed
to demonstrate the presence of cbl in the anti-p85
immunoprecipitate (data not shown). Stimulation of Nb2 cells with up to
200 ng/ml PRL did not change the amount of p85 associated with
cbl (data not shown).

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Figure 4. Association of cbl with the p85
Subunit of PI 3-Kinase
Nb2 or 32D/hPRLR cells were cultured as described in Fig. 1 , then
stimulated for 0 or 10 min with 10 nM PRL. The cells were
lysed with EB, immunoprecipitated with antiphosphotyrosine antibody
4G10 (lanes 1 and 2), anti-cbl (lanes 3 and 4), or
anti-p85 subunit of PI 3-kinase (Transduction Laboratory No. P13030),
and the immunoprecipitates were resolved on a 7% SDS polyacrylamide
gel. The top panel was probed with anti-p85 antiserum
(Transduction Laboratories Catalog No. P13030), and the
bottom panel was probed with anti-cbl
antiserum.
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The results obtained with Nb2 were confirmed by conducting the same
study on 32Dcl3 cells transfected with either the long form of the
human PRL receptor (hPRLR) or the Nb2 (intermediate) form of the rat
PRLR. The data in Fig. 4
, lanes 712, show the results obtained with
the 32D/hPRLR cells. Consistent with the results obtained with Nb2
cells, the constitutive association of p85 with cbl was
obtained (Fig. 4
, lanes 9 and 10). Compared with the results observed
with Nb2 cells, a higher percentage of p85 appeared to be
cbl-associated; approximately 5070% of the amount of p85
present in the anti-p85 immunoprecipitate was present in the
anti-cbl immunoprecipitate. Also consistent with the Nb2
cell study, p85 was detected in the antiphosphotyrosine
immunoprecipitate of both unstimulated and PRL-stimulated cells, and
there was a 1.5- to 2-fold increase in the amount of p85 present in the
antiphosphotyrosine immunoprecipitates of lysates of PRL-stimulated
32D/hPRLR cells compared to the amount in lysates of unstimulated
cells. Consistent results were observed in three independent
studies.
Under the conditions used, the anti-cbl antibody
precipitated all of the cbl protein present in the cell
extract; reimmunoprecipitation of the cell lysate with a second aliquot
of anti-cbl antibody did not precipitate any additional
protein (data not shown). Similar studies conducted with the anti-p85
antibody suggested that greater than 90% of the p85 was removed from
the cell lysate during the first immunoprecipitation (data not shown).
When a monoclonal antibody specific for the ß-isoform of p85 was
used, no association of p85 with cbl was noted (data not
shown). This is consistent with results reported elsewhere with the T
cell receptor (4). When cells were lysed with RIPA [150 mM
NaCl, 50 mM Tris (pH 7.4), 2 mM EGTA, 1%
Triton X-100, 0.25% sodium deoxycholate, 1 mM sodium
orthovanadate] instead of EB [50 mM NaCl, 10
mM Tris (pH 7.4), 5 mM EDTA, 50 mM
NaF, 1% Triton X-100, 1 mM sodium orthovanadate], no
association of p85 with cbl was detected, indicating that
this complex of proteins is very sensitive to the presence of
detergents (data not shown). These results are consistent with those
previously described for cbl after stimulation of myeloid
cells with interleukin-3 (17).
The p85 subunit of PI 3-kinase contains an SH3 domain and two SH2
domains that regulate the interaction of p85 with activated growth
factor receptors and other signaling molecules (34, 35). SH2 domains
mediate binding to phosphorylated tyrosine residues in a
sequence-specific context (36, 37), while SH3 domains bind to
proline-rich sequences (38). The constitutive association of p85 with
cbl suggested that this interaction was mediated by SH3
domains. This was examined with a binding assay in which the amount of
cbl that bound to GST fusion proteins encoding the SH3
domain and each of the two SH2 domains (GST-N-SH2 and GST-C-SH2, the
N-terminal and C-terminal SH2 domains of p85, respectively), was
examined. As can be seen in Fig. 5
, only the SH3 domain
of p85 was able to bind to cbl in lysates of either
unstimulated or PRL-stimulated Nb2 cells (Fig. 5
, lanes 7 and 8). No
cbl was observed to bind to GST alone, or to either the N-
or C-terminal SH2 domains. Stimulation of Nb2 cells with PRL did not
alter the amount of cbl that bound to the SH3 domain of p85.
These results are consistent with those described in Fig. 4
and suggest
a constitutive association of p85 with cbl.

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Figure 5. The SH3 Domain of p85 Binds to cbl
Unstimulated Nb2 cells, lysed in EB, were prepared as described in Fig. 1 . GST, GST-N-SH2, GST-C-SH2, or GST-SH3 was added to the cell lysates
at a final concentration of 2 µM. After a 1 h
incubation at 4 C on a rocking platform, 40 µl of a 50% suspension
of glutathione agarose beads were added for 20 min. The bound protein
complexes were washed three times with EB and solubilized in 30 µl of
2x SDS gel sample buffer, and the protein was resolved by
electrophoresis on a 7% SDS gel. Immunoblotting was with
anti-cbl antibody. The fusion protein used is indicated
at the top of each lane, and the position of
cbl is indicated on the right side of the
panel.
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To demonstrate that cbl immunoprecipitates contained PI
kinase activity in addition to the p85 subunit of PI 3-kinase, the PI
kinase assay was used to demonstrate the presence of enzymatic activity
in the various immunoprecipitates. Nb2 cells were stimulated with PRL
for 020 min and immunoprecipitated with either anti-cbl,
anti-p85 subunit, or a nonimmune rabbit serum. A very small amount of
PI kinase activity was detected in the nonimmune serum control;
however, the amount of activity did not increase after PRL stimulation
(Fig. 6
, lanes 9 and 10). The anti-p85 subunit antibody
detected a small amount of PI kinase activity in unstimulated cells,
and PRL stimulation induced a 4-fold increase in PI kinase
activity at 5 min (Fig. 6
, lanes 58). Anti-cbl
immunoprecipitates contained PI kinase activity that increased after
PRL stimulation (Fig. 6
, lanes 14). Increased PI kinase activity was
detectable as early as 2 min after PRL stimulation and was still
evident 20 min later. Densitometric analysis indicated that there was a
10-fold increase in PI kinase activity at 2 min compared to
unstimulated cells. The difference in the increase of PI kinase
activity at 2 min between the anti-cbl and anti-p85
immunoprecipitates may reflect the increased basal activity present in
the anti-p85 immunoprecipitate compared with the anti-cbl
immunoprecipitate. In addition to the expected product of
phosphatidylinositol phosphate (PIP), a second spot that migrated
halfway between the origin and PIP was observed (indicated by the
arrow on the right side of Fig. 6
). We suspect
that this spot may correspond to an oxidation product of PIP as the
amount of it increases directly in proportion to the amount of PIP.
Another spot of unknown identity was observed only in the reactions of
anti-p85 immunoprecipitates. A comparison of the amount of PI kinase
activity in anti-cbl immunoprecipitates at 5 and 20 min with
the amount in anti-p85 immunoprecipitates at the same time points
indicates that most, if not all, of the PI kinase activity is
cbl-associated after PRL stimulation. PI kinase activity was
observed in anti-phosphotyrosine immunoprecipitates; however, the
amount of activity did not increase after PRL stimulation (data not
shown).

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Figure 6. PRL-Induced Association of cbl with PI
3-Kinase Activity
Cells were stimulated for 020 min with 10 nM rat PRL,
lysed with EB, and immunoprecipitated with either
anti-cbl (lanes marked cbl), anti-p85
antibody (Transduction Laboratory No. P13030, lanes marked p85), or
nonimmune rabbit serum (lanes marked NIS). PI Kinase assay was
conducted as described in Materials and Methods. The
origin and the position of the reaction product, PIP, are indicated on
the right side of the figure. Lane numbers are indicated
across the top of the figure.
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DISCUSSION
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In this manuscript we have demonstrated the phosphorylation of
cbl after stimulation of Nb2 cells with PRL. The
phosphorylation of this protein was not anticipated because we have not
previously detected a protein of this size in our antiphosphotyrosine
immunoblots of either total cell lysates of PRL-stimulated cells or
antiphosphotyrosine immunoprecipitates of PRL-stimulated cells.
Tyrosine-phosphorylated JAK2 migrates with a slower electrophoretic
mobility and was readily separable from cbl. In the absence
of PRL stimulation, cbl was associated with the
SH2-containing adapter protein grb2, and the amount of grb2 complexed
with cbl did not change after PRL stimulation. The
constitutive association of grb2 with cbl is consistent with
our previous results obtained in studies on the phosphorylation of
cbl after stimulation of myeloid cells with interleukin-3
(17).
These studies have also described the constitutive association of the
p85 subunit of PI 3-kinase with cbl. This association was
detected in Nb2 cells and 32Dcl3 cells transfected with either the Nb2
form of the rat PRLR or the long form of the human PRLR. The percent of
p85 associated with cbl varied from 1070% of the amount
of p85 present in an anti-p85 immunoprecipitate, depending upon the
cell line examined. Binding studies with GST fusion proteins indicate
that the SH3 domain of p85 binds to cbl, which would explain
the constitutive association between these two proteins. This result is
consistent with studies describing the interaction between
cbl and p85 as mediated by the SH3 domain of p85 (5, 6, 14, 16). The association of cbl with the p85 subunit of PI
3-kinase has been observed in studies of the T cell receptor, the B
cell receptor, the Fc receptor, the EGF receptor, the interleukin-3
receptor, and the BCR-ABL oncogene (6, 7, 11, 17, 19). The fact that
multiple receptor systems appear to stimulate association of p85 and
cbl suggests that cbl may have a universal role
as an adapter protein in coupling the activation of PI 3-kinase to
activated growth factor receptors and oncogenes. The binding of the p85
subunit of PI 3-kinase to the receptor for platelet-derived growth
factor results in the tyrosine phosphorylation of p85 and the
activation of PI 3-kinase. We have not observed tyrosine
phosphorylation of p85 after PRL stimulation, suggesting that this is
not required to occur for activation of PI 3-kinase. Several studies
have indicated that the binding of p85 to cbl is mediated by
the binding of the SH2 domain(s) of p85 to tyrosine-phosphorylated
cbl (6, 7, 11, 19). Two major cbl phosphorylation
sites have been mapped using cbl purified from
v-abl-transformed cells (39). Both of these phosphorylation
sites fall into the pYXXP consensus class and would appear
to be binding sites for crk and crkL (36, 37, 39). The p85 subunit of PI 3-kinase binds to cbl in
BCR/ABL-and v-abl-transformed cells (19), suggesting that
either p85 binds to one of these phosphorylation sites, or that
additional sites of phosphorylation remain to be identified. It will be
interesting to determine whether cbl is phosphorylated at
these two sites in PRL-stimulated cells, and whether cbl
bearing tyrosine to phenylalanine mutations at these two sites will
function as a dominant-negative mutant in blocking PRL-stimulated
activation of PI 3-kinase. It is reasonable to expect that both the SH2
and SH3 domains of p85 are involved in the interaction of p85 with
cbl. Our model described below suggests the SH3 and SH2
domains play different roles in the interaction of p85 with
cbl and that both interactions are important.
While this work was in progress, another group described the
PRL-stimulated activation of PI 3-kinase in Nb2 cells (40). PI kinase
activity was observed in antiphosphotyrosine immunoprecipitates of Nb2
cells stimulated with 100 ng/ml ovine PRL; however, a substantial
amount of PI kinase activity was observed in immunoprecipitates of
unstimulated cells. No activation was observed at lower concentrations
of PRL. A modest increase in PI kinase activity was detected after 5
min; however, maximal activity was not observed until 2040 min of
stimulation. In contrast to our results, these investigators also
described the tyrosine phosphorylation of p85 after PRL stimulation
(40). We have consistently failed to observe the tyrosine
phosphorylation of p85, even when concentrations of rat PRL as high as
200 nM were used to stimulate the cells. We cannot account
for the difference between our results and those of Al-Sakkaf et
al. (40) since Nb2 cells were used in both studies. Belanga
et al. (41) have also recently described the phosphorylation
of the p85 subunit of PI 3-kinase in 293 cells transfected with an
epitope-tagged version of the rat PRLR after stimulation of these cells
with rat PRL (41). We have not observed the tyrosine phosphorylation of
p85 in either Nb2 cells or 32Dcl3 cells transfected with either the
hPRLR or the rat Nb2 form of the PRLR. We suspect that the
phosphorylation of p85 described by Belanga et al. may be
specific to the 293 cells used in their studies.
The model that we would favor is that grb2-cbl complexes,
bound by the SH3 domain of grb2 binding to cbl, exist in
unstimulated cells. A small amount of the cbl protein is
also complexed with p85; however, it is not clear whether the
cbl-p85 complexes also contain grb2. After binding of PRL to
the receptor, JAK2 becomes activated and phosphorylates the receptor at
multiple sites. The grb2-cbl complex may bind to one of
these phosphorylation sites, resulting in the phosphorylation of
cbl by JAK2 or perhaps by fyn. After the tyrosine
phosphorylation of cbl, one of the SH2 domains of the p85
subunit of PI 3-kinase presumably binds to a specific phosphotyrosine
residue in cbl, resulting in the activation of PI 3-kinase.
Our model predicts that both phosphotyrosine-independent and
phosphotyrosine-dependent interactions between p85 and cbl,
mediated by the SH3 and SH2 domains of p85, respectively, are critical
in the regulation of PI 3-kinase by cbl. Alternatively, PI
3-kinase could be activated by the binding of the fyn SH3
domain to a proline-rich sequence in p85 as has been described by
Pleiman et al. (42); however, this would not be consistent
with the association of PI kinase activity with cbl. This
model leads to several specific predictions: 1) that cbl
associates with the PRLR via a grb2 SH2 domain-binding site; 2) that
the SH3 domain of p85 binds to cbl; 3) that cbl
becomes tyrosine phosphorylated after PRL stimulation; 4) after
tyrosine phosphorylation of cbl, one of the SH2 domains of
p85 binds to this phosphorylation site, resulting in the catalytic
activation of PI 3-kinase; and 5) that fyn might be
responsible for the phosphorylation of cbl. Each of
these predictions can be tested by mapping the phosphorylation
sites in cbl and mutating these phosphorylation sites to
phenylalanine residues that cannot be phosphorylated. These tyrosine to
phenylalanine point mutants would be expected to block activation of PI
3-kinase and perhaps block PRL-induced mitogenesis.
 |
MATERIALS AND METHODS
|
---|
Cells and Cell Culture
The Nb2 cell line was obtained from D. Li-yuan Yu-Lee (Baylor
College of Medicine, Houston, TX) through the courtesy of Dr. Peter
Gout. The cells were maintained in RPMI 1640 media supplemented with
10% FCS, 10% horse serum, 1 mM L-glutamine,
100 U/ml penicillin, and 100 µg/ml streptomycin. All media components
were obtained from GIBCO/BRL (Gaithersburg, MD). Charcoal-stripped FCS
was obtained from HyClone (Logan, UT). Rat PRL, lots AFP-6452B and
AFP-3697A, was obtained from the National Hormone and Pituitary Program
(Rockville, MD).
Immunoprecipitation and Immunoblotting
Immunoprecipitation was as previously described (43). Cells were
lysed in either RIPA or with EB. Both lysis buffers were supplemented
with 100 U/ml kallikrein inhibitor (CalBiochem. La Jolla, CA). Rabbit
anti-p85 subunit of PI 3-kinase and antiphosphotyrosine monoclonal
antibody 4G10 coupled to agarose beads were obtained from Upstate
Biotechnology, Inc. (Lake Placid, NY). Antibodies to cbl,
grb2, and the p110 subunit of PI 3-kinase were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). An additional antibody to the p85
subunit of PI 3-kinase was obtained from Transduction Labs (Lexington,
KY). Immunoprecipitated proteins were resolved on SDS polyacrylamide
gels and electro-transferred to Immobilon membrane (Millipore, Bedford,
MA). Immunoblotting was conducted as described (17, 43) using the
Enhanced Chemiluminescence Lighting (ECL) system according to
manufacturers recommendations (Amersham, Arlington Heights, IL).
PI Kinase Assay
Reactions were a modification of previously described protocol
(17, 44, 45). The immunoprecipitated proteins were washed three times
with RIPA, twice with PAN [20 mM
piperazine-N,N'-bis[2-ethanesulfonic
acid] (pH 7.0), 20 µl/ml aprotinin, 100 mM NaCl], and
resuspended in 50 µl PAN. A 5-µl aliquot of each sample was removed
and placed in a new tube and 1 µl of 2 mg/ml PI in 4.5 mM
EGTA, 90% dimethylsulfoxide was added to each reaction. The tubes were
incubated at room temperature for 10 min before addition of the
reaction mixture containing ATP and incubation at 30 C for 15 min. The
final reaction mixture contained 20 mM HEPES (pH 7.4), 5
mM MgCl2, 0.45 mM EGTA, 10
µM ATP (5 µCi
-[32P]ATP), and 0.2
mg/ml PI. Reactions were terminated by the addition of 0.1 ml 1
M HCl and extracted with 0.2 ml CHCl3-methanol
(1:1). After the aqueous phase was discarded, the organic phase was
reextracted with 1 M HCl-methanol (1:1), and the organic
phase was dried in a Savant SpeedVac. The samples were dissolved in 10
µl CHCl3-methanol and spotted in Silica gel 60 plates (E.
Merck) that had been impregnated with sodium tartrate, and the plate
was developed in CHCl3-methanol-4 M
NH4OH (9:7:2). After chromatography, the plate was allowed
to dry before autoradiography.
GST Fusion Protein and Binding Assays
GST fusion proteins containing the amino-terminal SH2 domain,
the carboxyl-terminal SH2 domain, and the SH3 domain of p85 were kindly
provided by L. C. Cantley and B. Duckworth (Beth Isreal Hospital,
Boston, MA) (46). GST fusion proteins were purified as previously
described (17). Binding assays were conducted using lysates prepared in
RIPA from equal numbers of cells (2 x 107 cells per
binding assay) in 1 ml lysis buffer. Two nanomoles of the indicated GST
fusion protein were added to each lysate to yield a final concentration
of 2 µM. The remainder of the binding assay was conducted
as described previously (17).
Densitometry
Gels were scanned with a Hewlett Packard ScanJet IIcx/T scanner
(Palo Alto, CA) and images analyzed with Sigma Gel software (Sigma
Chemical Co, St. Louis, MO). Care was taken not to overexpose the film
such that the densitometer measurements were made in the linear range
of the film.
 |
ACKNOWLEDGMENTS
|
---|
Richard Klinghoffer and Andrius Kazlauskas kindly provided
assistance with PI kinase assays. GST fusion proteins encoding the SH3
and SH2 domains of p85 were kindly provided by Brian Duckworth and
Lewis Cantley. The authors also acknowledge the services of the
University of Colorado Cancer Center DNA Sequencing Core Facility in
support of this research. We thank Elizabeth Burton and Drs. Mary
Reyland and Arthur Gutierrez-Hartmann for their comments on this
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Steven M. Anderson, Department of Pathology, Box B-216, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, Colorado 80262.
This work was supported by NIH Grant DK-48879. The University of
Colorado Cancer Center is supported by NIH Grant CA-46934.
Received for publication November 1, 1996.
Revision received May 15, 1997.
Accepted for publication May 19, 1997.
 |
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