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University of Colorado Health Sciences Center Department of Pathology Denver, Colorado 80262
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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 1–4). 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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, 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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).
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 5–6). The
amount of tyrosine phosphorylation remained fairly constant over a
range of 10–100 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
9–14% was observed in three different studies of Nb2 cells) of the
amount observed in anti-grb2 immunoprecipitates (Fig. 3, compare lanes
9–12). When lanes 9–12 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 130,000–140,000 (Fig. 3, lanes 1 and 2). This protein remains to be identified.
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- 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 10–20% 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 20–30% 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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, lanes 7–12, 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 50–70% 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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, 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 5–8). Anti-cbl
immunoprecipitates contained PI kinase activity that increased after
PRL stimulation (Fig. 6, lanes 1–4). 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 10–70% 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 20–40 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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
manufacturer’s 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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receptors. J Biol Chem 270:9115–9120This article has been cited by other articles:
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K. M. McAveney, M. L. Book, P. Ling, J. Chebath, and L.-y. Yu-Lee Association of 2',5'-Oligoadenylate Synthetase with the Prolactin (PRL) Receptor: Alteration in PRL-Inducible Stat1 (Signal Transducer and Activator of Transcription 1) Signaling to the IRF-1 (Interferon-Regulatory Factor 1) Promoter Mol. Endocrinol., February 1, 2000; 14(2): 295 - 306. [Abstract] [Full Text]
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 R Hooshmand-Rad, L Hajkova, P Klint, R Karlsson, B Vanhaesebroeck, L Claesson-Welsh, and C. Heldin The PI 3-kinase isoforms p110(alpha) and p110(beta) have differential roles in PDGF- and insulin-mediated signaling J. Cell Sci., January 1, 2000; 113(2): 207 - 214. [Abstract] [PDF]
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M. Bartkiewicz, A. Houghton, and R. Baron Leucine Zipper-mediated Homodimerization of the Adaptor Protein c-Cbl. A ROLE IN c-Cbl's TYROSINE PHOSPHORYLATION AND ITS ASSOCIATION WITH EPIDERMAL GROWTH FACTOR RECEPTOR J. Biol. Chem., October 22, 1999; 274(43): 30887 - 30895. [Abstract] [Full Text] [PDF]
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R. C. H. Lee, J. A. Walters, M. E. Reyland, and S. M. Anderson Constitutive Activation of the Prolactin Receptor Results in the Induction of Growth Factor-independent Proliferation and Constitutive Activation of Signaling Molecules J. Biol. Chem., April 9, 1999; 274(15): 10024 - 10034. [Abstract] [Full Text] [PDF]
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A. Saci, S. Pain, F. Rendu, and C. Bachelot-Loza Fc Receptor-mediated Platelet Activation Is Dependent on Phosphatidylinositol 3-Kinase Activation and Involves p120cbl J. Biol. Chem., January 22, 1999; 274(4): 1898 - 1904. [Abstract] [Full Text] [PDF]
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S. Hunter, E. A. Burton, S. C. Wu, and S. M. Anderson Fyn Associates with Cbl and Phosphorylates Tyrosine 731 in Cbl, A Binding Site for Phosphatidylinositol 3-Kinase J. Biol. Chem., January 22, 1999; 274(4): 2097 - 2106. [Abstract] [Full Text] [PDF]
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T. Zhu, E. L. K. Goh, D. LeRoith, and P. E. Lobie Growth Hormone Stimulates the Formation of a Multiprotein Signaling Complex Involving p130Cas and CrkII. RESULTANT ACTIVATION OF c-Jun N-TERMINAL KINASE/STRESS-ACTIVATED PROTEIN KINASE (JNK/SAPK) J. Biol. Chem., December 11, 1998; 273(50): 33864 - 33875. [Abstract] [Full Text] [PDF]
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 C. Bole-Feysot, V. Goffin, M. Edery, N. Binart, and P. A. Kelly Prolactin (PRL) and Its Receptor: Actions, Signal Transduction Pathways and Phenotypes Observed in PRL Receptor Knockout Mice Endocr. Rev., June 1, 1998; 19(3): 225 - 268. [Abstract] [Full Text]
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K. M. Hill, Y. Huang, S.-C. Yip, J. Yu, J. E. Segall, and J. M. Backer N-terminal Domains of the Class IA Phosphoinositide 3-Kinase Regulatory Subunit Play a Role in Cytoskeletal but Not Mitogenic Signaling J. Biol. Chem., May 4, 2001; 276(19): 16374 - 16378. [Abstract] [Full Text] [PDF]
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E. Hamilton, K. M. Miller, K. M. Helm, W. Y. Langdon, and S. M. Anderson Suppression of Apoptosis Induced by Growth Factor Withdrawal by an Oncogenic Form of c-Cbl J. Biol. Chem., March 16, 2001; 276(12): 9028 - 9037. [Abstract] [Full Text] [PDF]
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