From the Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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We have investigated the interaction
between Cbl and the Src-related tyrosine kinase Fyn. Fyn was observed
to be constitutively associated with Cbl in lysates of several
different cell types including the interleukin-3-dependent
murine myeloid cell line 32Dcl3, and the prolactin-dependent
rat thymoma cell line Nb2. Binding studies indicated that Cbl could
bind to glutathione S-transferase (GST) fusion proteins
encoding the unique, Src homology domain 3 (SH3), and SH2 domains of
Fyn, Hck, or Lyn. Fusion proteins encoding either the SH3 or SH2
domains of Fyn bound to Cbl as effectively as the fusion protein
encoding the unique, SH3, and SH2 domains of Fyn. The Fyn SH2 domain
bound to both tyrosine-phosphorylated and nonphosphorylated Cbl,
implying that this interaction might be phosphotyrosine-independent.
Binding of the Fyn SH2 domain to Cbl was not disrupted by the addition
of phosphotyrosine, phosphoserine, or phosphothreonine. A GST fusion
protein encoding the proline-rich region of Cbl bound to Fyn present in
a total cell lysate. Far Western blot analysis also indicated that the
SH3 domain of Fyn bound preferentially to the proline-rich region of
Cbl. The addition of [ Adapter proteins play a critical role in the regulation and
activation of signaling molecules that lie downstream of various cell
surface receptors. Small adapter molecules (such as Shc, Grb2, Nck, and
Crk) have been extensively studied primarily because their simple
structure has allowed detailed analysis of their functions and
identification of the proteins with which they interact. This has led
to the important observation that both Grb2 and Shc play a critical
role in the activation of the GTP exchange factor son of
sevenless which in turn regulates the activation of Ras (1-5).
Likewise, an analysis of Crk has yielded insights into the proteins
that are associated with different Crk family members (6-10), although
the precise role for Crk in signaling events has remained elusive.
Recent studies have led to the realization that a second family of
large adapter proteins also exists, which are likely to also play a
critical role in the regulation of signal transduction pathways.
Proteins that belong to this family include IRS-11 (11), IRS-2 (12), Cbl
(13), and Cas (9, 14, 15). These proteins are all relatively large in
size, and contain numerous tyrosine residues, which could serve as
binding sites for multiple SH2-containing signaling molecules. Although
the precise role for these larger adapter molecules in signaling
processes is not known, it is clear that some of these large adapter
proteins (IRS-1 and Cbl) can interact with signaling molecules such as
phosphatidylinositol 3-kinase (PI 3-kinase) (16-22), which is
important in mitogenesis and/or suppression of apoptosis (23, 24).
We have previously described the phosphorylation of Cbl following
stimulation of cells with either interleukin-3 (IL-3) or prolactin
(PRL) (18, 19). A time- and dose-dependent phosphorylation of Cbl was observed in cells stimulated with either cytokine (18, 19).
Although the constitutive association of the p85 subunit of PI 3-kinase
with Cbl was observed in the cell lines used in these studies (18, 19),
a cytokine-induced increase in Cbl-associated PI 3-kinase activity was
observed following stimulation of cells with either IL-3 or PRL (18,
19). This suggested that Cbl may play a critical role in cytokine
receptor signaling events since, as noted above, PI 3-kinase is thought
to be important in mitogenesis and/or suppression of apoptosis. The
phosphorylation of Cbl has been observed following activation of
numerous receptors, including the T-cell receptor (25-28), the B-cell
receptor (29, 30), the Fc receptor (31, 32), the epidermal growth
factor receptor (20, 22, 33), erythropoietin receptor (34, 35), the
IL-3 receptor (18), and the prolactin receptor (PRLR) (19). Numerous
signaling molecules have been observed to associate with Cbl including
the Src-like kinases Fyn and Lyn (26, 27, 29, 31, 36), ZAP-70, and/or
Syk (32, 37, 38), Grb2 (10, 22, 28-30, 39), Crk (10, 25, 30, 40), Shc
(30), and PI 3-kinase (20, 21, 26, 28, 29, 41, 42).
An important question to be addressed is which tyrosine kinase(s)
regulate the phosphorylation of Cbl in response to cytokine stimulation. Cytokines such as PRL and IL-3 result in the activation of
one or more members of the Janus family of tyrosine kinases (43-46),
and one or more members of the Src family of tyrosine kinases (47-49).
Therefore, either Janus or Src-like kinases could be responsible for
the phosphorylation of Cbl following the activation of cytokine
receptors. In this study we show that Fyn is constitutively associated
with Cbl in PRL-responsive cells, and that Fyn, but not JAK2, can
phosphorylate a fusion protein that contains the region of Cbl to which
the p85 subunit of PI 3-kinase appears to bind. This suggests that
Src-like kinases phosphorylate Cbl and thereby regulate the activation
of PI 3-kinase.
Cells and Cell Culture--
The Nb2 cell line was obtained from
Dr. 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% fetal calf serum, 10% horse serum, 1 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10
Nb2 and 32Dcl3 cells expressing various forms of the PRLR were cultured
for 16 h in media supplemented with 5% CSS to reduce the levels
of tyrosine-phosphorylated proteins, prior to stimulation with PRL for
the indicated periods of time.
Immunoprecipitation and Immunoblotting--
Cells to be
immunoprecipitated were lysed in either EB (50 mM NaCl, 10 mM Tris, pH 7.4, 5 mM EDTA, 50 mM
sodium fluoride, 1% Triton X-100, 1 mM sodium
orthovanadate with 100 units/ml kallikrein inhibitor) or RIPA buffer
(150 mM NaCl, 50 mM Tris, pH 7.4, 2 mM EGTA, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM sodium orthovanadate with 100 units/ml kallikrein
inhibitor), and the lysates clarified by spinning at 13,000 rpm in a
Savant RCF13K refrigerated centrifuge for 30 min. A 1-µg amount of
the indicated antibodies was added to a cell lysate made from 2 × 107 Nb2 or 2 × 107 32Dcl3 cells in a
final volume of 1 ml, and placed on a rocking platform for 1 h at
4 °C. The immune complexes were collected by adding 30 µl of
Pansorbin (Calbiochem, La Jolla, CA) to each immunoprecipitate for
1 h. The bound proteins were washed three times with lysis buffer
and the immunoprecipitated proteins resolved by SDS-polyacrylamide gel
electrophoresis. The resolved proteins were electrotransferred to
Immobilon membranes (Millipore, Bedford, MA). Detection of proteins by
immunoblotting was conducted using the enhanced chemiluminescence
lighting (ECL) system according to the manufacturer's recommendations
(Amersham Corp.). Agarose-conjugated polyclonal anti-Fyn antibody and
rabbit anti-JAK2 were obtained from Upstate Biotechnology, Inc (Lake
Placid, NY). A monoclonal antibody directed against Fyn was obtained
from Transduction Laboratories (Lexington, KY). Polyclonal antibodies
directed against Cbl and Hck were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Monoclonal antibody 4G10 directed
against phosphotyrosine was kindly provided by Dr. Brian Druker
(University of Oregon Health Sciences Center, Portland, OR). Nonimmune
rabbit serum was obtained from our own nonimmunized animals.
Immune Complex Protein Kinase Assay and
Re-immunoprecipitation--
Samples were immunoprecipitated as
described above, washed three times with either EB or RIPA buffer, and
once with kinase buffer (10 mM Tris, pH 7.0, 10 mM MgCl2, 100 µM sodium
orthovanadate). The pellets were resuspended in 20 µl of kinase
buffer containing 10 µCi of [
The phosphorylation of GST fusion proteins was examined by the addition
of 5 µg of the desired GST fusion protein to the immunoprecipitated kinase. The reaction was initiated by the addition of 20 µl of kinase
buffer containing [ GST Fusion Proteins and Binding Assays--
The origin of GST
fusion proteins encoding different regions of Fyn, Hck, and Lyn were
previously described (18, 50).
A series of GST fusion proteins that include different regions of Cbl
were prepared for this study (Fig. 4A). The names for these
fusion proteins and the amino acids included in the proteins are as
follows: GST-YRICH (tyrosine-rich region, amino acids 228-479); GST-N-YRICH (N-terminal half of GST-YRICH, amino acids 228-357); GST-C-YRICH (C-terminal half of GST-YRICH, amino acids 358-479); GST-PRO (proline-rich region, amino acids 475-694); GST-PRO2 (a longer
version of the proline-rich region, amino acids 475-730); GST-LZIP
(the C-terminal end of Cbl which includes the leucine zipper region,
amino acids 426-906, GST-LZIP-Y731F (GST-LZIP with Tyr731
mutated to a Phe). A cDNA clone of human c-Cbl was provided by W. Langdon (University of Western Australia, Perth, Australia) and was
used as the template to make all of these GST fusion proteins. Primers
were taken directly from the published DNA sequence of this cDNA.
Mutagenesis of individual codons was performed according to the
manufacturer's recommendations using the Ex-Site mutagenesis kit from
Stratagene (La Jolla, CA).
Purification of the GST fusion proteins was conducted as described
previously (18). Tyrosine-phosphorylated GST fusion proteins were
prepared in the TKB1 bacteria (Stratagene) as described previously (50). The TKB1 strain of Escherichia coli can be induced
with indole-acrylic acid to express the elk1 tyrosine
kinase, which will phosphorylate bacterial fusion proteins expressed in
the same cells. GST fusion proteins were expressed in TKB1 bacteria, elk1 expression induced, and the tyrosine-phosphorylated GST
fusion proteins purified as described (50). Anti-phosphotyrosine
immunoblotting with monoclonal antibody 4G10 demonstrated the
phosphorylation of fusion proteins expressed in TKB1 bacteria, however,
there was no detectable tyrosine phosphorylation of GST fusion proteins isolated from DH5
Binding assays were conducted by adding 2 nmol of the desired GST
fusion protein to a 1-mg total cellular protein lysate prepared in RIPA
buffer as described above, in a final volume of 1 ml. Following a 1-h
incubation at 4 °C on a rocking platform, 40 µl of
glutathione-Sepharose (Pharmacia Biotech, Piscataway, NJ) was added and
incubated for 1 h. The bound proteins were washed three times with
RIPA, resolved on SDS-polyacrylamide gels, and subjected to
immunoblotting as described above.
Far Western Blotting--
Far Western blotting was conducted as
described previously (50).
Fyn Is Constitutively Associated with Cbl--
There have been
numerous investigations that have demonstrated the association of Cbl
with many different signaling molecules. These include adapter
molecules such as Crk and Grb2 (10, 22, 25, 28-30, 39, 40), PI
3-kinase (20, 21, 26, 28, 29, 41, 42), and the tyrosine kinases Fyn,
Lyn, and Lck (26, 27, 29, 31, 36). Differences in proteins associated with Cbl may vary with the type of cell examined, or the receptor system examined. A fundamental question is which tyrosine kinase is
responsible for phosphorylation of Cbl in response to activation of a
particular receptor system. We have examined this question in the
context of the PRLR. We have previously demonstrated that PRL induces
the tyrosine phosphorylation of Cbl and that following PRL stimulation,
one can detect increased Cbl-associated PI kinase activity (19).
Prolactin has been shown to activate numerous tyrosine kinases
including JAK2 (43, 45, 52) and the Src-related kinase Fyn (47). We
have also observed the activation of several other Src-related kinases,
including Hck and Lyn, depending on the cell type
examined.2
Lysates of unstimulated and PRL-stimulated Nb2, 32D/Nb2, and 32D/hPRLR
cells were immunoprecipitated with anti-Cbl antibody and immunoblotted
with antibodies specific for different tyrosine kinases. The
constitutive association of Cbl with Fyn was observed in all three cell
lines (Fig. 1); PRL stimulation had no
effect upon the amount of Fyn present in the anti-Cbl
immunoprecipitates. The same immunoblot was reprobed with either
anti-Hck or anti-Lyn antibodies, however, we were unable to detect the
presence of either of these Src-like kinases in the anti-Cbl
immunoprecipitates (data not shown). This was despite the fact that PRL
can stimulate the activation of both Hck and Lyn in 32D/Nb2
cells.3 The association of
Cbl with JAK2 was also examined by the same approach and we were unable
to detect JAK2 in anti-Cbl immunoprecipitates (data not shown). Thus in
two different cell types (murine myeloid and rat thymoma) from two
different species, we have observed the constitutive association of Fyn
and Cbl.
Interaction between Cbl and Fyn Is Mediated by Both the SH3 and SH2
Domains of FYN--
The interaction between Cbl and different
Src-related tyrosine kinases was examined using a series of GST fusion
proteins that encode the unique, SH3, and SH2 domains of Fyn, Hck, and Lyn. Lysates were prepared from either unstimulated Nb2 cells or Nb2
cells stimulated with rPRL for 10 min, and the ability of these GST
fusion proteins to bind to Cbl was examined (Fig. 2). We have previously demonstrated that
Cbl is phosphorylated in a PRL-dependent manner (19). Cbl
was not observed to bind to GST alone (Fig. 2, lanes 1 and
2). In contrast, GST-FYN, GST-HCK, and GST-LYN all bound to
Cbl in lysates of both unstimulated and PRL-stimulated Nb2 cells (Fig.
2, lanes 3-8), which co-migrated with Cbl present in an
anti-Cbl immunoprecipitate (Fig. 2, lanes 9 and
10). There was no difference in the amount of Cbl that bound to any of these fusion proteins when lysates from unstimulated cells
were compared with binding reactions with lysates of PRL-stimulated cells.
The data presented in Fig. 2 suggest that the interaction between Cbl
and the fusion proteins encoding the unique, SH3, and SH2 domains of
Fyn, Hck, and Lyn might be mediated largely by phosphotyrosine-independent binding, since there was no change in
amount of Cbl that bound to these three proteins following tyrosine
phosphorylation of Cbl. SH2 domains bind to phosphotyrosine residues in
a sequence-specific context and the three amino acids C-terminal to the
tyrosine residue are most important in determining which SH2 domain
will bind to a specific phosphotyrosine residue (53, 54). In contrast,
SH3 domains bind to proline-rich motifs which often contain a
Pro-X-X-Pro motif (55-59). It is currently not
known what sequence motifs, if any, are recognized by the unique region
of Src-like kinases. Since Cbl contains a proline-rich region with
numerous Pro-X-X-Pro sequences, it would be
expected that the binding described in Fig. 2 would largely involve the SH3 domain and/or the unique region. To examine this point a series of
GST fusion proteins which contained either the unique, SH3, or SH2
domain of Fyn were used to determine which region(s) of Fyn was
required for binding to Cbl. Consistent with the results shown in Fig.
2, the addition of GST-FYN to lysates of either unstimulated or
PRL-stimulated Nb2 cells resulted in binding to Cbl, although GST alone
was not able to bind to Cbl (Fig.
3A, lanes 1-4). Both the SH3
and SH2 domains of Fyn were able to bind to Cbl present in lysates of
both unstimulated or PRL-stimulated cells (Fig. 3A, lanes
5-8). The unique domain of Fyn did not bind to Cbl in any of our
studies (data not shown). There was no difference in the amount of Cbl
that bound to the Fyn SH3 domain when lysates of unstimulated and
PRL-stimulated cells were compared (Fig. 3A, lanes 5 and
6), which is consistent with the expected binding of the SH3
domain to proline-rich sequences in Cbl. We were surprised to observe
that the SH2 domain of Fyn bound equally to Cbl present in lysates of
unstimulated and PRL-stimulated Nb2 cells (Fig. 3A, lanes 7 and 8). This result was unexpected since as noted above, SH2
domains are largely expected to bind to phosphorylated tyrosine
residues, and there is very little if any tyrosine-phosphorylated Cbl
in unstimulated cells (19).
The Fyn SH2 Domain Binds to Cbl in a Phosphotyrosine-independent
Manner--
To provide further evidence that GST-FYN and GST-FYN-SH2
bound to Cbl in a phosphotyrosine-independent manner, we examined the
ability of different phosphoamino acids to disrupt the binding of these
two fusion proteins to Cbl. GST-FYN and GST-FYN-SH2 fusion proteins
were added to lysates of unstimulated Nb2 cells in the presence of 30 mM phosphoserine, 30 mM phosphotyrosine, or 30 mM phosphothreonine, and the effect of these phosphoamino
acids upon Cbl binding examined by immunoblotting (Fig. 3B).
The solutions of the different phosphoamino acids were carefully
neutralized so as to prevent them from altering the pH of the binding
reaction. The addition of these three phosphoamino acids had no effect
upon the binding of either GST-FYN or GST-FYN-SH2 to Cbl (Fig.
3B). There was no significant change in the amount of Cbl
bound by either fusion protein. This provides evidence that the binding of the Fyn SH2 domain to Cbl may occur in a phosphotyrosine-independent manner. Furthermore, the inability of either phosphoserine or phosphothreonine to block the binding of GST-FYN-SH2 to Cbl indicates that the recognition of other phosphorylated amino acids by the Fyn SH2
domain does not explain the observed results.
The Proline-rich Region of Cbl Binds to Fyn--
The interaction
of Cbl with Fyn was also examined by using a series of GST fusion
proteins containing different regions of Cbl (Fig.
4A). These fusion proteins
were prepared in both nonphosphorylated and tyrosine-phosphorylated
forms by expressing the fusion proteins in either DH5
To provide further evidence that the proline-rich region of Cbl was
important for association with Fyn, far Western blotting was used to
demonstrate the direct binding of the Fyn SH3 domain to the
proline-rich region of Cbl. 10 µg of the different GST fusion
proteins was run on 10% SDS-polyacrylamide gel and the resolved
proteins were electrotransferred to an Immobilon membrane. The filter
was probed with a biotinylated GST-FYN-SH3 fusion protein (Fig.
5). The biotinylated GST-FYN-SH3 probe
was observed to bind to the GST-PRO and GST-PRO2 fusion proteins in
both the nonphosphorylated and tyrosine-phosphorylated forms (Fig. 5,
lanes 2-5). No binding was detected to GST (Fig. 5,
lane 1), or to the nonphosphorylated or
tyrosine-phosphorylated GST-LZIP (Fig. 5, lanes 6 and
7). No binding was detected to either the nonphosphorylated
or the tyrosine-phosphorylated GST-YRICH region (data not shown). A
second parallel blot was probed with a biotinylated GST-FYN-SH2 probe
and only minimal binding was detected to the GST-PRO fusion protein
(Fig. 5, lanes 11 and 12). The inability of the
GST-FYN-SH2 probe to bind to the GST-CBL fusion proteins indicates that
the binding observed in Fig. 5 does not represent nonspecific binding
of the GST proteins. The data presented in Figs. 4 and 5 suggest that
the major determinants regulating interaction of Cbl with Fyn are the
SH3 domain of Fyn and the proline-rich region of Cbl.
Fyn That Co-precipitates with Cbl Is Able to Phosphorylate
Cbl--
The immune complex protein kinase assay was used to determine
whether the Fyn associated with Cbl was able to phosphorylate Cbl.
Lysates of unstimulated and PRL-stimulated Nb2 cells were immunoprecipitated with nonimmune serum, anti-Cbl antiserum, or anti-Fyn antiserum, washed extensively, and [
To provide further evidence of the identity of these two proteins, the
phosphorylated proteins present in a second parallel kinase reaction
were boiled in 1% SDS to denature the proteins and disrupt protein
complexes, the primary antibody removed with Pansorbin, and the
phosphorylated proteins immunoprecipitated a second time with nonimmune
serum, anti-Cbl, or anti-Fyn antibodies. The anti-Cbl antibody
precipitated a protein with an Mr of 120,000 from both the anti-Cbl and anti-Fyn kinase assays (Fig. 6, lanes 9-12). The same proteins were not detected when the
phosphorylated proteins present in the anti-Cbl kinase reaction were
immunoprecipitated with nonimmune serum (Fig. 6, lanes 7 and
8). This indicates that Cbl was a substrate for protein
kinases present in both the anti-Cbl and the anti-Fyn
immunoprecipitates. Roughly equal amounts of phosphorylated Cbl were
immunoprecipitated from both the anti-Cbl and anti-Fyn immune complex
kinase assays, despite the fact that there was more phosphorylated
protein with a Mr of 120,000 in the original
anti-Cbl immunoprecipitate. This could suggest that the secondary
antibody is either limiting, or that a significant amount of the Cbl
protein could not be reprecipitated following boiling in 1% SDS. A
small amount of phosphorylated protein with a molecular weight
corresponding to that of Fyn could be detected in kinase reactions that
were re-precipitated with anti-Cbl antibody (Fig. 6, lane
11). Immunoprecipitation of the denatured anti-Fyn kinase reaction
with anti-Fyn antibody precipitated two phosphorylated proteins with
molecular weights that would correspond to the alternatively spliced
forms of Fyn (59,000 and 56,000) (Fig. 6, lanes 13 and 14). No phosphorylated protein corresponding to the 120,000 molecular weight protein was detected in the reactions re-precipitated
with anti-Fyn antibody (Fig. 6, lanes 13 and 14).
These data suggest that Fyn can phosphorylate Cbl, at least when the
two proteins are in a complex with each other.
Fyn Is Able to Phosphorylate a Region of Cbl to Which the p85
Subunit of PI 3-Kinase Binds in a Phosphotyrosine-dependent
Manner--
We have previously demonstrated that the p85 subunit of PI
3-kinase is constitutively associated with Cbl in both 32Dcl3 and Nb2
cells (18, 19). Following stimulation of these cells with IL-3 or PRL,
respectively, there is a cytokine-induced increase in Cbl-associated PI
3-kinase activity, implying that Cbl may function as an adapter
molecule that can regulate the activation of PI 3-kinase (18, 19).
Activation of PI 3-kinase is apparently regulated by the binding of the
C-terminal SH2 domain of the p85 subunit to a phosphorylated tyrosine
residue (62, 63). The SH2 domains of p85 have a distinct preference for
binding to pYXXM motifs, where pY is
phosphotyrosine, M is methionine, and X represents any amino
acid (53, 54). There are two potential binding sites for the p85 SH2
domain in Cbl; Tyr371 which is present in the sequence
YCEM, and Tyr731 which is present in the sequence YEAM.
We used the series of GST fusion proteins which contain different
regions of Cbl in binding assays to identify the region(s) of Cbl to
which p85 bound. Tyr371 is present in the GST-YRICH fusion
protein, and Tyr731 is present in the LZIP fusion protein.
The fusion proteins were prepared in the nonphosphorylated and tyrosine
phosphorylated versions and added to lysates of unstimulated Nb2 cells.
In lanes 1-9 of Fig. 7, the binding of p85 to a series of
GST proteins was examined by immunoblotting with anti-p85 antiserum
(Fig. 7). No binding of p85 was detected
to GST alone. Likewise, no binding of p85 was detected to either the
nonphosphorylated or the tyrosine-phosphorylated versions of the
GST-YRICH, GST-PRO, or GST-PRO2 regions of Cbl (Fig. 7, lanes
2-7). In contrast, the tyrosine-phosphorylated GST-LZIP region of
Cbl bound to p85 as determined by immunoblotting, although no binding
was detected to the nonphosphorylated form of the same fusion protein
(Fig. 7, lane 8 versus 9). This suggests that based upon the
anti-p85 immunoblotting, the major binding site for p85 is in a
C-terminal region of Cbl, between amino acids 426 and 906 which
includes Tyr731. It should be noted that p85 did not bind
to the GST-YRICH fusion protein, which includes Tyr371,
regardless of whether the fusion protein was tyrosine-phosphorylated or
not. This indicates that Tyr371 does not represent a
preferred binding site for the p85 subunit of PI 3-kinase.
As noted above, Tyr731 is present in a sequence context
that would be predicted to be a binding site for the SH2 domain of p85. To demonstrate this, a mutant of the GST-LZIP fusion protein was prepared in which Tyr731 was mutated to Phe. The GST-LZIP
fusion protein contains five different tyrosines, thus the Y731F mutant
protein should still be capable of being phosphorylated on tyrosine
residues. This fusion protein was prepared in nonphosphorylated and
tyrosine-phosphorylated forms, and the GST-LZIP-Y731F protein prepared
in the TKB1 bacteria was still found to contain phosphotyrosine as
determined by anti-phosphotyrosine immunoblotting (data not shown). The
ability of the tyrosine-phosphorylated GST-LZIP and GST-LZIP-Y731F
fusion proteins to bind to p85 was examined in a GST binding assay.
Consistent with the data shown in Fig. 7, the tyrosine-phosphorylated
GST-LZIP fusion protein bound to p85, however, the unphosphorylated
fusion protein did not (Fig. 7, lanes 11 and 12).
The binding of p85 to the tyrosine-phosphorylated GST-LZIP-Y731F fusion
protein was barely detectable (Fig. 7, lane 14) suggesting
that Tyr731 represents the major binding site for the p85
SH2 domain in Cbl. An anti-p85 immunoprecipitate confirmed the position
of the p85 subunit of PI 3-kinase (Fig. 7, lane 15).
The absence of p85 binding to the GST-PRO and GST-PRO2 fusion proteins
was somewhat surprising since this region of Cbl contains numerous
potential binding sites for SH3 domains of different proteins. To
determine whether there might be a small amount of PI 3-kinase bound to
other regions of Cbl, and to confirm the data shown in the GST binding
assays shown in Fig. 7, proteins bound to the same series of GST fusion
proteins were also used in a PI kinase assay. Our previous studies have
shown that this is a more sensitive method for detecting PI 3-kinase
than immunoblotting with anti-p85 antibody (Refs. 18 and 19 and data
not shown). PI kinase activity was noted in binding assays using both
the GST-PRO and GST-LZIP fusion proteins in both the unphosphorylated and tyrosine-phosphorylated forms. A small but detectable amount of PI
kinase activity was associated with the GST-PRO fusion protein and
there was a 3-fold increase in the amount of kinase activity associated
with the tyrosine-phosphorylated GST-PRO fusion protein (Fig. 7,
lanes 16 and 17). Likewise there was a small
amount of PI kinase activity associated with the unphosphorylated
GST-LZIP fusion protein, however, there was a 5-10-fold increase in
the amount of PI kinase activity associated with the
tyrosine-phosphorylated GST-LZIP fusion protein. In contrast, there was
no PI kinase activity associated with either the unphosphorylated or
the tyrosine-phosphorylated GST-LZIP-Y731F fusion protein (Fig. 7,
lanes 20 and 21). An anti-p85 immunoprecipitate
was used as the control for the PI kinase assay (Fig. 7, lane
22). No PI kinase activity was associated with GST or the
GST-YRICH fusion protein in either the unphosphorylated or
tyrosine-phosphorylated forms (data not shown). These data support the
results of the anti-p85 immunoblotting assay and provide further
evidence that Tyr731 represents the major binding site for
the SH2 domain of the p85 subunit of PI 3-kinase.
The data presented in Fig. 7 suggests that Tyr731
represents the likely phosphotyrosine-dependent binding
site for the p85 subunit of PI 3-kinase in Cbl, which regulates the
activation of PI 3-kinase. The ability of different kinases to
phosphorylate the GST-LZIP fusion protein was examined using the immune
complex protein kinase assay. Unstimulated and stimulated Nb2 cells
were immunoprecipitated with nonimmune serum, anti-Fyn antiserum, or
anti-JAK2 antiserum. The immune complexes were washed, GST, GST-LZIP,
or GST-LZIP-Y731F fusion proteins were added as substrates, and the
reaction initiated by the addition of kinase buffer containing
[ One of the critical issues regarding signal transduction by
cytokine receptors is the role of different tyrosine kinases in mitogenic signaling. The binding of ligands to cytokine receptor family
members has been reported to activate one or more members of the Janus
family of tyrosine kinases, and one or more members of the Src family
of tyrosine kinases. Studies with mutant cell lines that are resistant
to interferon have demonstrated that activation of Janus kinases is
critical in mediating the activation of STAT molecules and the
induction of interferon-induced transcription (64-66). In cells
stimulated to proliferate by cytokines, such as IL-3 and PRL, it is
clear that one or more members of the Janus family of kinases is
activated, which leads to the activation of one or more STAT family
members (43, 46, 52, 67-69). Although it is clear that the activation
of JAK2 is required for the transcription of specialized gene products,
such as induction of In this study we have described the interaction between the Src-like
kinase Fyn and the adapter protein Cbl. Our data indicate that Fyn and
Cbl are constitutively associated in two different cell lines derived
from two different tissues (myeloid and T-cell) from two different
species (mouse and rat, respectively). Similar data have also been
obtained in the human breast cancer cell line T47D (data not shown).
There was no evidence that JAK2 was associated with Cbl in any of these
cell lines. Binding studies indicated that the SH3 and SH2 domains of
Fyn are able to bind to Cbl and that both domains can apparently
interact in a phosphotyrosine-independent manner.
Other investigators have demonstrated that Src-like kinases are able to
bind to Cbl and that these interactions are mediated by the SH3 domain,
the SH2 domain, or both (26, 27, 29, 31, 36). It is logical that the
SH3 domain of Fyn could bind to Cbl in a phosphotyrosine-independent
manner, since SH3 domains recognize proline-rich sequences, and there
are several proline-rich regions in Cbl. The direct binding of the Fyn
SH3 domain to the proline-rich region of Cbl, as demonstrated by far
Western blot analysis, is consistent with this expectation. We were,
however, surprised to observe the constitutive,
phosphotyrosine-independent binding of the Fyn SH2 domain to Cbl.
Although other investigators have reported the ability of the Abl and
Lck SH2 domain to bind to proteins that do not contain phosphotyrosine
(71-73), there have been suggestions that this could be explained by
the ability of these SH2 domains to recognize peptides containing
either phosphoserine or phosphothreonine (74). The inability of free
phosphoserine or phosphothreonine to block the binding of the Fyn SH2
domain to Cbl suggests that it may be able to recognize other motifs as
well. The ability of the Fyn SH2 domain to bind to Cbl in a phosphotyrosine-independent manner is consistent with our previously reported results (18).
We have also previously reported that although the p85 subunit of PI
3-kinase is constitutively associated with Cbl in both 32Dcl3 and Nb2
cells, and that stimulation with either IL-3 or PRL results in a
cytokine-induced increase in Cbl-associated PI kinase activity (18,
19). This has suggested to us that Cbl may function as an adapter or
scaffold molecule that could link cytokine receptors to the activation
of PI 3-kinase. Although it would be expected that the SH3 domain of
p85 could bind to the proline-rich region of Cbl, we could not detect
this interaction when the ability of GST-PRO to bind to p85 was
examined by immunoblotting with anti-p85 antiserum (Fig. 7). It has
been suggested by other investigators that the SH3 domain of p85 could
be bound in an intramolecular fashion to one of two proline-rich
sequences in the p85 subunit (75), and thus the SH3 domain of p85 may
not be able to bind to other molecules. The major binding site for p85
in Cbl appears to be Tyr731 (Fig. 7), which is present in
the sequence pYEAM, and would be expected to represent a binding site
for the SH2 domains of p85 (53). The association of the p85 subunit of
PI 3-kinase with the p110 subunit is thought to suppress the kinase
activity of the catalytic p110 subunit (76). The binding of the p85
subunit to a phosphopeptide results in the activation of the catalytic subunit (76). Thus it would be expected that the binding of the
C-terminal SH2 domain of p85 to phosphorylated Tyr731 might
be expected to lead to the catalytic activation of the enzyme.
In this study, we have also demonstrated that Fyn was able to
phosphorylate the Cbl protein with which it co-precipitated. In
addition, we have demonstrated that Fyn was able to phosphorylate a GST
fusion protein that encoded a region of Cbl that contained five
tyrosine residues, including Tyr731. Mutation of
Tyr731 to a Phe resulted in a substantial reduction in
phosphorylation of this protein. This latter result suggests that
Tyr731 is a preferred phosphorylation site for Src-like
kinases. We were unable to demonstrate that JAK2 could phosphorylate
the same GST-LZIP fusion protein that contained Tyr731.
These data suggest that the phosphorylation of at least one critical
tyrosine residue in Cbl, Tyr731, is mediated by Src-like
kinases and not by JAK2. Furthermore, the phosphorylation of Cbl at
this site represents one means by which the activation of PI 3-kinase
may be regulated. Thus Cbl, Fyn, and PI 3-kinase form a tripartite
complex that may be critical in cytokine-mediated signaling. Consistent
with this hypothesis is the observation that the yeast two-hybrid
system has demonstrated the direct interaction of Cbl and Lyn, and that
a tripartite complex of Cbl, Lyn, and p85 can be detected when
catalytically activate Lyn is expressed in these cells (77). While this
manuscript was in preparation, Feshchenko et al. (78)
demonstrated that Fyn, Yes, and Syk were able to phosphorylate Cbl, and
that tyrosines 700, 731, and 774 represented major sites of
phosphorylation. The results of this study are consistent with those
reported here, however, our data suggests that Tyr731
represents the major site of phosphorylation since Fyn does not phosphorylate GST-LZIP-Y731F to an appreciable extent (Fig. 8). The
GST-LZIP fusion protein does not include Tyr700, although
it does contain Tyr774. The previous study also focused
upon the T-cell receptor, while ours utilized the PRLR, a member of the
cytokine receptor superfamily. Deckert et al. (79) have also
described the association of both Syk and Fyn with Cbl, however, they
have suggested that Fyn functions as an adapter that facilitates the
interaction of Syk with Cbl, and that Syk phosphorylates Cbl.
It is our hypothesis that Cbl represents one of the major substrates
for Src-like kinases activated downstream of cytokine receptors, and
that phosphorylation of Cbl may represent one means by which PI
3-kinase is regulated. We further hypothesize that activation of PI
3-kinase is important in regulating the mitogenic response of cells to
cytokines such as PRL and IL-3. Alternatively, this complex of
signaling molecules could be important in cytokine-induced suppression
of apoptosis. The activation of PI 3-kinase has been shown to lead to
the activation of the Akt kinase (24, 80-84), which can apparently
suppress apoptosis through the phosphorylation of the pro-apoptotic
molecule Bad (85, 86). The question that remains to be addressed is
whether the Cbl-dependent activation of PI 3-kinase is
important in regulating the activation of Akt and thereby suppressing
apoptosis or whether the PI 3-kinase-dependent activation
of Akt is regulated in a different manner.
-32P]ATP to either anti-Cbl
immunoprecipitates or anti-Fyn immunoprecipitates resulted in the
phosphorylation of both Cbl and Fyn as demonstrated by
immunoprecipitation of the phosphorylated proteins with specific antisera. Fyn directly phosphorylated a GST fusion protein containing the C-terminal region of Cbl (GST-CBL-LZIP). In contrast,
immunoprecipitated JAK2 was not able to phosphorylate this same region
of Cbl. The GST-CBL-LZIP fusion protein contains a binding site for the
SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase, which mapped to Tyr731, which is present in the sequence YEAM.
Mutation of Tyr731 in GST-CBL-LZIP eliminated binding of
the p85 subunit of phosphatidylinositol 3-kinase and substantially
reduced the phosphorylation of this fusion protein by Fyn, despite the
presence of four other tyrosine residues in this fusion protein. These
data are consistent with the hypothesis that Cbl represents a substrate
for Src-like kinases that are activated in response to the engagement
of cell surface receptors, and that Src-like kinases are responsible
for the phosphorylation of a tyrosine residue in Cbl that may regulate
activation of phosphatidylinositol 3-kinase.
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
4 M
-mercaptoethanol. The 32Dcl3 cell line was obtained from Dr. Joel
Greenberger (University of Pittsburgh, Pittsburgh, PA), and its
cultivation has been described recently (18). Characterization of
32Dcl3 cells expressing the long form of the human PRLR and the Nb2
form of the rat PRLR were described previously (19). Charcoal-stripped
fetal calf serum (CSS) was obtained from HyClone (Logan, UT) and fetal
calf serum was from Summit Biotechnology (Fort Collins, CO). All other
media components were from Life Technologies, Inc. (Gaithersburg, MD).
Human (lot AFP-3855A), ovine (lot AFP-10677C), and rat (lot AFP6452B)
prolactin were obtained from the National Hormone and Pituitary Program
(Rockville, MD).
-32P]ATP (catalog
number BLU502A, 3000 Ci/mmol; New England Nuclear, Boston, MA), and
incubated at room temperature for 10 min. In the case of kinase
reactions that were to be directly analyzed by gel electrophoresis, the
reactions were terminated by the addition of 2 × SDS sample
buffer, and heated at 100 °C for 10 min. Reactions to be denatured
and re-immunoprecipitated were washed once with EB, resuspended in 100 µl of 1% SDS, 50 mM Hepes, pH 7.4, 150 mM
NaCl, and boiled for 10 min. The volume was brought to 1 ml by the
addition of 900 µl of 1% Triton X-100, 0.25% sodium deoxycholate, 50 mM Hepes, pH 7.4, 150 mM NaCl, 100 units/ml
kallikrein inhibitor, 1 mM sodium orthovanadate. A 30-µl
volume of Pansorbin was added to remove the remaining primary antibody,
and the tube incubated at 4 °C on a rocking platform for 30 min. The
Pansorbin-bound IgG was pelleted, and the supernatant fluid was removed
and placed in a siliconized tube. The desired antibody was added, and
incubated overnight at 4 °C on a rocking platform. A 30-µl volume
of Pansorbin was added for 1 h. The immune complexes were washed
three times with EB, dissolved in 2 × SDS sample buffer, heated
at 100 °C for 10 min, and resolved on the same 7.5%
SDS-polyacrylamide gel as the kinase reaction products.
-32P]ATP, and the reaction
incubated at room temperature for 5 min. The reaction was terminated by
the addition of a equal volume of 2 × SDS sample buffer, the
samples boiled, and the reaction products resolved by electrophoresis
on a 10% SDS-polyacrylamide gel. The location of the GST fusion
proteins was determined by staining the gel with Coomassie Brilliant
Blue. A Molecular Dynamics Storm 780 PhosphorImager was used to
quantitate protein phosphorylation, and data was analyzed with
ImageQuant software.
cells. The stoichiometry with which the different tyrosine residues present in the GST fusion proteins were
phosphorylated was not addressed in this study.
RESULTS
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Fig. 1.
Constitutive association of Cbl with
Fyn. Nb2 (lanes 1-3), 32D/Nb2 (lanes 4-6),
and 32D/hPRLR (lanes 7-9) cells were cultured overnight in
medium containing 2% CSS, concentrated, then stimulated with either
rPRL (Nb2 and 32D/Nb2) or oPRL for 0 (lanes marked ) or 10 min (lanes
marked +). The cells were lysed and immunoprecipitated as described
with anti-Cbl antiserum. The immunoprecipitated proteins were resolved
by electrophoresis on a 7.5% SDS-polyacrylamide gel, the proteins
transferred to an Immobilon membrane, and the immunoblot probed with
anti-Fyn monoclonal antibody. Positive controls consist of whole cell
lysates from each of the cell lines (lanes marked "W").
The position of the 68,000 molecular weight marker is indicated on the
left side of the panel and the position of Fyn is indicated
by the arrowhead on the right side of the
panel.
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Fig. 2.
Binding of Cbl to GST fusion proteins
encoding the unique, SH3 and SH2 domains of Fyn, Hck, and Lyn. Nb2
cells were cultured overnight in media supplemented with 2% CSS, then
stimulated with 100 ng/ml rPRL for 0 (lanes marked ) or 10 (lanes
marked +) min. 2 nmol of GST (lanes 1 and 2),
GST-FYN (lanes 3 and 4), GST-HCK (lanes
5 and 6), or GST-LYN (lanes 7 and
8) were added to each cell lysates in a final volume of 1 ml. As a control, unstimulated and PRL-stimulated cell lysates were
also immunoprecipitated with anti-Cbl antiserum. The bound proteins
were resolved on a 7.5% SDS-polyacrylamide gel, transferred to
Immobilon membrane, and immunoblotted with anti-Cbl antibody.
Lane numbers are indicated at the bottom of each
lane.
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Fig. 3.
The SH2 domain of Fyn binds to Cbl in a
phosphotyrosine-independent manner. A, A binding assay
using different GST fusion proteins was conducted as described in Fig.
2. Lysates of unstimulated (lanes marked ) and PRL-stimulated (lanes
marked +) Nb2 cells were used in these studies. The bound proteins were
resolved on an 8% SDS-polyacrylamide gel, transferred to an Immobilon
membrane, and probed with anti-Cbl antiserum. Only the portion of the
gel corresponding to Cbl is shown. Lane numbers are
indicated at the bottom of the panel and the position of Cbl
is indicated on the left side of the panel. B, binding
assays were conducted as described in the legend to Fig. 2. The GST-FYN
(lanes 1-4) and GST-FYN-SH2 (lanes 5-8) fusion
proteins were added to lysates of unstimulated Nb2 cells. Each cell
lysate contained 2 × 107 cells and 2 nmol of the
indicated fusion protein in 1.5 ml. 30 mM phosphoserine was
added to lanes 2 and 6, 30 mM
phosphotyrosine was added to lanes 3 and 7, and
30 mM phosphothreonine was added to lanes 4 and
8. Immunoblot analysis was with anti-Cbl antiserum.
Lane numbers are indicated at the bottom of the
panel.
or TKB1
strains of E. coli, respectively. Anti-phosphotyrosine immunoblotting indicated that GST fusion proteins prepared in TKB1
cells were tyrosine phosphorylated, while those prepared in DH5
bacteria did not contain phosphotyrosine (data not shown). All of the
GST-Cbl fusion proteins contained multiple tyrosine residues, however,
no attempt was made to determine the stoichiometry with which the
different tyrosine residues were phosphorylated. The nonphosphorylated
and phosphorylated fusion proteins were added to lysates of
unstimulated 32Dcl3 cells, and the amount of Fyn that bound to the
different fusion proteins examined by anti-Fyn immunoblotting (Fig.
4B). We did not detect the binding of Fyn to GST,
GST-N-YRICH, GST-C-YRICH, or GST-LZIP (Fig. 4B, lanes 1-5,
8, and 9). Fyn was only observed to bind to GST-PRO fusion proteins in both the unphosphorylated or tyrosine-phosphorylated forms (Fig. 4B, lanes 6 and 7). There did not
appear to be a significant change in the amount of Fyn that bound to
the nonphosphorylated GST-PRO fusion proteins compared with that which
bound to the tyrosine-phosphorylated form. Similar results were
obtained with lysates prepared from unstimulated Nb2 cells (data not
shown). These data indicate that the proline-rich region of Cbl may be responsible for the constitutive association of Cbl with Fyn.
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Fig. 4.
The proline-rich region of Cbl binds to
Fyn. A, a series of GST fusion proteins were prepared
which encoded different regions of Cbl. The amino acid numbers that
mark the boundaries of each of the different proteins are indicated,
alone with the name of the protein. The linear diagram of Cbl notes
structural motifs present in the protein. B, the indicated
GST fusion proteins were prepared in the nonphosphorylated or tyrosine
phosphorylated form by expression in either the DH5 or the TKB1
strains of E. coli, respectively. Lysates of unstimulated
32Dcl3 cells were prepared from cells that had been cultured overnight
in media containing CSS. 2 nmol of GST (lane 1),
GST-Cbl-N-YRICH (lane 2), tyrosine-phosphorylated
GST-Cbl-N-YRICH (lane 3), GST-Cbl-C-YRICH (lane
4), tyrosine-phosphorylated GST-Cbl-C-YRICH (lane 5),
GST-PRO (lane 6), tyrosine-phosphorylated GST-PRO
(lane 7), GST-LZIP (lane 8), and
tyrosine-phosphorylated GST-LZIP (lane 9) were added to each
cell lysate in a final volume of 1 ml. The bound proteins were resolved
on a 10% SDS-polyacrylamide gel, transferred to Immobilon membrane,
and immunoblotted with anti-Fyn monoclonal antibody. The position of
Fyn is indicated by the arrowhead on the right
side of the panel. Lane numbers are indicated at the
bottom.
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Fig. 5.
The SH3 domain of Fyn binds directly to the
proline-rich region of Cbl. The direct binding of the SH3 and SH2
domains of Fyn to different regions of Cbl was examined by far Western
blot analysis. A 10 µg amount of the indicated Cbl fusion proteins
was run on an SDS-polyacrylamide gel and electrotransferred to an
Immobilon membrane. The filter was probed with either a biotinylated
GST-FYN-SH3 fusion protein (panel A) or a biotinylated
GST-FYN-SH2 fusion protein (panel B), and the binding of the
probes was detected by ECL. Lanes 1 and 10 contain GST; lanes 2 and 11, GST-PRO; lanes
3 and 12, pGST-PRO; lanes 4 and
13, GST-PRO2; lanes 5 and 14,
pGST-PRO2; lanes 6 and 15, GST-LZIP; and
lanes 7 and 16, pGST-LZIP; lanes 8 and
17, GST-LZIP-Y731F; and lanes 9 and
18, pGST-LZIP-Y731F. The positions of prestained molecular
weight markers are indicated on the left side of each panel,
and the positions of the indicated GST fusion proteins are indicated on
the right side of the panel. Lane numbers are
indicated at the bottom of each panel.
-32P]ATP
was added to the immune complexes to identify proteins that could be
phosphorylated. A protein with a molecular weight of approximately
120,000 was observed to be phosphorylated in the anti-Cbl
immunoprecipitates from both unstimulated and PRL-stimulated cells,
although the extent of phosphorylation was significantly higher in
immunoprecipitates from PRL-stimulated cells (Fig.
6, lane 3 versus lane 4).
Anti-Fyn immunoprecipitates from unstimulated and PRL-stimulated Nb2
cells contained phosphorylated proteins with molecular weights of
approximately 120,000, 59,000, and 56,000. The former protein
co-migrated with the 120,000 Mr protein present in the anti-Cbl immunoprecipitates and the latter two proteins correspond to the known sizes of the two different splicing variants of
Fyn (60, 61). The 120,000, 59,000, and 56,000 Mr
proteins were not observed in kinase assays performed with nonimmune
antiserum control immunoprecipitates (Fig. 6, lanes 1 and
2).
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Fig. 6.
Phosphorylation of Cbl in anti-Fyn immune
complexes. Nb2 cells were cultured overnight in medium containing
2% CSS, concentrated, then stimulated with rPRL for either 0 (lanes
marked ) or 10 min (lanes marked +). The cells were lysed and
immunoprecipitated as described with either nonimmune serum
(NIS), anti-Cbl antiserum, or agarose-conjugated anti-Fyn
antibody. The immunoprecipitated proteins were washed extensively and
then phosphorylated in the immune complex protein kinase assay. One set
of reactions was terminated by the addition of 2 × SDS sample
buffer and the phosphorylated proteins were resolved by electrophoresis
on a 7.5% SDS-polyacrylamide; NIS, control kinase
reactions, lanes 1 and 2; anti-Cbl kinase
reactions, lanes 3 and 4; and anti-Fyn kinase
reactions, lanes 5 and 6. The second set of
reactions were terminated by bringing the volume of the reaction to 0.1 ml and a final concentration of 1% SDS, and boiling the reaction for
10 min (lanes 7-14). These reactions were then
re-immunoprecipitated with NIS, anti-Cbl antiserum, or anti-Fyn
antiserum. The antibodies used for the first and second set of
immunoprecipitations are indicated at the top of each pair
of lanes. The positions of prestained molecular weight markers are
indicated on the left side of the figure, and the positions
of both Cbl and Fyn are indicated by the arrowheads in the
center of the figure.
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Fig. 7.
The p85 subunit of PI 3-kinase binds to
Tyr731 of Cbl. Lysates of unstimulated Nb2 cells were
used in binding assays with various GST-fusion proteins encoding
different regions of Cbl (see Fig. 1 for the location of the different
fusion proteins). The series of GST fusion proteins were prepared in
DH5 bacteria and these proteins did not contain phosphotyrosine as
indicated by anti-phosphotyrosine immunoblotting (data not shown). A
second series of GST fusion proteins were prepared in the TKB1 strain
of E. coli which can be induced to express the
Elk1 tyrosine kinase resulting in the production of
tyrosine-phosphorylated fusion proteins (identified as
pGST-XXX). A, a 2 nmol amount of each GST fusion
protein was added to 1 mg of total cellular protein in a final volume
of 1 ml and the binding assay conducted as described under "Materials
and Methods." The fusion proteins used were: GST (lanes 1 and 10), GST-YRICH (lane 2), pGST-YRICH
(lane 3), GST-PRO (lane 4), pGST-PRO (lane
5), GST-PRO2 (lane 6), pGST-PRO2 (lane 7);
GST-LZIP (lanes 8 and 11), pGST-LZIP (lanes
9 and 12), GST-LZIP-Y731F (lane 13), and
pGST-LZIP-Y731F (lane 14). An anti-p85 immunoprecipitate is
shown in lane 15. The bound proteins were washed to remove
nonspecifically bound proteins, the bound proteins resolved by SDS-gel
electrophoresis, and subjected to immunoblotting with an anti-p85
antibody. The names of the fusion proteins are indicated at the
top of each lane, lane numbers at the
bottom of each lane, and the position of p85 is indicated by
the arrow on the right side of the panel.
B, the binding of PI kinase to the same series of GST fusion
proteins was examined using the PI kinase assay.
-32P]ATP. The phosphorylated reaction products were
resolved by SDS-gel electrophoresis and the position of the GST fusion
proteins determined by staining the gels with Coomassie Brilliant Blue.
A Molecular Dynamics Storm 780 PhosphorImager was used to quantitate
the extent of substrate phosphorylation (Fig.
8). Compared with the nonimmune serum
control of unstimulated cells, there was a 10.7-fold increase in the
phosphorylation of the GST-LZIP fusion protein by the anti-Fyn immunoprecipitate (Fig. 8). PRL stimulation increased the background level of GST-LZIP phosphorylation observed in the nonimmune serum control, however, the kinase activity of the anti-Fyn immunoprecipitate was still 6-fold greater (Fig. 8). Relative to the GST-LZIP
phosphorylation observed with the anti-Fyn immunoprecipitate of
unstimulated cells, there was a 1.6-fold increase following PRL
stimulation (Fig. 8). The GST-LZIP-Y731F fusion protein was still
phosphorylated by the anti-Fyn immunoprecipitate, however, quantitation
of the amount of incorporated radioactivity was one-third of that
observed with the GST-LZIP fusion protein. Quantitation of the amount
of radioactivity incorporated into the GST-LZIP and GST-LZIP-Y731F fusion proteins when anti-JAK2 immunoprecipitates were used revealed that the amount of radioactivity was less than or equal to the amount
of phosphorylation observed with the nonimmune serum controls. These
data suggest that under the conditions used in this study, Fyn but not
JAK2 is the major kinase that phosphorylates Cbl, and that
Tyr731 represents a preferred phosphorylation site for Fyn
in the C-terminal region of Cbl.
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Fig. 8.
Fyn but not JAK2, is able to phosphorylate a
GST fusion protein that contains Tyr731 of Cbl. Nb2
cells were cultured overnight in media containing charcoal-stripped
serum to allow signaling molecules to return to their basal state. The
cells were then washed, concentrated, and either left unstimulated, or
stimulated with 50 nM rPRL for 10 min. The cells were lysed
in RIPA buffer and subjected to immunoprecipitation with nonimmune
serum, anti-Fyn antibody, or anti-JAK2 antibody. The immune complexes
were collected with Pansorbin and washed to remove nonspecifically
bound proteins. All buffer was removed from the Pansorbin pellet and 5 µg of the indicated GST fusion protein was added to each pellet, and
the pellet and GST fusion protein were mixed by vortexing. The
reactions were initiated by the addition of 20 µl of kinase buffer
containing 10 µCi of [ -32P]ATP, and the reactions
incubated for 5 min at room temperature. The reaction products were
resolved by electrophoresis on a 10% polyacrylamide gel. The positions
GST, GST-LZIP, and GST-LZIP-Y731F fusion proteins were determined by
staining the gel with Coomassie Brilliant Blue. The phosphorylation of
the GST fusion proteins was quantitated with a Molecular Dynamics
PhosphorImager, and the data analyzed with ImageQuant software. The
relative PhosphorImager units are plotted versus the GST
fusion proteins examined. Nonimmune serum controls are shown in the
open bars, anti-Fyn immunoprecipitates in the dark
filled bars, and anti-JAK2 immunoprecipitates in the gray
bars.
DISCUSSION
-casein by PRL (70), there is no evidence that
activation of JAK2 and STAT molecules are required for mitogenesis. It
has been our general hypothesis that the activation of Src-like kinases
is critical in the activation of mitogenic signaling cascades that lie
downstream of cytokine receptors.
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FOOTNOTES |
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* This work was supported by Grants CA45241, DK48845, and DK48878 from the National Institutes of Health and The University of Colorado Cancer Center DNA Sequencing Core supported by Grant CA46934 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pathology,
Box B-216, University of Colorado Health Sciences Center, 4200 East
Ninth Ave., Denver, CO 80262. Tel.: 303-315-4787; Fax: 303-315-6721;
E-mail: steve.anderson{at}uchsc.edu.
The abbreviations used are: IRS-1, insulin-regulated substrate-1; CSS, charcoal-stripped serum; GST, glutathione S-transferase; hPRL, human prolactin; IL-3, interleukin-3; IRS-2, insulin-regulated substrate-2; JAK2, Janus kinase 2; PI 3-kinase, phosphatidylinositol 3'-kinase; PRL, prolactin; PRLR, prolactin receptor; rPRL, rat prolactin; SH2, Src homology 2; SH3, Src homology 3; STAT, signal transducer and activator of transcription.
2 S. M. Anderson, unpublished data.
3 S. M. Anderson, unpublished data.
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REFERENCES |
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