Fyn Associates with Cbl and Phosphorylates Tyrosine 731 in Cbl, A Binding Site for Phosphatidylinositol 3-Kinase*

Seija Hunter, Elizabeth A. Burton, Steven C. Wu, and Steven M. AndersonDagger

From the Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado 80262

    ABSTRACT
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Abstract
Introduction
References

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 [gamma -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

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.

    MATERIALS AND METHODS

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 10-4 M beta -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).

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 [gamma -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.

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 [gamma -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.

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 DH5alpha cells. The stoichiometry with which the different tyrosine residues present in the GST fusion proteins were phosphorylated was not addressed in this study.

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).

    RESULTS

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.


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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.

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.


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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.

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).


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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.

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 DH5alpha 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 DH5alpha 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 DH5alpha 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.

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.


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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.

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 [gamma -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.

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.


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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 DH5alpha 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.

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 [gamma -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 [gamma -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

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 beta -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.

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.

    FOOTNOTES

* 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.

Dagger 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.

    REFERENCES
Top
Abstract
Introduction
References

  1. Rozakis-Adcock, M., McGlade, J., Mbamalu, G., Pelicci, G., Daly, R., Li, W., Batzer, A., Thomas, S., Brugge, J. S., Pelicci, P. G., Schlessinger, J., and Pawson, T. (1992) Nature 360, 689-692[CrossRef][Medline] [Order article via Infotrieve]
  2. Gale, N. W., Kaplan, S., Lowenstein, E. J., Schlessinger, J., and Bar-Sagi, D. (1993) Nature 363, 88-92[CrossRef][Medline] [Order article via Infotrieve]
  3. Egan, S. E., Giddings, B. W., Brooks, M. W., Buday, L., Sizeland, A. M., and Weinberg, R. A. (1993) Nature 363, 45-51[CrossRef][Medline] [Order article via Infotrieve]
  4. Rozakis-Adcock, M., Fernley, R., Wade, J., Pawson, T., and Bowtell, D. (1993) Nature 363, 83-85[CrossRef][Medline] [Order article via Infotrieve]
  5. Li, N., Batzer, A., Daly, R., Yajnik, V., Skolnik, E., Chardin, P., Bar-Sagi, D., Margolis, B., and Schlessinger, J. (1993) Nature 363, 85-88[CrossRef][Medline] [Order article via Infotrieve]
  6. Sattler, M., Salgia, R., Okuda, K., Uemura, N., Durstin, M. A., Pisick, E., Xu, G., Li, J.-L., Prasad, K. V., and Griffin, J. D. (1996) Oncogene 12, 839-846[Medline] [Order article via Infotrieve]
  7. Ingham, R. J., Krebs, D. L., Barbazuk, S. M., Turck, C. W., Hirai, H., Matsuda, M., and Gold, M. R. (1996) J. Biol. Chem. 271, 32306-32314[Abstract/Free Full Text]
  8. Matsuda, M., Ota, S., Tanimura, R., Nakamura, H., Matuoka, K., Takenawa, T., Nagashima, K., and Kurata, T. (1997) J. Biol. Chem. 271, 14468-14472[Abstract/Free Full Text]
  9. Harte, M. T., Hildebrand, J. D., Burnham, M. R., Bouton, A. H., and Parsons, J. T. (1996) J. Biol. Chem. 271, 13649-13655[Abstract/Free Full Text]
  10. Khwaja, A., Hallberg, B., Warne, P. H., and Downward, J. (1996) Oncogene 12, 2491-2498[Medline] [Order article via Infotrieve]
  11. White, M. F., and Kahn, C. R. (1994) J. Biol. Chem. 269, 1-4[Free Full Text]
  12. Sun, X. J., Wang, L.-M., Zhang, Y., Yenush, L., Myers, M. G., Glasheen, E., Lane, W. S., Pierce, J. H., and White, M. F. (1995) Nature 337, 173-177
  13. Blake, T. J., Shapiro, M., Morse, H. C., and Langdon, W. Y. (1991) Oncogene 6, 653-657[Medline] [Order article via Infotrieve]
  14. Sakai, R., Iwamatsu, A., Hirano, N., Ogawa, S., Tanaka, T., Nishida, J., Yazaki, Y., and Hirai, H. (1994) J. Biol. Chem. 269, 32740-32746[Abstract/Free Full Text]
  15. Burnham, M. R., Harte, M. T., Richardson, A., Parsons, J. T., and Bouton, A. H. (1996) Oncogene 12, 2467-2472[Medline] [Order article via Infotrieve]
  16. Parrizas, M., Saltiel, A. R., and LeRoith, D. (1997) J. Biol. Chem. 272, 154-161[Abstract/Free Full Text]
  17. Yamamoto-Honda, R., Honda, Z., Ueki, K., Tobe, K., Kaburagi, Y., Takahashi, Y., Tamemoto, H., Suzuki, T., Itoh, K., Akanuma, Y., Yazaki, Y., and Kadowaki, T. (1996) J. Biol. Chem. 271, 28677-28681[Abstract/Free Full Text]
  18. Anderson, S. M., Burton, E. A., and Koch, B. L. (1997) J. Biol. Chem. 272, 739-745[Abstract/Free Full Text]
  19. Hunter, S., Koch, B. L., and Anderson, S. M. (1997) Mol. Endocrinol 11, 1213-1222[Abstract/Free Full Text]
  20. Soltoff, S. P., and Cantley, L. C. (1996) J. Biol. Chem. 271, 563-567[Abstract/Free Full Text]
  21. Hartley, D., and Corvera, S. (1996) J. Biol. Chem. 271, 21939-21943[Abstract/Free Full Text]
  22. Fukazawa, T., Miyake, S., Band, V., and Band, H. (1996) J. Biol. Chem. 271, 14554-14559[Abstract/Free Full Text]
  23. Yao, R., and Cooper, G. M. (1995) Science 267, 2003-2006[Medline] [Order article via Infotrieve]
  24. Kennedy, S. G., Wagner, A. J., Conzen, S. D., Jordan, J., Bellacosa, A., Tsichlis, P. N., and Hay, N. (1997) Genes Dev. 11, 701-713[Abstract]
  25. Sawasdikosol, S., Chang, J.-H., Pratt, J. C., Wolf, G., Shoelson, S. E., and Burakoff, S. J. (1996) J. Immunol. 157, 110-116[Abstract]
  26. Fukazawa, T., Reedquist, K. A., Trub, T., Soltoff, S., Panchamoorthy, G., Druker, B., Cantley, L., Shoelson, S. E., and Band, H. (1995) J. Biol. Chem. 270, 19141-19150[Abstract/Free Full Text]
  27. Donovan, J. A., Wange, R. L., Langdon, W. Y., and Samelson, L. E. (1994) J. Biol. Chem. 269, 22921-22924[Abstract/Free Full Text]
  28. Meisner, H., Conway, B. R., Hartley, D., and Czech, M. P. (1995) Mol. Cell. Biol. 15, 3571-3578[Abstract]
  29. Panchamoorthy, G., Fukazawa, T., Miyake, S., Soltoff, S., Reedquist, K., Druker, B., Shoelson, S., Cantley, L., and Band, H. (1996) J. Biol. Chem. 271, 3187-3194[Abstract/Free Full Text]
  30. Smit, L., van Der Horst, G., and Borst, J. (1996) Oncogene 13, 381-389[Medline] [Order article via Infotrieve]
  31. Tanaka, S., Neff, L., Baron, R., and Levy, J. B. (1995) J. Biol. Chem. 270, 14347-14351[Abstract/Free Full Text]
  32. Marcilla, A., Rivero-Lezcano, O. M., Agarwal, A., and Robbins, K. C. (1995) J. Biol. Chem. 270, 9115-9120[Abstract/Free Full Text]
  33. Galisteo, M. L., Dikic, I., Batzer, A. G., Langdon, W. Y., and Schlessinger, J. (1995) J. Biol. Chem. 270, 20242-20245[Abstract/Free Full Text]
  34. Odai, H., Sasaki, K., Iwamatsu, A., Hanazono, Y., Tanaka, T., Mitani, K., Yazaki, Y., and Hirai, H. (1995) J. Biol. Chem. 270, 10800-10805[Abstract/Free Full Text]
  35. Barber, D. L., Mason, J. M., Fukazawa, T., Reedquist, K. A., Druker, B. J., Band, H., and D'Andrea, A. D. (1997) Blood 89, 3166-3174[Abstract/Free Full Text]
  36. Luo, X., and Sando, J. J. (1997) J. Biol. Chem. 272, 12221-12228[Abstract/Free Full Text]
  37. Fitzer-Attas, C. J., Schindler, D. G., Waks, T., and Eshhar, Z. (1997) J. Biol. Chem. 272, 8551-8557[Abstract/Free Full Text]
  38. Lupher, M. L., Reedquist, K., Miyake, S., Langdon, W. Y., and Band, H. (1997) J. Biol. Chem. 271, 24063-24068[Abstract/Free Full Text]
  39. Campbell, K. S., Hager, E. J., Friedrich, J., and Cambier, J. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3982[Abstract]
  40. Sattler, M., Salgia, R., Shrikhande, G., Verma, S., Pisick, E., Prasad, K. V. S., and Griffin, J. D. (1997) J. Biol. Chem. 272, 10248-10253[Abstract/Free Full Text]
  41. Kretzner, L., Blackwood, E. M., and Eisenman, R. N. (1992) Nature 359, 426-429[CrossRef][Medline] [Order article via Infotrieve]
  42. Hannemann, J., Hara, T., Kawai, M., Miyajima, A., Ostertag, W., and Stocking, C. (1995) Mol. Cell. Biol. 15, 2402-2412[Abstract]
  43. Rui, H., Kirken, R. A., and Farrar, W. L. (1994) J. Biol. Chem. 269, 5364-5368[Abstract/Free Full Text]
  44. Yamasaki, K., Taga, T., Hirata, Y., Yawata, H., Kawanishi, Y., Seed, B., Taniguchi, T., Hirano, T., and Kishimoto, T. (1988) Science 241, 825-828[Medline] [Order article via Infotrieve]
  45. DaSilva, L., Howard, O. M. Z., Rui, H., Kirken, R. A., and Farrar, W. L. (1994) J. Biol. Chem. 269, 18267-18270[Abstract/Free Full Text]
  46. Silvennoinen, O., Witthuhn, B. A., Quelle, F. W., Cleveland, J. L., Yi, T., and Ihle, J. N. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8429-8433[Abstract/Free Full Text]
  47. Clevenger, C. V., and Medaglia, M. V. (1994) Mol. Endocrinol. 8, 674-681[Abstract]
  48. Torigoe, T., O'Connor, R., Santoli, D., and Reed, J. C. (1992) Blood 80, 617-624[Abstract]
  49. Anderson, S. M., and Jorgensen, B. (1995) J. Immunol. 155, 1660-1670[Abstract]
  50. Burton, E. A., Hunter, S., Wu, S. C., and Anderson, S. M. (1997) J. Biol. Chem. 272, 16189-16195[Abstract/Free Full Text]
  51. Deleted in proof
  52. Lebrun, J.-J., Ali, S., Sofer, L., Ullrich, A., and Kelly, P. A. (1994) J. Biol. Chem. 269, 14021-14026[Abstract/Free Full Text]
  53. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778[Medline] [Order article via Infotrieve]
  54. Songyang, Z., Shoelson, S. E., McGlade, J., Olivier, P., Pawson, T., Bustelo, X. R., Barbacid, M., Sabe, H., Hanafusa, H., Yi, T., Ren, R., Baltimore, D., Patnofsky, S., Feldman, R. A., and Cantley, L. C. (1994) Mol. Cell. Biol. 14, 2777-2785[Abstract]
  55. Ren, R., Mayer, B. J., Cicchetti, P., and Baltimore, D. (1993) Science 259, 1157-1161[Medline] [Order article via Infotrieve]
  56. Rickles, R. J., Botfield, M. C., Weng, Z., Taylor, J. A., Green, O. M., Brugge, J. S., and Zoller, M. J. (1994) EMBO J. 13, 5598-5604[Abstract]
  57. Rickles, R. J., Botfield, M. C., ZHou, X.-M., Hanery, P. A., Brugge, J. S., and Zoller, M. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10909-10913[Abstract]
  58. Sparks, A. B., Quilliam, L. A., Thorn, J. M., Der, C. J., and Kay, B. K. (1994) J. Biol. Chem. 269, 23853-23956[Abstract/Free Full Text]
  59. Cheadle, C., Ivashchenko, Y., South, V., Searfoss, G. H., French, S., Howk, R., Ricca, G. A., and Jaye, M. (1994) J. Biol. Chem. 269, 24034-24039[Abstract/Free Full Text]
  60. Kawakami, Y., Furue, M., and Kawakami, T. (1989) Oncogene 4, 389-391[Medline] [Order article via Infotrieve]
  61. Davidson, D., Chow, L. M. L., Fournel, M., and Veillette, A. (1992) J. Exp. Med. 175, 1483-1492[Abstract]
  62. Klippel, A., Escobedo, J. A., Fantl, W. J., and Williams, L. T. (1992) Mol. Cell. Biol. 12, 1451-1459[Abstract]
  63. Fantl, W. J., Escobedo, J. A., Martin, G. A., Turck, C. W., del Rosario, M., McCormick, F., and Williams, L. T. (1992) Cell 69, 413-423[Medline] [Order article via Infotrieve]
  64. Watling, D., Guschin, D., Muller, M., Silvennoinen, O., Witthuhn, B. A., Quelle, F. W., Rogers, N. C., Schindler, C., Stark, G. R., and Kerr, I. M. (1993) Nature 366, 166-170[CrossRef][Medline] [Order article via Infotrieve]
  65. Muller, M., Briscoe, J., Laxton, C., Guschin, D., Ziemiecki, A., Silvennoinen, O., Harpur, A. G., Barbieri, G., Witthuhn, B. A., Schindler, C., Pellegrini, S., Wilks, A. F., Ihle, J. N., Stark, G. R., and Kerr, I. M. (1993) Nature 366, 129-135[CrossRef][Medline] [Order article via Infotrieve]
  66. Qureshi, S. A., Leung, S., Kerr, I. M., Stark, G. R., and Darnell, J. E., Jr. (1996) Mol. Biol. Cell 16, 288-293
  67. Larner, A. C., David, M., Feldman, G. M., Igarashi, K.-I., Hackett, R. H., Webb, D. S. A., Sweitzer, S. M., Petricoin, E. F., and Finbloom, D. S. (1993) Science 261, 1730-1733[Medline] [Order article via Infotrieve]
  68. David, M., Petricoin, E. F., III, Igarashi, K.-I., Feldman, G. M., Finbloom, D. S., and Larner, A. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7174-7178[Abstract]
  69. Gilmour, K. C., and Reich, N. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6850-6854[Abstract]
  70. Gao, J., Hughes, J. P., Auperin, B., Buteau, H., Edery, M., Zhuang, H., Wojchowski, D. M., and Horseman, N. D. (1996) Mol. Endocrinol. 10, 847-856[Abstract]
  71. Raffe, G. D., Parmar, K., and Rosenberg, N. (1996) J. Biol. Chem. 271, 4640-4645[Abstract/Free Full Text]
  72. Joung, I., Strominger, J. L., and Shin, J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5991-5995[Abstract/Free Full Text]
  73. Park, I., Chung, J., Walsh, C. T., Yan, Y., Strominger, J. L., and Shin, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12338-12342[Abstract]
  74. Pendergast, A. M., Muller, A. J., Havlik, M. H., Maru, Y., and Witte, O. (1991) Cell 66, 161-171[Medline] [Order article via Infotrieve]
  75. Vanhaesebroeck, B., Leevers, S. J., Panayotou, G., and Waterfield, M. D. (1997) Trends Biochem. Sci. 22, 267-272[CrossRef][Medline] [Order article via Infotrieve]
  76. Yu, J., Zhang, Y., McIlroy, J., Rordorf-Nikolic, T., Orr, G. A., and Backer, J. M. (1998) Mol. Cell. Biol. 18, 1379-1387[Abstract/Free Full Text]
  77. Dombrosky-Ferlan, P. M., and Corey, S. J. (1997) Oncogene 14, 2019-2024[CrossRef][Medline] [Order article via Infotrieve]
  78. Feshchenko, E. A., Langdon, W. Y., and Tsygankov, A. Y. (1998) J. Biol. Chem. 273, 8323-8331[Abstract/Free Full Text]
  79. Deckert, M., Elly, C., Altman, A., and Liu, Y.-C. (1998) J. Biol. Chem. 273, 8867-8874[Abstract/Free Full Text]
  80. Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R. J., Reese, C. B., and Cohen, P. (1997) Current Biol. 7, 261-269[Medline] [Order article via Infotrieve]
  81. Stokoe, D., Stephens, L. R., Copeland, T., Gaffney, P. R. J., Reese, C. B., Painter, G. F., Holmes, A. B., McCormick, F., and Hawkins, P. T. (1997) Science 277, 567-570[Abstract/Free Full Text]
  82. Franke, T. F., Kaplan, D. R., Cantley, L. C., and Toker, A. (1997) Science 275, 665-668[Abstract/Free Full Text]
  83. Datta, K., Bellacosa, A., Chan, T. O., and Tsichlis, P. N. (1996) J. Biol. Chem. 271, 30835-30839[Abstract/Free Full Text]
  84. Franke, T. F., Yang, S.-I., Chan, T. O., Datta, K., Kazlauskas, A., Morrison, D. K., Kaplan, D. R., and Tsichlis, P. N. (1995) Cell 81, 727-736[Medline] [Order article via Infotrieve]
  85. Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M. E. (1997) Cell 91, 231-241[Medline] [Order article via Infotrieve]
  86. Del Peso, L., Gonzalez-Garcia, M., Page, C., Herrera, R., and Nunez, G. (1997) Science 278, 687-689[Abstract/Free Full Text]


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