Abstract

Transformation of hematopoietic cells by the Bcr-abl oncoprotein leads to constitutive tyrosine phosphorylation of a number of cellular polypeptides that function in normal growth factor-dependent cell proliferation. Recent studies have shown that the CrkL adaptor protein and the Cbl protooncoprotein are constitutively tyrosine phosphorylated and form a preformed complex in cells expressing Bcr-abl. In the current study, we have examined cytokine-dependent tyrosine phosphorylation of Cbl and its association with Crk proteins. Erythropoietin (EPO) and interleukin-3 induced a dose and time-dependent tyrosine phosphorylation of Cbl in both EPO-dependent Ba/F3 and DA-3 transfectants, and the erythroid cell line HCD-57. Furthermore, once phosphorylated, Cbl associated with Crk adaptor proteins. Of the three Crk isoforms expressed in hematopoietic cells (CrkL, CrkII, and CrkI), tyrosine phosphorylated Cbl binds preferentially to CrkL and CrkII. The amount of Cbl associated with CrkL and CrkII exceeded the fraction of Cbl associated with Grb2 indicating that unlike other receptor systems, the Cbl-Crk association represents the dominant complex of Cbl in growth factor-stimulated hematopoietic cells. In factor-dependent hematopoietic cell lines, CrkL constitutively associated with the guanine nucleotide release factor, C3G, which is known to interact via Crk src-homology 3 (SH3) domains. Our data suggest that the inducible Cbl-Crk association is a proximal component of a signaling pathway downstream of multiple cytokine receptors.

ERYTHROPOIETIN (EPO), the primary regulator of erythropoiesis,1,2 provides both proliferative and differentiative signals to erythroid progenitors. Through binding of its cognate ligand, the 66 kD EPO receptor (EPO-R) elicits several signal transduction cascades. EPO stimulation of factor-dependent cell lines leads to a rapid activation of the cytoplasmic tyrosine kinase, JAK2,3,4 which is weakly associated with the juxtamembrane domain of the EPO-R. JAK2 then phosphorylates itself and individual tyrosine residues of the EPO-R cytoplasmic region. These phosphotyrosine residues subsequently serve as docking sites for proteins containing src-homology 2 (SH2) domains including STAT55-8 and Shp2.9,10 EPO and interleukin-3 (IL-3) activate the Ras/Raf1/MAP kinase pathway by recruitment of Grb2, either directly through binding to the receptor or indirectly, via adaptor molecules such as Shc11-15 or Shp2.9,15 The SH2 domain of Grb2 binds to a YXN motif16,17 and its SH3 domains are constitutively bound to the guanine nucleotide release factor (GNRF ), Son of Sevenless (Sos).18-24 This interaction then allows Sos to convert Ras to its GTP-bound active form.

Bcr-abl–mediated transformation of hematopoietic cells results in deregulated tyrosine kinase activity and constitutive assembly of tyrosine phosphorylated signaling complexes normally observed only after mitogenic growth factor stimulation. Examples of constitutively tyrosine phosphorylated substrates include STAT5,25 Shc,26 Shp2,27 Paxillin,28 and Vav.29 Analysis of additional tyrosine phosphorylated substrates in Bcr-abl transformed cells have recently identified p120Cbl30-32 and CrkL.30-36 Cbl was originally described as the transforming oncogene of the Cas NS-1 retrovirus resulting in pre-B–cell lymphomas and myelogenous leukemia in mice.37 However, p120cbl, the product of c-Cbl,38 is nontransforming. We and others have recently identified Cbl as a target of tyrosine phosphorylation in response to stimulation through a number of cell surface receptors including the T-cell receptor,39-42 B-cell receptor,43-47 epidermal growth factor (EGF ) receptor,48-53 colony stimulating factor-1 receptor,54,55 and FcγRII/RIII receptor.54,56 Interestingly, GM-CSF and EPO also affect tyrosine phosphorylation of Cbl in UT-7 cells.57 

Crk proteins are the cellular homologues of v-crk, which was originally described as an oncogene from the avian retroviruses CT1058 and ASV-1.59 Three Crk protein variants are expressed in hematopoietic cells: CrkI (28 kD)60; an alternatively spliced CrkII (40 and 42 kD)60; and CrkL (36 kD).61 CrkII and CrkL contain an amino terminal SH2 domain followed by two SH3 domains, whereas the carboxy terminal SH3 domain of CrkII is not found in CrkI. Crk proteins are adaptors with no known catalytic activity. As Grb2 mediates SH3 dependent interaction with Sos, CrkL binds C3G, a unique GNRF. Recent studies performed in COS cells suggest that C3G can catalyze GTP exchange of a distinct Ras family member, Rap1.62 

We and others have recently shown that a prominent complex is induced between tyrosine phosphorylated Cbl and the SH2 domain of Crk proteins on antigen receptor stimulation of T cells63-65 and EGF stimulation of mammary epithelial cells.53 Therefore, recent results that GM-CSF and EPO activated the tyrosine phosphorylation of Cbl57 and that tyrosine phosphorylated Cbl formed a complex with CrkL in Bcr-abl transformed cells30-32 prompted us to investigate if such a complex was induced by normal hematopoietic growth factor stimulation. Using EPO- and IL-3–dependent hematopoietic cell lines, we demonstrate that these cytokines also induce tyrosine phosphorylation of Cbl which promotes complex formation with CrkL and CrkII. CrkL was constitutively associated with the GNRF, C3G. In contrast to other receptor systems, growth factor stimulation did not induce a detectable tyrosine phosphorylation of Grb2-associated Cbl. These results suggest a distinct role for Crk-Cbl complexes in hematopoietic growth factor signaling pathways.

MATERIALS AND METHODS

Cells and cell culture.Ba/F3 and DA-3 cells (generously provided by J. Ihle, Memphis, TN) were maintained in RPMI 1640 medium supplemented with 10% (vol/vol) fetal calf serum (FCS) and 5% conditioned medium from WEHI-3 cells (IL-3 medium). Ba/F3-EPO-R and DA-3-EPO-R cells were maintained in RPMI 1640 medium supplemented with 10% (vol/vol) FCS and 0.5 U/mL of human erythropoietin (Kirin Brewery, Tokyo, Japan). HCD-57 cells were cultured in IMDM supplemented with 30% FCS and 0.1 U/mL of human recombinant EPO (Kirin Brewery, Tokyo, Japan).

Analysis of Cbl tyrosine phosphorylation and identification of associated proteins.For cytokine depletion, cell lines were incubated in RPMI 1640/1% bovine serum albumin (BSA) (no supplemental growth factor) for an 8-hour period and then stimulated for various periods with either no factor, IL-3 (Kirin Brewery), or EPO (Kirin Brewery) at 37°C. Cell lysates were prepared in 50 mmol/L Tris-HCl (pH 8.0) 150 mmol/L NaCl, 1.0% Triton X-100 plus phosphatase and protease inhibitors as previously described.66 Immunoprecipitations were performed with the following rabbit polyclonal antibodies: anti-Cbl,64 anti-CrkL, anti-CrkII, anti-Grb2, or anti-C3G (all from Santa Cruz Biotechnology, Santa Cruz, CA). The anti-Crk monoclonal antibody (Transduction Laboratories, Lexington, KY) recognizes CrkI and CrkII.64 Immune complexes were isolated with Protein A-Sepharose, washed three times with 50 mmol/L Tris HCl (pH 8.0), 150 mmol/L sodium chloride, 0.1% Triton X-100, 10 mmol/L sodium pyrophosphate, 10 mmol/L sodium fluoride, 5 mmol/L EDTA, and 1 mmol/L sodium orthovanadate, and prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously.66 

For immunodepletion experiments, 2 mg of DA-3-EPO-R lysate was incubated in the presence of 1 μg anti-CrkII antibody and Protein A-Sepharose overnight. The supernatant was then incubated with 1 μg of anti-CrkI/II antibody for 1 hour, followed by a 1-hour incubation with Protein A-Sepharose. The immunoprecipitations were washed and analyzed by SDS-PAGE as described above.

Following electrophoretic transfer of proteins to nitrocellulose, the membranes were blocked and incubated with the anti-phosphotyrosine monoclonal antibody, 4G10, washed in 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L sodium chloride, 0.1% Triton X-100 (TBST), followed by horseradish peroxidase (HRP) conjugated sheep antimouse immunoglobulin G (Amersham, Arlington Heights, IL), followed by washing in TBST. After enhanced chemiluminescence detection, the membrane was stripped by incubation in 62.5 mmol/L Tris-HCl (pH 6.8), 2% (wt/vol) sodium dodecyl sulfate, 100 mmol/L β-mercaptoethanol for 1 hour at 55°C. Membranes were blocked and reprobed with the following antibodies: anti-Cbl (Santa Cruz Biotechnology), anti-CrkL, anti-CrkII, anti-Crk, anti-Grb2, or anti-C3G. Incubations were performed with the relevant secondary reagent, either HRP-Protein A (Amersham) or HRP-sheep antimouse IgG and the membrane was washed before enhanced chemiluminescence detection.

RESULTS

Cbl is tyrosine phosphorylated in response to IL-3 and EPO.Our initial experiments using two-dimensional electrophoresis demonstrated that EPO induces the tyrosine phosphorylation of several 120-kD proteins (data not shown). To investigate whether Cbl represented one of these phosphoproteins, we used anti-Cbl immunoprecipitations to assess the tyrosine phosphorylation of Cbl in cell lines expressing the endogenous EPO-R or transfected EPO-R transfectants (Fig 1). IL-3 (lanes 2 and 5) and EPO (lanes 3 and 6) activated tyrosine phosphorylation of Cbl in Ba/F3-EPO-R or DA-3-EPO-R cells. There was an elevated level of uninduced tyrosine phosphorylation in Ba/F3-EPO-R cells (lane 1). EPO also stimulated the tyrosine phosphorylation of Cbl in an erythroid cell line, HCD-57, expressing the endogenous EPO-R polypeptide (lane 8). DA-3-EPO-R cells were used throughout the remainder of this study due to a lower level of endogenous phosphorylation and more robust activation. These results confirm and extend the results of previous studies of EPO and GM-CSF induction of Cbl tyrosine phosphorylation in human UT-7 cells.57 

Fig. 1.

EPO and IL-3 activate tyrosine phosphorylation of Cbl. Ba/F3 (lanes 1-3), DA-3-EPO-R (lanes 4-6), and HCD-57 (lanes 7-10) cells were depleted of cytokine for 4 hours and stimulated with no factor (lanes 1, 4, 7, and 9), 50 U of murine IL-3 (lanes 2 and 5) or 50 U/mL of human EPO (lanes 3, 6, 8, and 10) for 15 minutes. Following cell lysis, an immunoprecipitation was performed with an anti-Cbl polyclonal antibody. Lysate controls are shown in lanes 9 and 10. Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with an anti-phosphotyrosine (pTyr) monoclonal antibody 4G10. Molecular mass standards are indicated.

Fig. 1.

EPO and IL-3 activate tyrosine phosphorylation of Cbl. Ba/F3 (lanes 1-3), DA-3-EPO-R (lanes 4-6), and HCD-57 (lanes 7-10) cells were depleted of cytokine for 4 hours and stimulated with no factor (lanes 1, 4, 7, and 9), 50 U of murine IL-3 (lanes 2 and 5) or 50 U/mL of human EPO (lanes 3, 6, 8, and 10) for 15 minutes. Following cell lysis, an immunoprecipitation was performed with an anti-Cbl polyclonal antibody. Lysate controls are shown in lanes 9 and 10. Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with an anti-phosphotyrosine (pTyr) monoclonal antibody 4G10. Molecular mass standards are indicated.

Cbl tyrosine phosphorylation displays dose and time-dependence in DA-3-EPO-R cells.The dose-dependence of Cbl tyrosine phosphorylation was next examined (Fig 2A). DA-3-EPO-R cells were depleted of cytokine and then stimulated with increasing concentrations of either IL-3 or EPO. Immunoprecipitations were performed with an anti-Cbl antibody, followed by detection with a monoclonal anti-phosphotyrosine antibody, 4G10. Increasing concentrations of IL-3 or EPO resulted in tyrosine phosphorylation of Cbl. Cbl tyrosine phosphorylation was observed at cytokine concentrations as low as 2 U/mL IL-3 or 0.5 U/mL EPO.

Fig. 2.

Dose-dependent activation and time-dependent activation of Cbl tyrosine phosphorylation in DA-3-EPO-R cells. (A) DA-3-EPO-R cells were depleted of cytokine for 8 hours and then stimulated with no added factor (lanes 1, 7, and 13), or various concentrations of IL-3 (lanes 2-6 and 14) or EPO (lanes 8-12 and 15) for 15 minutes as shown. Following cell lysis, an immunoprecipitation was conducted with anti-Cbl polyclonal antibody. Lysate controls corresponding to 50 U/mL stimulation are shown in lanes 13-15. Western blot analysis using the monoclonal anti-phosphotyrosine 4G10 antibody was performed (pTyr immunoblot). Molecular mass standards are indicated. (B) DA-3-EPO-R cells were depleted of cytokine for 8 hours and then stimulated with no added factor (lanes 1 and 8), 50 U/mL IL-3 (lanes 2-7), or 50 U/mL EPO (lanes 9-14) for various times as shown. Following cell lysis, an immunoprecipitation was conducted with anti-Cbl polyclonal antibody. Western blotting using the monoclonal anti-phosphotyrosine 4G10 was performed (pTyr immunoblot). The membrane was then stripped and reprobed with a Cbl polyclonal antibody. Molecular mass standards are indicated.

Fig. 2.

Dose-dependent activation and time-dependent activation of Cbl tyrosine phosphorylation in DA-3-EPO-R cells. (A) DA-3-EPO-R cells were depleted of cytokine for 8 hours and then stimulated with no added factor (lanes 1, 7, and 13), or various concentrations of IL-3 (lanes 2-6 and 14) or EPO (lanes 8-12 and 15) for 15 minutes as shown. Following cell lysis, an immunoprecipitation was conducted with anti-Cbl polyclonal antibody. Lysate controls corresponding to 50 U/mL stimulation are shown in lanes 13-15. Western blot analysis using the monoclonal anti-phosphotyrosine 4G10 antibody was performed (pTyr immunoblot). Molecular mass standards are indicated. (B) DA-3-EPO-R cells were depleted of cytokine for 8 hours and then stimulated with no added factor (lanes 1 and 8), 50 U/mL IL-3 (lanes 2-7), or 50 U/mL EPO (lanes 9-14) for various times as shown. Following cell lysis, an immunoprecipitation was conducted with anti-Cbl polyclonal antibody. Western blotting using the monoclonal anti-phosphotyrosine 4G10 was performed (pTyr immunoblot). The membrane was then stripped and reprobed with a Cbl polyclonal antibody. Molecular mass standards are indicated.

The time dependence of Cbl activation in DA-3-EPO-R cells was next tested (Fig 2B). Tyrosine phosphorylation of Cbl occurred as early as 1 minute after cytokine stimulation and was shown to dissipate by 90 minutes.

Cytokines induce tyrosine phosphorylation of Cbl and binding to CrkL and CrkII.We next examined EPO- and IL-3–dependent association of the Crk family of proteins with tyrosine phosphorylated Cbl. DA-3-EPO-R cells were depleted of cytokine and then stimulated with IL-3 or EPO for increasing time periods. Lysates were then immunoprecipitated with peptide-specific antibodies to CrkL (Fig 3) and CrkII (Fig 4). A 120-kD phosphoprotein displayed time-dependent association with CrkL in response to either IL-3 or EPO stimulation. Stripping and reprobing this blot with a peptide-specific anti-Cbl antibody identified this protein as Cbl (Cbl immunoblot). Equal amounts of CrkL were immunoprecipitated in this experiment as demonstrated by stripping and reprobing the blot with a peptide-specific CrkL antibody (CrkL immunoblot). The CrkL antibody was immune specific since no CrkII or CrkI signal was observed in the CrkL immunoprecipitations as detected by reprobing the blot with the relevant antibodies (data not shown).

Fig. 3.

EPO and IL-3 activate the formation of Cbl/CrkL complexes. DA-3-EPO-R cells were depleted of cytokine for 8 hours and stimulated with no factor (lanes 6 and 12), 50 U/mL of murine IL-3 (lanes 1-5 and 13) or 50 U/mL of human EPO (lanes 7-11 and 14) for various periods of time as shown. Following cell lysis, an immunoprecipitation was performed with a CrkL polyclonal antibody. Lysate controls from 15 minute stimulations are shown in lanes 12-14. Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with 4G10 monoclonal anti-phosphotyrosine antibody followed by HRP-Sheep antimouse IgG. The blot was then stripped and reprobed with either Cbl or CrkL polyclonal antibodies. Molecular mass standards are indicated.

Fig. 3.

EPO and IL-3 activate the formation of Cbl/CrkL complexes. DA-3-EPO-R cells were depleted of cytokine for 8 hours and stimulated with no factor (lanes 6 and 12), 50 U/mL of murine IL-3 (lanes 1-5 and 13) or 50 U/mL of human EPO (lanes 7-11 and 14) for various periods of time as shown. Following cell lysis, an immunoprecipitation was performed with a CrkL polyclonal antibody. Lysate controls from 15 minute stimulations are shown in lanes 12-14. Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with 4G10 monoclonal anti-phosphotyrosine antibody followed by HRP-Sheep antimouse IgG. The blot was then stripped and reprobed with either Cbl or CrkL polyclonal antibodies. Molecular mass standards are indicated.

Fig. 4.

EPO and IL-3 activate the formation of Cbl/CrkII complexes. DA-3-EPO-R cells were depleted of cytokine for 8 hours and stimulated with no factor (lanes 6 and 12), 50 U/mL of murine IL-3 (lanes 1-5 and 13) or 50 U/mL of human EPO (lanes 7-11 and 14) for various periods of time as shown. Following cell lysis, an immunoprecipitation was performed with a CrkII polyclonal antibody. Lysate controls from 15 minute stimulations are shown in lanes 12-14. Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with 4G10 monoclonal anti-phosphotyrosine antibody followed by HRP-Sheep antimouse IgG. The blot was then stripped and reprobed with either Cbl or CrkII polyclonal antibodies. Molecular mass standards are indicated.

Fig. 4.

EPO and IL-3 activate the formation of Cbl/CrkII complexes. DA-3-EPO-R cells were depleted of cytokine for 8 hours and stimulated with no factor (lanes 6 and 12), 50 U/mL of murine IL-3 (lanes 1-5 and 13) or 50 U/mL of human EPO (lanes 7-11 and 14) for various periods of time as shown. Following cell lysis, an immunoprecipitation was performed with a CrkII polyclonal antibody. Lysate controls from 15 minute stimulations are shown in lanes 12-14. Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with 4G10 monoclonal anti-phosphotyrosine antibody followed by HRP-Sheep antimouse IgG. The blot was then stripped and reprobed with either Cbl or CrkII polyclonal antibodies. Molecular mass standards are indicated.

Similarly, Cbl displayed a time-dependent association with CrkII (Fig 4). The amount of a 120-kD phosphoprotein, Cbl, associated with CrkII increased over time on IL-3 and EPO stimulation (Cbl immunoblot). Equal amounts of CrkII were immunoprecipitated in this experiment as shown by reprobing with a peptide-specific anti-CrkII antibody (CrkII immunoblot). The CrkII antibody was specific as no CrkL or CrkI were immunoprecipitated in this experiment (data not shown).

Variable binding of activated Cbl to CrkI and CrkII.We next assessed the relative binding of tyrosine phosphorylated Cbl to CrkI and Crk II (Fig 5). For these studies we used a monoclonal antibody that recognizes both CrkI and CrkII (anti-CrkI/II). Again, IL-3 or EPO stimulated the tyrosine phosphorylation of Cbl (lanes 1-3). An anti-CrkII specific antibody (lanes 4-6) or the anti-CrkI/II antibody (lanes 10-12) immunoprecipitated the tyrosine phosphorylated Cbl polypeptide. Supernatants that had been immunodepleted with the CrkII antibody (lanes 4-6) were next reimmunoprecipitated with the anti-CrkI/II antibody (lanes 7-9). The amount of co-immunoprecipitated tyrosine phosphorylated Cbl in this second immunoprecipitation was markedly reduced when the lysates were precleared with the CrkII-specific antibody (compare lanes 7-9 to lanes 4-6). The pTyr blot was purposefully overexposed to reveal the signal in lanes 7-9. While approximately half of the CrkII was immunoprecipitated with the CrkII specific antibody (CrkI/II immunoblot, lanes 4-6), most of the tyrosine phosphorylated Cbl was found in this immune complex (pTyr immunoblot, lanes 4-6). Taken together, these data demonstrate that activated Cbl binds selectively to CrkII, not CrkI, following cytokine induction.

Fig. 5.

Tyrosine phosphorylated Cbl preferentially associates with CrkII. DA-3-EPO-R cells were depleted of cytokine for 8 hours and stimulated with no factor (lanes 1, 4, 7, and 10), 50 U/mL of murine IL-3 (lanes 1, 5, 8, and 11) or 50 U/mL of human EPO (lanes 3, 6, 9, and 12) for 15 minutes. Following cell lysis, an immunoprecipitation was performed with either an anti-Cbl antibody (lanes 1-3), an anti-CrkII antibody (lanes 4-6), and anti-CrkII antibody followed by an anti-Crk antibody (lanes 7-9), or an anti-Crk antibody (lanes 10-12). The anti-Crk antibody recognizes both CrkI and CrkII. Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with 4G10 monoclonal anti-phosphotyrosine antibody and then stripped and reprobed with either a Cbl polyclonal antibody or a Crk monoclonal antibody. Molecular mass standards are indicated.

Fig. 5.

Tyrosine phosphorylated Cbl preferentially associates with CrkII. DA-3-EPO-R cells were depleted of cytokine for 8 hours and stimulated with no factor (lanes 1, 4, 7, and 10), 50 U/mL of murine IL-3 (lanes 1, 5, 8, and 11) or 50 U/mL of human EPO (lanes 3, 6, 9, and 12) for 15 minutes. Following cell lysis, an immunoprecipitation was performed with either an anti-Cbl antibody (lanes 1-3), an anti-CrkII antibody (lanes 4-6), and anti-CrkII antibody followed by an anti-Crk antibody (lanes 7-9), or an anti-Crk antibody (lanes 10-12). The anti-Crk antibody recognizes both CrkI and CrkII. Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with 4G10 monoclonal anti-phosphotyrosine antibody and then stripped and reprobed with either a Cbl polyclonal antibody or a Crk monoclonal antibody. Molecular mass standards are indicated.

Cbl-Crk association exceeds Cbl-Grb2 association in vivo.Cbl is constitutively associated with the SH3 domains of Grb2 and this fraction of Cbl is a prominent target of tyrosine phosphorylation after stimulation of T and B lymphocyte antigen receptors.41,45 Cbl was also shown to associate with Grb2 in vitro using GST fusions and in Cbl immunoprecipitations in cytokine-dependent UT-7 cells but tyrosine phosphorylation of this fraction was not assessed.57 In order to compare the amount of Cbl associated with either Grb2 or CrkL in DA-3-EPO-R cells, immunoprecipitations were performed with a peptide-specific Grb2 antibody and a CrkL antibody (Fig 6). Immunoprecipitation with a peptide-specific Cbl antibody confirmed that IL-3 or EPO stimulation of DA-3-EPO-R cells increased Cbl tyrosine phosphorylation (lanes 2 and 3) and enhanced association of Cbl with CrkL (lanes 5 and 6) and CrkII (data not shown). Grb2 immunoprecipitated Shc (52 kD) and Shp2 (68 kD) after IL-3 or EPO stimulation (lanes 8 and 9) and the EPO-R (72 kD) after EPO stimulation (lane 9), as previously demonstrated.9,11,15 An additional unknown 145-kD phosphoprotein, most likely SHIP,67-69 a recently identified inositol 5′-phosphatase was observed to co-immunoprecipitate in both the CrkL and Grb2 immunoprecipitates (lanes 3-9). However, no 120 kD phosphoproteins were observed in anti-Grb2 immunoprecipitates (lanes 7-9). The 72-kD EPO-R was shown to co-immunoprecipitate with CrkL (lane 6) and Grb2 (lane 9) EPO stimulation of DA-3-EPO-R cells. Stripping and reprobing the nitrocellulose with an anti-Grb2 antibody demonstrated that a low, but detectable amount of Grb2 co-immunoprecipitated with Cbl (Grb2 immunoblot, lanes 1-3). IL-3 or EPO-dependent association of Cbl with CrkL or CrkII was also observed in Ba/F3-EPO-R cells (data not shown). Thus, while a number of Grb2-associated polypeptides were prominently tyrosine phosphorylated, Cbl was not among these. Moreover, the fraction of Cbl associated with Crk proteins far exceeded the amount of Cbl constitutively bound to Grb2.

Fig. 6.

Cytokine induced Cbl/Crk complexes exceed Cbl/Grb2 complexes in DA-3-EPO-R cells. DA-3-EPO-R cells were depleted of cytokine for 8 hours and stimulated with no factor (lanes 1, 4, and 7), 50 U/mL of murine IL-3 (lanes 2, 5, and 8) or 50 U/mL of human EPO (lanes 3, 6, and 9) for 15 minutes. Following cell lysis, an immunoprecipitation was performed with either an anti-Cbl polyclonal antibody (lanes 1-3), an anti-CrkL polyclonal antibody (lanes 4-6), or an anti-Grb2 polyclonal antibody (lanes 7-9). Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with 4G10 monoclonal anti-phosphotyrosine antibody and then stripped and reprobed with either Cbl or Grb2 polyclonal antibodies. The exposure periods of the pTyr immunoblot are 3 minutes (lanes 1-6) and 1 minute (lanes 7-9). The migration of EPO-R, Shp2, and Shc were determined by stripping and reprobing the membrane with specific antibodies (data not shown). Molecular mass standards are indicated.

Fig. 6.

Cytokine induced Cbl/Crk complexes exceed Cbl/Grb2 complexes in DA-3-EPO-R cells. DA-3-EPO-R cells were depleted of cytokine for 8 hours and stimulated with no factor (lanes 1, 4, and 7), 50 U/mL of murine IL-3 (lanes 2, 5, and 8) or 50 U/mL of human EPO (lanes 3, 6, and 9) for 15 minutes. Following cell lysis, an immunoprecipitation was performed with either an anti-Cbl polyclonal antibody (lanes 1-3), an anti-CrkL polyclonal antibody (lanes 4-6), or an anti-Grb2 polyclonal antibody (lanes 7-9). Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with 4G10 monoclonal anti-phosphotyrosine antibody and then stripped and reprobed with either Cbl or Grb2 polyclonal antibodies. The exposure periods of the pTyr immunoblot are 3 minutes (lanes 1-6) and 1 minute (lanes 7-9). The migration of EPO-R, Shp2, and Shc were determined by stripping and reprobing the membrane with specific antibodies (data not shown). Molecular mass standards are indicated.

CrkL constitutively associates with the guanine nucleotide release factor, C3G.Previous studies have shown that the Crk SH3 domain binds to a unique guanine nucleotide release factor, C3G.70 Therefore, we tested the association of CrkL and C3G in DA-3-EPO-R cells (Fig 7). When the blot was probed with an anti-C3G antibody, a 130-140 kD protein was observed in the CrkL immunoprecipitation which co-migrated with C3G (C3G immunoblot, lanes 7-9). However, C3G was not detected to co-immunoprecipitate with Cbl (C3G immunoblot, lanes 1-3). Stripping and reprobing the blot with an anti-Cbl antibody confirmed that Cbl associates with CrkL (Fig 7, lanes 5 and 6) but not with C3G (lanes 7-9). CrkL failed to associate with either Cbl or C3G when the membrane was reprobed with a peptide-specific CrkL antibody (data not shown). Interestingly, C3G appeared to migrate with a slower mobility after IL-3 or EPO stimulation (lanes 4-6). This probably corresponds to serine or threonine phosphorylation as previously described for the related GNRF, Sos.71,72 

Fig. 7.

CrkL constitutively associates with C3G. DA-3-EPO-R cells were depleted of cytokine for 8 hours and stimulated with no factor (lanes 1, 4, and 7), 50 U/mL of murine IL-3 (lanes 2, 5, and 8) or 50 U/mL of human EPO (lanes 3, 6, and 9) for 15 minutes. Following cell lysis, an immunoprecipitation was performed with either an anti-Cbl antibody (lanes 1-3), an anti-CrkL antibody (lanes 4-6), or an anti-C3G antibody (lanes 7-9). Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with 4G10 monoclonal anti-phosphotyrosine antibody and then reprobed with either a Cbl or C3G polyclonal antibodies. The assignment of EPO-R and Shc was determined by stripping and reprobing the membrane with peptide-specific antibodies (data not shown). Molecular mass standards are indicated.

Fig. 7.

CrkL constitutively associates with C3G. DA-3-EPO-R cells were depleted of cytokine for 8 hours and stimulated with no factor (lanes 1, 4, and 7), 50 U/mL of murine IL-3 (lanes 2, 5, and 8) or 50 U/mL of human EPO (lanes 3, 6, and 9) for 15 minutes. Following cell lysis, an immunoprecipitation was performed with either an anti-Cbl antibody (lanes 1-3), an anti-CrkL antibody (lanes 4-6), or an anti-C3G antibody (lanes 7-9). Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with 4G10 monoclonal anti-phosphotyrosine antibody and then reprobed with either a Cbl or C3G polyclonal antibodies. The assignment of EPO-R and Shc was determined by stripping and reprobing the membrane with peptide-specific antibodies (data not shown). Molecular mass standards are indicated.

There was an EPO-dependent association of the 72 kD phosphoprotein with CrkL (Fig 6; pTyr immunoblot, lane 6; Fig 7, pTyr immunoblot, lane 6) and with C3G on long exposures of the anti-phosphotyrosine immunoblot (data not shown). Stripping and reprobing these blots with an anti-EPO-R antibody confirmed the identity of this phosphoprotein (data not shown). The EPO-R contains a sequence motif Y484SHP, which represents a potential binding site for the Crk SH2 domain.73 However, the EPO-R was only shown to associate with CrkL (Figs 6 and 7) and not with CrkII (Figs 4 and 5). Whether this presents differences in antibody affinity or individual Crk SH2 domain selectivity remains to be investigated.

DISCUSSION

Cbl has emerged as an important signal transduction protein downstream of a number of tyrosine kinase associated cell surface receptors. Recent analyses have focused on delineating the association of Cbl with other signaling proteins, since Cbl possesses a large proline-rich region that mediates binding to SH3 domains and a number of potential tyrosine phosphorylation sites. One phosphorylation-dependent interaction of significant interest is the complex of Cbl with Crk proteins, which was observed constitutively in Bcr-abl transformed cells 30-32 and on receptor stimulation of B47 and T lymphocytes63,64,74 and EGF-dependent mammary cells.53 Given that Bcr-abl–induced phosphorylation commonly involves substrates normally involved in growth factor signaling we have examined the association of Cbl and Crk in IL-3 and EPO-dependent hematopoietic cells.

In the current work, we show that IL-3 and EPO stimulation of cytokine-dependent hematopoietic cells activates tyrosine phosphorylation of Cbl. In other experiments, Cbl was phosphorylated in response to IL-2, IL-15, and EPO but not IL-4 stimulation of CTLL-EPO-R cells (data not shown). Tyrosine phosphorylation of Cbl resulted in its association with CrkL and CrkII. IL-3 and EPO dependent Cbl tyrosine phosphorylation displays rapid induction similar to other substrates of cytokine-dependent tyrosine phosphorylation. These data complement those describing constitutive association of Cbl and CrkL in cell lines transformed with Bcr-abl30-32 and the T-cell receptor dependent binding of Cbl and Crk adaptor proteins.63,64,74 

Ras activation is mediated by the interaction of the adaptor protein Grb2 with the GNRF, Sos, resulting in the conversion of Ras to its GTP-bound form.18-24 A parallel signaling pathway has been suggested which couples the Crk adaptor proteins to the GNRF, C3G, which acts to convert Rap1 to an activated state.62 Here we show a stable association of C3G with CrkL. Although a ternary Cbl-CrkL-C3G complex could not be detected in IL-3 or EPO-dependent cells (Fig 7), such a complex was observed in T-cell receptor stimulated lymphocytes engineered to overexpress Cbl.64 Therefore, the ability to detect a ternary Cbl-CrkL-C3G complex may be dependent on the expression level of these proteins, antibody specificity, or stability of this complex under the lysis and/or immunoprecipitation conditions used in this study. The cytokine-dependent stimulation of Rap1 remains to be tested.

Cbl binds to SH3 domains of Grb239,41,45,49,53,55,63 and thereby forms a constitutive complex distinct from the Grb2-Sos complex. Notably, this pool of Cbl is a target of rapid tyrosine phosphorylation on stimulation of T-39,41 and B-lymphocyte antigen receptors45 and the EGF receptor49,53 Surprisingly, here we show that Grb2-associated Cbl is a substantially smaller pool compared with the amount of Cbl that is associated with Crk proteins. More importantly, Grb2 associated Cbl was not detectably tyrosine phosphorylated on IL-3 or EPO stimulation. Thus, Cbl-Crk is the predominant in vivo association in factor-dependent hematopoietic cells as compared with the Cbl-Grb2 complex. Earlier published in vitro studies completed with bacterially expressed fusion proteins must be interpreted with caution. Given that Grb2 and Crk proteins preferentially associate with Sos and C3G, respectively, it appears that selective Crk-C3G complex formation may regulate the balance of nucleotide exchange factors on cytokine stimulation. In this regard it is noteworthy that the C3G target, Rap1, has been shown to down-regulate Ras activity.62 

It is unclear how the EPO-R signals to Cbl. We have not obtained any evidence that Cbl associates with the EPO-R. However, tyrosine phosphorylated Cbl binds CrkL and/or CrkII. Both the EPO-R (Y484SHP) and Cbl (Y360LFP, Y681MTP, and Y774DVP) contain putative binding sites for the Crk SH2 domain.73 In vitro mixing experiments using the isolated SH2 domain of Crk expressed as a GST fusion protein demonstrate that in vitro Crk can bind both to Cbl and the EPO-R (data not shown). Cbl Y774 is thought to mediate Crk binding based on phosphopeptide competition studies completed in the analysis of T-cell receptor activation.64,65 Crk proteins bind to both EPO-R and Cbl in vitro; however, in vivo the predominant complex appears to be Cbl-Crk. Mutagenesis of the corresponding tyrosines of Cbl and the EPO-R will be necessary to better address these questions. Alternatively, the EPO-R could signal to Cbl by novel adaptor proteins.

Taken together, our data support two parallel and interrelated signal transduction pathways downstream of the EPO-R (Fig 8). EPO-R activation results in the dose- and time-dependent tyrosine phosphorylation of Cbl. Once phosphorylated, Cbl can interact with complexes of Crk adaptor proteins, which associate with a specific GNRF, C3G. It is unclear how Cbl couples to the EPO-R. However, immunoprecipitation experiments demonstrate that only a small fraction of Cbl associates with Grb2 in DA-3 Cells. Crk proteins can potentially interact with the tyrosine phosphorylated EPO-R through an SH2-dependent association. However, immunoprecipitation experiments reveal that CrkL, CrkII, and CrkI associate with Cbl. Future experiments will address whether EPO activates Rap1.

Fig. 8.

EPO activates the Cbl-Crk-C3G cascade. EPO activates multiple signaling cascades within hematopoietic cells. A critical adaptor protein necessary for Ras activation, Grb2, can bind constitutively to Cbl. However, the predominant complex following cytokine activation is the Cbl-Crk complex. The preferred complexes are indicated by bold lines and larger arrowheads. Dashed lines indicate associations that occur in vitro, based on immunoprecipitation experiments (ie, Grb2-Cbl and EPO-R-Crk), but may have lesser significance in vivo. It remains unclear how EPO-R couples to Cbl and whether EPO-R activates Rap1.

Fig. 8.

EPO activates the Cbl-Crk-C3G cascade. EPO activates multiple signaling cascades within hematopoietic cells. A critical adaptor protein necessary for Ras activation, Grb2, can bind constitutively to Cbl. However, the predominant complex following cytokine activation is the Cbl-Crk complex. The preferred complexes are indicated by bold lines and larger arrowheads. Dashed lines indicate associations that occur in vitro, based on immunoprecipitation experiments (ie, Grb2-Cbl and EPO-R-Crk), but may have lesser significance in vivo. It remains unclear how EPO-R couples to Cbl and whether EPO-R activates Rap1.

Cbl tyrosine phosphorylation has been demonstrated in several systems suggesting that it plays a vital role in mitogenesis and/or other cellular responses to receptor stimulation. The present findings suggest an approach to assess the role of Cbl-Crk association in mitogenesis through overexpression of various mutant Cbl and Crk proteins in factor-dependent hematopoietic cell lines.

ACKNOWLEDGMENT

We acknowledge the kind gift of IL-15 from David Cosman and Judy Giri, Immunex Corp, Seattle, WA. We thank Bernard Mathey-Prevot, Martin Carroll, Cheryl Miller, and Sonya Penfold for helpful comments on the manuscript.

Supported by the National Institutes of Health Grant No. RO1 DK 43889-01 to A.D.D. and AR6308 to H.B. and American Cancer Society Grant No. IM-770 to H.B. D.L.B. is a Special Fellow of the Leukemia Society of America and is also supported by the Medical Research Council of Canada. K.A.R. is a predoctoral fellow of the Howard Hughes Medical Institute and Ryan Foundation. A.D.D. is a Scholar of the Leukemia Society of America.

Address reprint requests to Alan D. D'Andrea, MD, Dana-Farber Cancer Institute, Pediatric Oncology, 44 Binney St, Boston, MA 02115.

REFERENCES

REFERENCES
1
Wu
H
Liu
X
Jaenisch
R
Lodish
HF
Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor.
Cell
83
1995
59
2
Lin
C-S
Lim
S-K
D'Agati
V
Constantini
F
Differential effects of an erythropoietin receptor gene disruption on primitive and definitive erythropoiesis.
Genes Dev
10
1996
154
3
Witthuhn
BA
Quelle
FW
Silvennoinen
O
Yi
T
Tang
B
Miura
O
Ihle
JN
JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin.
Cell
74
1993
227
4
Barber
DL
D'Andrea
AD
Erythropoietin and interleukin-2 activate distinct JAK kinase family members.
Mol Cell Biol
14
1994
6506
5
Damen
JE
Wakao
H
Miyajima
A
Krosl
J
Humphries
RK
Cutler
RL
Krystal
G
Tyrosine 343 in the erythropoietin receptor positively regulates erythropoietin-induced cell proliferation and Stat5 activation.
EMBO J
14
1995
5557
6
Gobert
S
Chretien
S
Gouilleux
F
Muller
O
Pallard
C
Dusanter-Fourt
I
Groner
B
Lacombe
C
Gisselbrecht
S
Mayeux
P
Identification of tyrosine residues within the intracellular domain of the erythropoietin receptor crucial for STAT5 activation.
EMBO J
15
1996
2434
7
Quelle
FW
Wang
D
Nosaka
T
Thierfelder
WE
Stravopodis
D
Weinstein
Y
Ihle
JN
Erythropoietin induces activation of Stat5 through association with specific tyrosine on the receptor that are not required for a mitogenic response.
Mol Cell Biol
16
1996
1622
8
Klingmuller
U
Bergelson
S
Hsiao
JG
Lodish
HF
Multiple tyrosine residues in the cytosolic domain of the erythropoietin receptor promote activation of STAT5.
Proc Natl Acad Sci USA
93
1996
8324
9
Tauchi
T
Feng
GS
Shen
R
Hoatlin
M
Bagby
GJ
Kabat
D
Lu
L
Broxmeyer
HE
Involvement of SH2-containing phosphotyrosine phosphatase Syp in erythropoietin receptor signal transduction pathways.
J Biol Chem
270
1995
5631
10
Tauchi
T
Damen
JE
Toyama
K
Feng
G-S
Broxmeyer
HE
Krystal
G
Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis.
Blood
87
1996
4495
11
Damen
JE
Liu
L
Cutler
RL
Krystal
G
Erythropoietin stimulates the tyrosine phosphorylation of Shc and its association with Grb2 and a 145-Kd tyrosine phosphorylated protein.
Blood
82
1993
2296
12
Cutler
RL
Liu
L
Damen
JE
Krystal
G
Multiple cytokines induce the tyrosine phosphorylation of Shc and its association with Grb2 in hemopoietic cells.
J Biol Chem
268
1993
21463
13
Miura
Y
Miura
O
Ihle
JN
Aoki
N
Activation of the mitogen-activated protein kinase pathway by the erythropoietin receptor.
J Biol Chem
269
1994
29962
14
He
TC
Jiang
N
Zhuang
H
Wojchowski
DM
Erythropoietin-induced recruitment of Shc via a receptor phosphotyrosine-independent, Jak2-associated pathway.
J Biol Chem
270
1995
11055
15
Barber
DL
Corless
CN
Xia
K
Roberts
TM
D'Andrea
AD
Erythropoietin activates Raf1 by a Shc independent pathway in CTLL-EPO-R cells.
Blood
89
1997
55
16
Songyang
Z
Shoelson
SE
McGlade
J
Olivier
P
Pawson
T
Bustelo
XR
Barbacid
M
Sabe
H
Hanafusa
H
Yi
T
Ren
R
Baltimore
D
Ratnofsky
S
Feldman
RA
Cantley
LC
Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav.
Mol Cell Biol
14
1994
2777
17
Lanfrancone
L
Pelicci
G
Brizzi
MF
Arouica
MG
Casciari
C
Giuli
S
Pegoraro
L
Pawson
T
Pelicci
PG
Overexpression of Shc proteins potentiates the proliferative response to the granulocyte-macrophage colony-stimulating factor and recruitment of Grb2/SoS and Grb2/p140 complexes to the beta receptor subunit.
Oncogene
10
1995
907
18
Egan
SE
Giddings
BW
Brooks
MW
Buday
L
Sizeland
AM
Weinberg
RA
Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation.
Nature
363
1993
45
19
Rozakis-Adcock
M
Fernley
R
Wade
J
Pawson
T
Bowtell
D
The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.
Nature
363
1993
83
20
Li
N
Batzer
A
Daly
R
Yajnik
V
Skolnik
E
Chardin
P
Bar
SD
Margolis
B
Schlessinger
J
Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling.
Nature
363
1993
85
21
Gale
NW
Kaplan
S
Lowenstein
EJ
Schlessinger
J
Bar-Sagi
D
Grb2 mediates the EGF-dependent activation of guanine nucleotide exchange on Ras.
Nature
363
1993
88
22
Buday
L
Downward
J
Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor.
Cell
73
1993
611
23
Baltensperger
K
Kozma
LM
Cherniack
AD
Klarlund
JK
Chawla
A
Banerjee
U
Czech
MP
Binding of the Ras activator Son of Sevenless to insulin receptor substrate-1 signaling complexes.
Science
260
1993
1950
24
Skolnik
EY
Batzer
A
Li
N
Lee
CH
Lowenstein
E
Mohammadi
M
Margolis
B
Schlessinger
J
The function of GRB2 in linking the insulin receptor to Ras signaling pathways.
Science
260
1993
1953
25
Carlesso
N
Frank
DA
Griffin
JD
Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl.
J Exp Med
183
1996
811
26
Matsuguchi
T
Salgia
R
Hallek
M
Eder
M
Druker
B
Ernst
TJ
Griffin
JD
Shc phosphorylation in myeloid cells is regulated by granulocyte macrophage colony-stimulating factor, interleukin-3, and steel factor and is constitutively increased by p210BCR/ABL.
J Biol Chem
269
1994
5016
27
Tauchi
T
Feng
GS
Shen
R
Song
HY
Donner
D
Pawson
T
Broxmeyer
HE
SH2-containing phosphotyrosine phosphatase Syp is a target of p210Bcr/Abl tyrosine kinase.
J Biol Chem
269
1994
15381
28
Salgia
R
Uemura
N
Okuda
K
Li
J-L
Pisick
E
Sattler
M
Jong
RD
Druker
B
Heisterkamp
N
Chen
LB
Groffen
J
Griffen
JD
CRKL links p210BCR/ABL with paxillin in chronic myelogenous leukemia cells.
J Biol Chem
270
1995
29145
29
Matsuguchi
T
Inhorn
R
Carlesso
N
Xu
G
Druker
B
Griffin
JD
Tyrosine phosphorylation of p95VAV in myeloid cells is regulated by GM-CSF, IL-3, and steel factor and is constitutively increased by p210 Bcr/Abl.
EMBO J
14
1995
257
30
de Jong
R
ten Hoeve
J
Heisterkamp
N
Groffen
J
Crkl is complexed with tyrosine-phosphorylated Cbl in Ph-positive leukemia.
J Biol Chem
270
1995
21468
31
Ribon
V
Hubbell
S
Herrera
R
Saltiel
AR
The product of the cbl oncogene forms stable complexes in vivo with endogenous Crk in a tyrosine phosphorylation-dependent manner.
Mol Cell Biol
16
1996
45
32
Sattler
M
Salgia
R
Okuda
K
Uemura
N
Durstin
MA
Pisick
E
Xu
G
Li
J-L
Prasad
KV
Griffin
JD
The proto-oncogene product p120CBL and the adaptor proteins CRKL and c-CRK like c-ABL, p190 BCR/ABL and p210BCR/ABL to the phosphatidylinositol-3′ kinase pathway.
Oncogene
12
1996
839
33
ten Hoeve
J
Kaartinen
V
Fioretos
T
Haataja
L
Voncken
JW
Heisterkamp
N
Groffen
J
Cellular interactions of CRKL, and SH2-SH3 adaptor protein.
Cancer Res
54
1994
2563
34
ten Hoeve
J
Arlinghaus
RB
Guo
JQ
Heisterkamp
N
Groffen
J
Tyrosine phosphorylation of CRKL in Philadelphia+ leukemia.
Blood
84
1994
1731
35
Oda
T
Heaney
C
Hagopian
JR
Okuda
K
Griffin
JD
Druker
BJ
Crkl is the major tyrosine-phosphorylated protein in neutrophils from patients with chronic myelogenous leukemia.
J Biol Chem
269
1994
32740
36
Nichols
GL
Raines
MA
Vera
JC
Lacomis
L
Tempst
P
Golde
DW
Identification of CRKL as the constitutively phosphorylated 39-kD tyrosine phosphoprotein in chronic myelogenous leukemia cells.
Blood
84
1994
2912
37
Langdon
WY
Hartley
JW
Klinken
SP
Ruscetti
SK
Morse
III HC
v-cbl, an oncogene from a dual-recombinant murine retrovirus that induces early B-lineage lymphomas.
Proc Natl Acad Sci USA
86
1989
1168
38
Blake
TJ
Shapiro
M
Morse
III HC
Langdon
WY
The sequences of the human and mouse c-cbl proto-oncogenes show v-cbl was generated by a large truncation encompassing a proline-rich domain and a leucine zipper-like motif.
Oncogene
6
1991
653
39
Donovan
JA
Wange
RL
Langdon
WY
Samelson
LE
The protein product of the c-cbl protooncogene is the 120-kDa tyrosine-phosphorylated protein in Jurkat cells activated via the T cell antigen receptor.
J Biol Chem
269
1994
22925
40
Meisner
H
Conway
BR
Hartley
D
Czech
MP
Interactions of Cbl with Grb2 and phosphatidylinositol 3′-kinase in activated Jurkat cells.
Mol Cell Biol
15
1995
3571
41
Fukazawa
T
Reedquist
KA
Trub
T
Soltoff
S
Panchamoorthy
G
Druker
B
Cantley
L
Shoelson
SE
Band
H
The SH3 domain-binding T cell tyrosyl phosphoprotein p120. Demonstration of its identity with the c-cbl protooncogene product and in vivo complexes with Fyn, Grb2, and phosphatidylinositol 3-kinase.
J Biol Chem
270
1995
19141
42
Fournel
M
Davidson
D
Weil
R
Veillette
A
Association of tyrosine protein kinase Zap-70 with the protooncogene product p120c-cbl in T lymphocytes.
J Exp Med
183
1996
301
43
Cory
GO
Lovering
RC
Hinshelwood
S
MacCarthy
ML
Levinsky
RJ
Kinnon
C
The protein product of the c-cbl protooncogene is phosphorylated after B cell receptor stimulation and binds the SH3 domain of Bruton's tyrosine kinase.
J Clin Invest
87
1991
915
44
Kim
TJ
Kim
Y-T
Pillai
S
Association of activated phosphatidylinositol 3-kinase with p120cbl in antigen receptor-ligated B cells.
J Biol Chem
270
1995
27504
45
Panchamoorthy
G
Fukazawa
T
Miyake
S
Soltoff
S
Reedquist
K
Druker
B
Shoelson
S
Cantley
L
Band
H
p120cbl is a major substrate of tyrosine phosphorylation upon B cell antigen receptor stimulation, and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc adaptors, and the p85 subunit of phosphatidylinositol 3-kinase.
J Biol Chem
271
1996
3187
46
Tezuka
T
Umemori
H
Fusaki
N
Yagi
T
Takata
M
Kurosaki
T
Yamamoto
T
Physical and functional association of the cbl protooncogene product with an src-family protein tyrosine kinase, p53/p56lyn, in the B cell antigen receptor-mediated signaling.
J Exp Med
183
1996
675
47
Smit
L
Horst
Gvd
Borst
J
Sos, vav and C3G participate in B cell receptor-induced signaling pathways and differentially associate with Shc-Grb2, Crk, and Crk-L adaptors.
J Biol Chem
271
1996
8564
48
Galisteo
ML
Dikic
I
Batzer
AG
Langdon
WY
Schlessinger
J
Tyrosine phosphorylation of the c-cbl proto-oncogene protein product and association with epidermal growth factor (EGF ) receptor upon EGF, stimulation.
J Biol Chem
270
1995
20242
49
Meisner
H
Czech
MP
Coupling of the proto-oncogene product c-Cbl to the epidermal growth factor receptor.
J Biol Chem
270
1995
25332
50
Soltoff
SP
Cantley
LC
p120cbl is a cytosolic adapter protein that associates with phosphoinositide 3-kinase in response to epidermial growth factor in PC12 and other cells.
J Biol Chem
271
1996
563
51
Bowtell
DDL
Langdon
WY
The protein product of the c-cbl oncogene rapidly complexes with the EGF receptor and is tyrosine phosphorylated following EGF stimulation.
Oncogene
11
1995
1561
52
Levkowitz
G
Klapper
LN
Tzahar
E
Freywald
A
Sela
M
Yarden
Y
Coupling of the c-Cbl protooncogene product to ErbB-1/EGF-receptor but not to other ErbB proteins.
Oncogene
12
1996
1117
53
Fukazawa
T
Miyake
S
Band
V
Band
H
Tyrosine phosphorylation of Cbl upon epidermal growth factor (EGF ) stimulation and its association with EGF receptor and downstream signalling proteins.
J Biol Chem
271
1996
14554
54
Tanaka
S
Neff
L
Baron
R
Levy
JB
Tyrosine phosphorylation and translocation of the c-cbl protein after activation of tyrosine kinase signaling pathways.
J Biol Chem
270
1995
14347
55
Wang
Y
Yeung
Y-G
Langdon
WY
Stanley
ER
c-cbl is transiently tyrosine-phosphorylated, ubiquintinated, and membrane-targeted following CSF-1 stimulation of macrophages.
J Biol Chem
271
1996
17
56
Marcilla
A
Riverolezcano
OM
Agarwal
A
Robbins
KC
Identification of the major tyrosine kinase substrate in signaling complexes formed after engagement of Fcγ receptors.
J Biol Chem
270
1995
9115
57
Odai
H
Sasaki
K
Iwamatsu
A
Hanazono
Y
Tanaka
T
Mitani
K
Yazaki
Y
Hirai
H
The proto-oncogene product c-Cbl becomes tyrosine phosphorylated by stimulation with GM-CSF or Epo and constitutively binds to the SH3 domain of Grb2/Ash in human hematopoietic cells.
J Biol Chem
270
1995
10800
58
Mayer
BJ
Hamaguchi
M
Hanafusa
H
A novel viral oncogene with structural similarity to phospholipase C.
Nature
332
1988
272
59
Tsuchie
H
Chang
CH
Yoshida
M
Vogt
PK
A newly isolated avian sarcoma virus, ASV-1, carries the crk oncogene.
Oncogene
6
1991
653
60
Matsuda
M
Tanaka
S
Nagata
S
Kojima
A
Kurata
T
Shibuya
M
Two species of human CRK cDNA encode proteins with distinct biological activities.
Lancet
337
1991
987
61
ten Hoeve
J
Morris
C
Heisterkamp
N
Groffen
J
Isolation and chromosomal localization of CRKL, a human crk-like gene.
Oncogene
8
1993
2853
62
Gotoh
T
Hattori
S
Nakamura
S
Kitayama
H
Noda
M
Takai
Y
Kaibuchi
K
Matsui
H
Hatas
O
Takahashi
H
Kurata
T
Matsuda
M
Identification of Rap1 as a target for the Crk SH2 domain-binding guanine nucleotide-releasing factor C3G.
Mol Cell Biol
15
1995
6746
63
Buday
L
Khwaja
A
Sipeki
S
Farago
A
Downward
J
Interactions of Cbl with two adaptor proteins, Grb2 and Crk, upon T cell activation.
J Biol Chem
271
1996
6159
64
Reedquist
KA
Fukazawa
T
Panchamoorthy
G
Langdon
WY
Shoelson
SE
Druker
BJ
Band
H
Stimulation through the T cell receptor induces Cbl association with Crk proteins and guanine nucleotide exchange protein C3G.
J Biol Chem
271
1996
8435
65
Sawasdikosol
S
Chang
J-H
Pratt
JC
Wolf
G
Shoelson
SE
Burakoff
SJ
Tyrosine-phosphorylated Cbl binds to Crk after T cell activation.
J Immunol
151
1996
110
66
Barber
DL
DeMartino
JC
Showers
MO
D'Andrea
AD
A dominant negative erythropoietin (EPO) receptor inhibits EPO-dependent growth and blocks F-gp55-dependent transformation.
Mol Cell Biol
14
1994
2257
67
Damen
JE
Liu
L
Rosten
P
Humphries
RK
Jefferson
AB
Majerus
PW
Krystal
G
The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5-phosphatase.
Proc Natl Acad Sci USA
93
1996
1689
68
Kavanaugh
WM
Pot
DA
Chin
SM
Deuter-Reinhard
M
Jefferson
AB
Norris
FA
Masiarz
FR
Cousens
LS
Majerus
PW
Williams
LT
Multiple forms of an inositol polyphosphate 5-phosphatase form signaling complexes with Shc and Grb2.
Curr Biol
6
1996
438
69
Lioubin
MN
Algate
PA
Tsai
S
Carlberg
K
Aebersold
R
Rohrschneider
LR
p150Ship, a signal transduction molecule with inositol polyphospate-5-phosphatase activity.
Genes Dev
10
1996
1084
70
Tanaka
S
Morishita
T
Hashimoto
Y
Hattori
S
Nakamura
S
Shibuya
M
Matuoka
K
Takenawa
T
Kurata
T
Nagashima
K
Matsuda
M
C3G, a guanine nucleotide-releasing protein expressed ubiquitously, binds to the Src homology 3 domains of Crk and Grb2/Ash proteins.
Proc Natl Acad Sci USA
91
1994
3443
71
Cherniack
AD
Klarlund
JK
Conway
BR
Czech
MP
Disassembly of Son-of-sevenless proteins from Grb2 during p21ras desensitization by insulin.
J Biol Chem
270
1995
1485
72
Waters
SB
Yamauchi
K
Pessin
JE
Insulin-stimulated disassociation of the SOS-Grb2 complex.
Mol Cell Biol
15
1995
2791
73
Songyang
Z
Shoelson
SE
Chaudhuri
M
Gish
G
Pawson
T
Haser
WG
King
F
Roberts
T
Ratnofsky
S
Lechleider
RJ
Neel
BG
Birge
RB
Fajardo
JE
Chou
MM
Hanafusa
H
Schaffhausen
B
Cantley
LC
SH2 domains recognize specific phosphopeptide sequences.
Cell
72
1993
767
74
Sawasdikosol
S
Ravichandran
KS
Lee
KK
Chang
JH
Burakoff
SJ
Crk interacts with tyrosine-phosphorylated p116 upon T cell activation.
J Biol Chem
270
1995
2893