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Phosphorylation of Cbl following Stimulation with Interleukin-3 and Its Association with Grb2, Fyn, and Phosphatidylinositol 3-Kinase*

  • Steven M. Anderson
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    From the University of Colorado Health Sciences Center, Department of Pathology, Denver, Colorado 80262
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  • Elizabeth A. Burton
    Affiliations
    From the University of Colorado Health Sciences Center, Department of Pathology, Denver, Colorado 80262
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  • Becky L. Koch
    Affiliations
    From the University of Colorado Health Sciences Center, Department of Pathology, Denver, Colorado 80262
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  • Author Footnotes
    * This research was supported in part by the Council for Tobacco Research, U. S. A. grant 2895A. The University of Colorado Cancer Center, which provided some core facilities used in this research, is supported by a grant by the NIH (CA46934). 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.
Open AccessPublished:January 10, 1997DOI:https://doi.org/10.1074/jbc.272.2.739
      We have demonstrated that a 120-kDa protein, identified as Cbl, becomes rapidly phosphorylated on tyrosine residues following stimulation of factor-dependent cells with interleukin-3 (IL-3). Little or no phosphorylation of Cbl was observed in the absence of IL-3 stimulation and phosphorylation is maximal by 20-30 min after IL-3 stimulation. Association of Cbl with Grb2 was noted in unstimulated cells, and the amount of Cbl associated with Grb2 increased following IL-3 stimulation. The p85 subunit of phosphatidylinositol 3-kinase was constitutively associated with Cbl. Approximately 10% of the PI kinase activity present in anti-phosphotyrosine immunoprecipitates was present in anti-Cbl immunoprecipitates of IL-3-stimulated cells. The constitutive association of Cbl with Fyn was also observed. Cbl was observed to bind to bacterial fusion proteins encoding the unique, SH3, and SH2 domains of Fyn, Hck, and Lyn. The SH2 domain of Fyn alone was able to bind Cbl to nearly the same extent as did the fusion protein encoding the unique, SH3, and SH2 domains. This was not the case for the SH2 domain of Hck, however, as binding of the Hck fusion protein to Cbl appeared to require multiple domains. The binding of the fusion proteins to Cbl occurred regardless of whether Cbl was tyrosine-phosphorylated or not, and the binding could not be disrupted by the addition of 30 mM free phosphotyrosine. These data suggest the unexpected conclusion that the Fyn SH2 domain may bind to Cbl in a phosphotyrosine-independent manner.

      INTRODUCTION

      Stimulation of type I cytokine family receptors with their ligands results in the rapid tyrosine phosphorylation of multiple cellular proteins including the receptors themselves. Since these receptors do not encode intrinsic protein-tyrosine kinase activity (
      • Itoh N.
      • Yonehara S.
      • Schreurs J.
      • Gorman D.M.
      • Maruyama K.
      • Ishii A.
      • Yahara I.
      • Arai K.
      • Miyajima A.
      ,
      • Gorman D.M.
      • Itoh N.
      • Kitamura T.
      • Schreurs J.
      • Yonehara S.
      • Yahara I.
      • Arai K.
      • Miyajima A.
      ,
      • Kitamura T.
      • Sato N.
      • Arai K.
      • Miyajima A.
      ,
      • Hayashida K.
      • Kitamura T.
      • Gorman D.M.
      • Arai K.
      • Yokota T.
      • Miyajima A.
      ,
      • Ihle J.N.
      • Witthuhn B.A.
      • Quelle F.W.
      • Yamamoto K.
      • Thierfelder W.E.
      • Kreider B.L.
      • Silvennoinen O.
      ,
      • Boutin J.-M.
      • Jolicoeur C.
      • Okamura H.
      • Gagnon J.
      • Edery M.
      • Shirota M.
      • Banville D.
      • Dusanter-Fourt I.
      • Djiane J.
      • Kelly P.A.
      ,
      • Kelly P.A.
      • Djiane J.
      • Postel-Vinay M.-C.
      • Edery M.
      ,
      • D'Andrea A.D.
      • Fasman G.D.
      • Lodish H.F.
      ), the activation of nonreceptor tyrosine kinases, such as the Janus and Src families of tyrosine kinases, is critical in signaling events. Investigations utilizing cell lines lacking specific Janus family members has indicated that they are critical in signal transduction response to cytokines (
      • Watling D.
      • Guschin D.
      • Muller M.
      • Silvennoinen O.
      • Witthuhn B.A.
      • Quelle F.W.
      • Rogers N.C.
      • Schindler C.
      • Stark G.R.
      • Kerr I.M.
      ,
      • 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.
      • Kerr I.M.
      ). Similar studies have not been conducted to date with Src-like kinases. In addition to understanding which tyrosine kinases are activated, the identification of downstream signaling molecules is fundamental to understanding signal transduction. We have been investigating the activation of Src-like kinases in signal transduction by the IL-3
      The abbreviations used are: IL-3
      interleukin-3
      GST
      glutathione S-transferase
      GM-CSF
      granulocyte-macrophage colony-stimulating factor
      PI
      phosphatidylinositol
      PI 3-kinase
      phosphatidylinositol 3'-kinase
      SH2
      Src homology 2
      SH3
      Src homology 3
      PIPES
      1,4-piperazinediethanesulfonic acid
      RIPA
      radioimmune precipitation buffer.
      receptor. Following IL-3 stimulation of the murine myeloid cell line 32D cl3, we have observed the activation of three Src-like kinases: Fyn, Hck, and Lyn (
      • Anderson S.M.
      • Jorgensen B.
      ). As part of a study to examine the interaction of Src-like kinases with the β subunit of the IL-3 receptor, we observed that a tyrosine-phosphorylated protein of 120 kDa bound to bacterial fusion proteins containing the unique, SH3, and SH2 domains of Fyn, Hck, and Lyn.
      E. A. Burton, S. Hunter, S. C. Wu, and S. M. Anderson, submitted for publication.
      The current study was initiated to determine whether this protein was Cbl.
      The Cbl protein represents the cellular homologue of the oncogene present in the Cas-NS-1 retrovirus (
      • Langdon W.Y.
      • Hartley J.W.
      • Klinken S.P.
      • Ruscetti S.K.
      • Morse H.C.
      ). Sequence analysis of the Cbl cDNA revealed that the protein contains 913 amino acids, a putative nuclear localization sequence in its N-terminal region, a “RING finger” motif typical of numerous DNA-binding proteins, and several proline-rich sequences in its C-terminal half that may serve as SH3 domain binding sites (
      • Langdon W.Y.
      • Hartley J.W.
      • Klinken S.P.
      • Ruscetti S.K.
      • Morse H.C.
      ). In spite of the presence of a nuclear localization signal and a DNA-binding motif, there is no evidence that Cbl is present in the nucleus or that it binds to DNA (
      • Langdon W.Y.
      • Hartley J.W.
      • Klinken S.P.
      • Ruscetti S.K.
      • Morse H.C.
      ,
      • Blake T.J.
      • Heath K.G.
      • Langdon W.Y.
      ). Recent studies have demonstrated that Cbl becomes tyrosine-phosphorylated following stimulation of the following receptors: the T-cell receptor (
      • Hartley D.
      • Meisner H.
      • Corvera S.
      ,
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ,
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ), the B-cell receptor (
      • Panchamoorthy G.
      • Fukazawa T.
      • Miyake S.
      • Soltoff S.
      • Reedquist K.
      • Druker B.
      • Shoelson S.
      • Cantley L.
      • Band H.
      ,
      • Smit L.
      • van Der Horst G.
      • Borst J.
      ), the Fc receptor (
      • Tanaka S.
      • Neff L.
      • Baron R.
      • Levy J.B.
      ,
      • Marcilla A.
      • Rivero-Lezcano O.M.
      • Agarwal A.
      • Robbins K.C.
      ), the epidermal growth factor receptor (
      • Soltoff S.P.
      • Cantley L.C.
      ,
      • Galisteo M.L.
      • Dikic I.
      • Batzer A.G.
      • Langdon W.Y.
      • Schlessinger J.
      ,
      • Levkowitz G.
      • Klapper L.N.
      • Tzahar E.
      • Freywald A.
      • Sela M.
      • Yarden Y.
      ,
      • Fukazawa T.
      • Miyake S.
      • Band V.
      • Band H.
      ), the erythropoietin receptor (
      • Odai H.
      • Sasaki K.
      • Iwamatsu A.
      • Hanazono Y.
      • Tanaka T.
      • Mitani K.
      • Yazaki Y.
      • Hirai H.
      ), and the receptor for granulocyte-macrophage colony-stimulating factor (
      • Odai H.
      • Sasaki K.
      • Iwamatsu A.
      • Hanazono Y.
      • Tanaka T.
      • Mitani K.
      • Yazaki Y.
      • Hirai H.
      ). The Cbl protein is also phosphorylated in cells expressing either v-Abl or BCR-ABL (
      • Ribon V.
      • Hubbell S.
      • Herrera R.
      • Saltiel A.R.
      ,
      • Sattler M.
      • Salgia R.
      • Okuda K.
      • Uemura N.
      • Durstin M.A.
      • Pisick E.
      • Xu G.
      • Li J.-L.
      • Prasad K.V.
      • Griffin J.D.
      ). In receptor-stimulated cells, Cbl has been observed to associate with a variety of proteins by either co-immunoprecipitation studies or binding to bacterial fusion proteins. Association has been observed with PI 3-kinase in a phosphotyrosine-dependent manner via SH2 and SH3 domains (
      • Hartley D.
      • Meisner H.
      • Corvera S.
      ,
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ,
      • Soltoff S.P.
      • Cantley L.C.
      ,
      • Fukazawa T.
      • Miyake S.
      • Band V.
      • Band H.
      ,
      • Sattler M.
      • Salgia R.
      • Okuda K.
      • Uemura N.
      • Durstin M.A.
      • Pisick E.
      • Xu G.
      • Li J.-L.
      • Prasad K.V.
      • Griffin J.D.
      ), with the SH3 domain of Lyn (
      • Tanaka S.
      • Neff L.
      • Baron R.
      • Levy J.B.
      ,
      • Marcilla A.
      • Rivero-Lezcano O.M.
      • Agarwal A.
      • Robbins K.C.
      ), with the SH2 and SH3 domains of Fyn (
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ,
      • Tanaka S.
      • Neff L.
      • Baron R.
      • Levy J.B.
      ), with the SH2 domains of Crk (
      • Sattler M.
      • Salgia R.
      • Okuda K.
      • Uemura N.
      • Durstin M.A.
      • Pisick E.
      • Xu G.
      • Li J.-L.
      • Prasad K.V.
      • Griffin J.D.
      ), and with Grb2 via one of its SH3 domains (
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ,
      • Smit L.
      • van Der Horst G.
      • Borst J.
      ,
      • Fukazawa T.
      • Miyake S.
      • Band V.
      • Band H.
      ,
      • Odai H.
      • Sasaki K.
      • Iwamatsu A.
      • Hanazono Y.
      • Tanaka T.
      • Mitani K.
      • Yazaki Y.
      • Hirai H.
      ). The association of Cbl with the p85 subunit of PI 3-kinase and Grb2 suggests that phosphorylation of Cbl may regulate activation of PI 3-kinase and Ras. It is not clear whether Cbl can associate with all of these molecules in the same cell, or in different cell types. In this report, we demonstrate that Cbl is the 120-kDa phosphoprotein observed in IL-3-stimulated cells. Cbl was observed to associate with Grb2, Fyn, and PI 3-kinase. The association of Cbl with Grb2 following cytokine stimulation is consistent with a previous report (
      • Odai H.
      • Sasaki K.
      • Iwamatsu A.
      • Hanazono Y.
      • Tanaka T.
      • Mitani K.
      • Yazaki Y.
      • Hirai H.
      ); however, the described interaction Cbl with Fyn and PI 3-kinase following cytokine stimulation is novel to this report. The association of Cbl with Fyn may be mediated by the SH2 domain of Fyn binding to Cbl in a phosphotyrosine-independent manner.

      DISCUSSION

      In this paper we have described the phosphorylation of Cbl on tyrosine following stimulation of factor-dependent 32D cl3 cells with IL-3. Association of Cbl with three other molecules involved in signal transduction was noted; Grb2, Fyn, and the p85 subunit of PI 3-kinase. IL-3 stimulation increased the amount of Grb2 associated with Cbl; however, it did not appear to alter the amount of Fyn or p85 associated with Cbl. Activated PI kinase activity was associated with Cbl following IL-3 stimulation; however, the Cbl-associated activity was only 10% of the activity noted in anti-phosphotyrosine or anti-p85 immunoprecipitates. The association of Cbl with Src-like kinases, the p85 subunit of PI 3-kinase, or PI kinase activity following stimulation of cytokine family receptors has not been described before and is novel to this report. Our results are consistent with a previous publication demonstrating tyrosine phosphorylation of Cbl following stimulation of factor-dependent cells with erythropoietin or GM-CSF (
      • Odai H.
      • Sasaki K.
      • Iwamatsu A.
      • Hanazono Y.
      • Tanaka T.
      • Mitani K.
      • Yazaki Y.
      • Hirai H.
      ). In that work, the constitutive association of Grb2 with Cbl, via the SH3 domain of Grb2, was noted.
      Using GST fusion proteins in binding assays, we have determined that the SH2 domain of Fyn can bind to Cbl in a phosphotyrosine-independent manner. Observations supporting this conclusion include: (a) GST-FYN-SH2 bound to non-phosphorylated Cbl, and (b) 30 mM phosphotyrosine did not block this binding. While several other investigators have demonstrated that binding of proteins to Cbl is mediated by SH2 domains, none have investigated whether these interactions are phosphotyrosine-dependent. The phosphotyrosine-independent binding of the v-Abl SH2 domain to Shc has been described (
      • Raffel G.D.
      • Parmar K.
      • Rosenberg N.
      ). In addition, a 62-kDa protein has been described that binds the SH2 domain of Lck in a phosphotyrosine-independent manner (
      • Joung I.
      • Strominger J.L.
      • Shin J.
      ,
      • Park I.
      • Chung J.
      • Walsh C.T.
      • Yan Y.
      • Strominger J.L.
      • Shin J.
      ). The possibility that these binding interactions represent the binding of SH2 domains to phospholipid-modified proteins remains to be investigated (
      • Rameh L.E.
      • Chen C.-S.
      • Cantley L.
      ).
      Activation of PI 3-kinase by cytokines such as IL-3 and GM-CSF has been reported by several investigators (
      • Wang L.-M.
      • Keegan A.D.
      • Paul W.E.
      • Heidaran M.A.
      • Gutkind J.S.
      • Pierce J.H.
      ,
      • Corey S.
      • Eguinoa A.
      • Puyana-Theall K.
      • Bolen J.B.
      • Cantley L.
      • Mollinedo F.
      • Jackson T.R.
      • Hawkins P.T.
      • Stephens L.R.
      ,
      • Gold M.R.
      • Duronio V.
      • Saxena S.P.
      • Schrader J.W.
      • Aebersold R.
      ). Although Wang et al. (
      • Wang L.-M.
      • Keegan A.D.
      • Paul W.E.
      • Heidaran M.A.
      • Gutkind J.S.
      • Pierce J.H.
      ) noted the low level tyrosine-phosphorylation of p85, other investigators, including this report, have not observed phosphorylation of p85 following stimulation with IL-3 or GM-CSF (
      • Gold M.R.
      • Duronio V.
      • Saxena S.P.
      • Schrader J.W.
      • Aebersold R.
      ,
      • Jücker M.
      • Feldman R.A.
      ). The fact that PI 3-kinase activity is detected in anti-phosphotyrosine immunoprecipitates (this report and
      • Gold M.R.
      • Duronio V.
      • Saxena S.P.
      • Schrader J.W.
      • Aebersold R.
      ), suggests that this enzyme is complexed to other tyrosine-phosphorylated proteins. Candidate tyrosine-phosphorylated proteins to which p85/PI 3-kinase might associate include the β subunit, Cbl, Src-like kinases, and a protein referred to as p80 (
      • Jücker M.
      • Feldman R.A.
      ). Numerous investigators have described the association of PI 3-kinase (and p85) with Cbl following activation of the EGF receptor, the T-cell receptor, and the B-cell receptor (
      • Hartley D.
      • Meisner H.
      • Corvera S.
      ,
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ,
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ,
      • Panchamoorthy G.
      • Fukazawa T.
      • Miyake S.
      • Soltoff S.
      • Reedquist K.
      • Druker B.
      • Shoelson S.
      • Cantley L.
      • Band H.
      ,
      • Soltoff S.P.
      • Cantley L.C.
      ,
      • Galisteo M.L.
      • Dikic I.
      • Batzer A.G.
      • Langdon W.Y.
      • Schlessinger J.
      ,
      • Fukazawa T.
      • Miyake S.
      • Band V.
      • Band H.
      ,
      • Kretzner L.
      • Blackwood E.M.
      • Eisenman R.N.
      ). The same association has also been observed with BCR-ABL (
      • Ribon V.
      • Hubbell S.
      • Herrera R.
      • Saltiel A.R.
      ,
      • Sattler M.
      • Salgia R.
      • Okuda K.
      • Uemura N.
      • Durstin M.A.
      • Pisick E.
      • Xu G.
      • Li J.-L.
      • Prasad K.V.
      • Griffin J.D.
      ). Our investigation suggests that approximately 10% of the PI kinase activity in an anti-phosphotyrosine immunoprecipitate can be found in an anti-Cbl immunoprecipitate. This suggests that the majority of PI 3-kinase is associated with other tyrosine-phosphorylated proteins. To date no investigators have described the association of p85 directly with the β subunit, and the β subunit does not appear to have the predicted consensus sequence to which the SH2 domain of p85 might bind.
      The association of PI 3-kinase with Src-like kinases is known to occur. Corey et al. (
      • Corey S.
      • Eguinoa A.
      • Puyana-Theall K.
      • Bolen J.B.
      • Cantley L.
      • Mollinedo F.
      • Jackson T.R.
      • Hawkins P.T.
      • Stephens L.R.
      ) demonstrated that activated PI 3-kinase was associated the Lyn and Yes kinases following GM-CSF stimulation. Pleiman et al. (
      • Pleiman C.M.
      • Hertz W.M.
      • Cambier J.C.
      ) have shown that the binding of the SH3 domain of Fyn or Lyn to a proline-rich sequence in p85 results in the activation of PI 3-kinase. The SH3 domain of Fyn was also demonstrated to bind to p85 following activation of the T-cell receptor (
      • Prasad K.V.S.
      • Janssen O.
      • Kapeller R.
      • Raab M.
      • Cantley L.C.
      • Rudd C.E.
      ). These results clearly indicate that Src-like kinases may regulate PI 3-kinase activation.
      Jücker and Feldman (
      • Jücker M.
      • Feldman R.A.
      ) have described the association of a 76-85-kDa tyrosine-phosphorylated protein, referred to as p80, with p85 following IL-3 or GM-CSF stimulation of human TF-1 cells. They have also determined the p80 is highly associated with Src-like kinases (Src, Yes, and Lyn) (
      • Jücker M.
      • Feldman R.A.
      ). These investigators, however, did not determine whether active PI 3-kinase is associated with p80. We have observed the co-immunoprecipitation of several tyrosine-phosphorylated proteins with p85, including one that might correspond to p80; however, the major band we have observed has a molecular mass of 95-100 kDa. The identity of p80, as well as other tyrosine-phosphorylated proteins, and their role in regulating PI 3-kinase remain an important question for future studies.
      We propose a model in which Cbl functions as an adaptor protein similar to insulin-regulated substrate-1. Unphosphorylated Cbl is associated with molecules such as Grb2 and p85, which could link it to the Ras/Raf/MAP kinase and the PI 3-kinase pathways. Following phosphorylation, Cbl may serve as a binding site to which other signaling molecules bind and become activated. While it may play a role in regulating PI 3-kinase, it is clear that other proteins are also involved. It remains to be determined whether these other proteins, such as Src-like kinases, function independently of Cbl, or whether they are transiently associated with Cbl.

      Acknowledgments

      We thank John Cambier and Clifford Lowell for providing some of the GST expression plasmids used in this study. Richard Klinghoffer and Andrius Kazlauskas kindly provided assistance with PI kinase assays. We also acknowledge the services of the University of Colorado Cancer Center DNA Sequencing Core Facility and Antibody Core Facility in support of this research. The PhosphorImager used in this study is part of the Molecular Biology Core of the University of Colorado Cancer Center. The University of Colorado Cancer Center is supported by National Institutes of Health Grant CA46934. We also thank Drs. Mary Reyland and Julie Gelderloos for their comments on the manuscript.

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