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Formation of c-Cbl·Phosphatidylinositol 3-Kinase Complexes on Lymphocyte Membranes by a p56lck-independent Mechanism*

  • David Hartley
    Affiliations
    Program in Molecular Medicine and Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
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  • Silvia Corvera
    Correspondence
    To whom correspondence should be addressed: Program in Molecular Medicine and Dept. of Cell Biology, University of Massachusetts Medical School, 55 Lake Ave. N., Worcester, MA 01655. Tel.: 508-856-6898; Fax: 508-856-4289;
    Affiliations
    Program in Molecular Medicine and Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
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  • Author Footnotes
    * This study was supported in part by National Institutes of Health Grant DK40330 (to S. C.). 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:September 06, 1996DOI:https://doi.org/10.1074/jbc.271.36.21939
      The proto-oncogene c-Cbl was originally identified as a cellular homologue of the transforming protein expressed by the murine Cas NS-1 retrovirus. The full-length c-Cbl protein is a predominantly cytoplasmic protein, abundant in lymphoid cells, and potentially involved in signal transduction in several cell types. The specific signal transduction pathways in which c-Cbl participates, and its precise role in these pathways, are unclear. Previous studies from our laboratory have shown that c-Cbl is the predominant tyrosine-phosphorylated protein bound to the p85 subunit of phosphatidylinositol (PI) 3-kinase on T lymphocyte and B lymphocyte activation. To further understand the properties of c-Cbl and the significance of its interactions with PI 3-kinase, we have further studied the cellular biological and biochemical responses of c-Cbl to stimulation in lymphoid cells. We show that stimulation induces the association of a highly tyrosine-phosphorylated pool of c-Cbl with lymphocyte membranes and with a detergent-insoluble particulate fraction. Immunoprecipitation of c-Cbl from subcellular fractions reveals that p85 is predominantly associated with the c-Cbl pool recovered from the membrane fraction, despite the fact that this pool represents a small amount of total cellular c-Cbl. The formation of c-Cbl·PI 3-kinase complexes on lymphocyte membranes did not depend on the catalytic activity of PI 3-kinase since it was unaltered by the treatment of cells with wortmannin prior to stimulation. Interestingly, c-Cbl tyrosine phosphorylation and the formation of c-Cbl·PI 3-kinase complexes were also observed in a mutant Jurkat cell line, JCaM1.6, lacking p56lck expression. Because p56lck is critical for mitogenic signal transduction in response to T cell receptor activation, our results suggest that the activation of c-Cbl and the formation of c-Cbl·PI 3-kinase complexes occur upstream or independently of mitogenic signal transduction pathways in T cells.

      INTRODUCTION

      The proto-oncogene c-Cbl was originally identified as a cellular homologue of the transforming protein expressed by the murine Cas NS-1 retrovirus (
      • Blake T.J.
      • Shapiro M.
      • Morse III, H.C.
      • Langdon W.Y.
      ). The retroviral form, v-Cbl, was found to be expressed as a gag fusion with the C-terminal 355 amino acids of c-Cbl. v-Cbl contains a putative nuclear localization signal, has an ability to bind DNA, and was shown to reside in both the nucleus and cytoplasm. The full-length c-Cbl protein, however, has been characterized as a predominantly cytoplasmic protein that is particularly abundant in lymphoid cells (
      • Blake T.J.
      • Heath K.G.
      • Langdon W.Y.
      ).
      c-Cbl has many tyrosine residues, some of which become phosphorylated after the stimulation of lymphocytes through their antigen receptors (
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ). c-Cbl also contains a proline-rich region suitable for binding SH3-containing proteins. In fact, it has been reported that c-Cbl can bind the adapter molecules Grb-2 and Nck through its proline-rich region (
      • Meisner H.
      • Conway B.R.
      • Hartley D.
      • Czech M.P.
      ,
      • Rivero-Lezcano O.M.
      • Sameshima J.H.
      • Marcilla A.
      • Robbins K.C.
      ). Furthermore, in vitro studies demonstrate the potential association of c-Cbl with multiple nonreceptor tyrosine kinases (
      • Fukazawa T.
      • Reedquist K.A.
      • Panchamoorthy G.
      • Soltoff S.
      • Trub T.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ,
      • Cory G.O.C.
      • Lovering R.C.
      • Hinshelwood S.
      • MacCarthy-Morrough L.
      • Levinsky R.J.
      • Kinnon C.
      ). Recent work on the function of c-Cbl in Caenorhabditis elegans has suggested that it can act as a negative regulator of EGF
      The abbreviations used are: EGF
      epidermal growth factor
      PI phosphatidylinositol IL-2
      interleukin-2
      PAGE
      polyacrylamide gel electrophoresis.
      receptor signaling (
      • Yoon C.H.
      • Lee J.
      • Jongeward G.D.
      • Sternberg P.W.
      ). This evidence suggests that the role of c-Cbl is to function as a cytoplasmic intermediate in signal transduction pathways elicited through growth factor receptors. However, the specific signal transduction pathways in which c-Cbl participates and the precise role of c-Cbl in these pathways are unclear.
      Numerous cellular responses are elicited after stimulation of the T cell antigen receptor·CD3 complex. These responses include the induction of IL-2 secretion, the up-regulation of receptors for IL-2 and transferrin, and the initiation of cell proliferation (
      • Janeway Jr,
      • Goldstein P.
      ). Numerous proteins, of which c-Cbl is a prominent one, become tyrosine phosphorylated following T cell receptor activation (
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ). Thus, c-Cbl may be a component of the signal transduction pathways that lead to cell proliferation or adhesion or to both following T cell receptor activation.
      Previous studies from our laboratory and others have shown that, on stimulation, c-Cbl associates in vivo with the p85 subunit of PI 3-kinase (
      • Meisner H.
      • Conway B.R.
      • Hartley D.
      • Czech M.P.
      ,
      • Hartley D.
      • Meisner H.
      • Corvera S.
      ). In fact, c-Cbl is the predominant tyrosine-phosphorylated protein bound to p85 · p110 on lymphocyte stimulation (
      • Hartley D.
      • Meisner H.
      • Corvera S.
      ). These results suggest that c-Cbl and PI 3-kinase are elements of the same signal transduction pathway. Interestingly, PI 3-kinase does not appear to be involved in mediating mitogenic responses elicited by costimulation in T cells since this response is insensitive to wortmannin, a fungal toxin that completely blocks the activation of PI 3-kinase following T cell receptor activation (
      • Crooks M.E.C.
      • Littman D.R.
      • Carter R.H.
      • Fearon D.T.
      • Weiss A.
      • Stein P.H.
      ). PI 3-kinase activity, however, has been implicated in the activation of integrin binding to fibronectin in T cells (
      • Shimizu Y.
      • Mobley J.L.
      • Finkelstein L.D.
      • Chan S.H.
      ). These results suggest that c-Cbl and PI 3-kinase may function in a signal transduction pathway involved in integrin activation and cell adhesion. PI 3-kinase has also been found to be involved in the down-regulation of platelet-derived growth factor receptors expressed in HepG2 cells (
      • Joly M.
      • Kazlauskas A.
      • Fay F.S.
      • Corvera S.
      ). A role for c-Cbl·PI 3-kinase complexes in the down-regulation of yet unidentified signaling complexes in lymphoid cells may also exist.
      To further understand the properties of c-Cbl and the significance of its interactions with PI 3-kinase, we have further studied the cellular biological and biochemical responses of c-Cbl to stimulation in lymphoid cells. Our results indicate that a specific pool of c-Cbl is recruited from the cytosol to the membrane fraction after activation, where it forms a complex with PI 3-kinase. After activation, c-Cbl becomes associated with a detergent-insoluble fraction, possibly composed of cytoskeletal elements. These data support the possibility that c-Cbl·PI 3-kinase complexes operate at the membrane, perhaps to regulate the changes in cytoskeletal function that occur after T cell activation. Interestingly, the phosphorylation of c-Cbl and the recruitment of c-Cbl·PI 3-kinase complexes were also observed in a mutant cell line (JCaM1.6) that lacks p56lck, a tyrosine kinase which is critical for mitogenic signal transduction in response to T cell receptor activation (
      • Straus D.B.
      • Weiss A.
      ). These results suggest that the activation of c-Cbl and the formation of c-Cbl·PI 3-kinase complexes occur upstream or independently of mitogenic signal transduction pathways in T cells.

      DISCUSSION

      Previous work from our laboratory has shown that c-Cbl is the principal tyrosine-phosphorylated protein associated with the p85 subunit of PI 3-kinase following T and B cell activation (
      • Hartley D.
      • Meisner H.
      • Corvera S.
      ). Here we show that this association occurs concomitantly with the translocation of c-Cbl to membrane and insoluble/cytoskeletal fractions (Fig. 1). In addition, we show that phosphorylation of c-Cbl and formation of membrane-bound c-Cbl·p85 complexes occur by a mechanism independent of the nonreceptor tyrosine kinase p56lck.
      Others have shown by immunofluorescent methods that Fcγ receptor stimulation of a monocyte cell line induces a translocation of c-Cbl from a diffuse to a concentrated perinuclear staining (
      • Tanaka S.
      • Neff L.
      • Baron R.
      • Levy J.B.
      ). These authors have suggested that this staining is consistent with a translocation to the trans Golgi network. However, based on previous work on PI 3-kinase in other cell systems, it is equally likely to be an endosomal/lysosomal membrane compartment with which c-Cbl becomes associated (
      • Joly M.
      • Kazlauskas A.
      • Fay F.S.
      • Corvera S.
      ).
      The mechanism whereby c-Cbl associates with membrane fractions is not known. However, it is interesting to note that c-Cbl was found to be associated with membranes prior to stimulation (Figs. 2A and 3A). Under these conditions, c-Cbl is neither tyrosine phosphorylated to a large extent nor associated with p85. Thus, other regions in c-Cbl, such as the large proline-rich region, might be involved in determining its membrane association (
      • Blake T.J.
      • Heath K.G.
      • Langdon W.Y.
      ). This association of c-Cbl with membranes may in turn be modulated by posttranslational modification that alters the structure of the protein, such as tyrosine phosphorylation.
      Despite a high level of tyrosine phosphorylation of both cytosolic and membrane-associated c-Cbl, the membrane-derived fraction is associated with p85 to a much greater extent. We have previously shown that the association between p85 and c-Cbl is principally mediated through the SH2 domains of p85 (
      • Hartley D.
      • Meisner H.
      • Corvera S.
      ). When taken together, these results suggest that the phosphorylation of tyrosine residues on c-Cbl may be heterogeneous, and a critical phosphotyrosine residue is phosphorylated preferentially when c-Cbl is associated with the membrane fraction. Tyrosine 751 on c-Cbl is flanked by amino acids that comprise a consensus site for p85 SH2 binding. This tyrosine residue lies within the region to which a specific antisera against c-Cbl (R2) was generated (
      • Blake T.J.
      • Heath K.G.
      • Langdon W.Y.
      ). Interestingly, immunoprecipitation using the R2 antisera results in less coprecipitatation of p85 compared with other anti-cbl antibodies (data not shown), lending support to this site being the target for p85 binding to c-Cbl.
      The mechanism whereby the p85 binding sites become phosphorylated in response to stimulation is not known. In this paper, however, we find that phosphorylation of c-Cbl and its association with p85 are independent of p56lck. This result is significant because the stimulation-induced tyrosine phosphorylation of most proteins is abrogated in p56lck-deficient cells (
      • Straus D.B.
      • Weiss A.
      ). The fact that c-Cbl is one of a few exceptions suggests that a signal transduction pathway distinct from the p56lck pathway is involved in the activation of c-Cbl and the formation of c-Cbl·p85 complexes. This finding may be relevant in terms of understanding the physiological significance of the formation of c-Cbl·p85 complexes. Abrogation of p56lck leads to impaired early and late T cell responses, including induction of IL-2 production and mitogenesis. The finding that p56lck deficiency does not impair the formation of c-Cbl·p85 complexes indicates that these complexes are insufficient to signal a mitogenic response. The role of these complexes may not be directly related to mitogenic signaling but perhaps may be related to other important responses to stimulation, such as adhesion. The finding that inhibition of PI 3-kinase activity with wortmannin does not inhibit mitogenic signaling in lymphocytes, but does inhibit adhesion (
      • Shimizu Y.
      • Mobley J.L.
      • Finkelstein L.D.
      • Chan S.H.
      ), is consistent with this hypothesis.
      Alternatively, c-Cbl·p85 complexes may operate in pathways that involve down-regulation of yet unidentified membrane proteins. A role for PI 3-kinase activity in the down-regulation of receptor tyrosine kinases in other cell systems has been proposed (
      • Joly M.
      • Kazlauskas A.
      • Fay F.S.
      • Corvera S.
      ). Interestingly, C. elegans, a homologue of c-Cbl (
      • Yoon C.H.
      • Lee J.
      • Jongeward G.D.
      • Sternberg P.W.
      ), functions as a negative regulator of signals elicited through a homologue of the EGF receptor. Results by others have suggested that c-Cbl acts as a cytosolic adaptor protein for PI 3-kinase recruitment to the EGF receptor (
      • Soltoff S.P.
      • Cantley L.C.
      ). It is interesting to speculate on the possibility that c-Cbl·PI 3-kinase complexes in this system may operate as negative regulators by virtue of their ability to down-regulate receptor tyrosine kinases. Disruption of c-Cbl function would be predicted to result in enhanced mitogenesis or transformation. Interestingly, when 17 amino acids in the central region of c-Cbl are deleted, the protein becomes transforming (
      • Andoniou C.E.
      • Thien C.B.
      • Langdon W.Y.
      ). This protein may operate as a dominant negative of endogenous c-Cbl function. Continued work to determine the p85 binding site in c-Cbl as well as the regions of c-Cbl that determine its association with cellular membranes will be necessary to answer these questions.

      Acknowledgments

      We thank Dr. Wallace Y. Langdon for the information on and supply of antisera to c-Cbl. We also thank Dr. Arthur Weiss for the gift of the JCaM1.6 cells.

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