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Cbl-b, a RING-type E3 Ubiquitin Ligase, Targets Phosphatidylinositol 3-Kinase for Ubiquitination in T Cells*

  • Deyu Fang
    Affiliations
    From the Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Hong-Ying Wang
    Affiliations
    From the Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Nan Fang
    Affiliations
    From the Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Yoav Altman
    Affiliations
    From the Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Chris Elly
    Affiliations
    From the Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Yun-Cai Liu
    Correspondence
    To whom correspondence should be addressed: Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121
    Affiliations
    From the Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant RO1DK56558 (to Y.-C. L.).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:February 16, 2001DOI:https://doi.org/10.1074/jbc.M008901200
      Cbl-b is implicated in setting the threshold of T lymphocyte activation. In Cbl-b-deficient T cells, the activation of Vav, a guanine nucleotide exchange factor, is significantly enhanced. The molecular mechanism underlying Cbl-b-regulated Vav activation was unclear. Here it is shown that Cbl-b interacts with and induces ubiquitin conjugation to the p85 regulatory subunit of phosphatidylinositol 3-kinase, an upstream regulator of Vav. A functional RING finger of Cbl-b was essential for p85 ubiquitination. However, a loss of function mutation at the well-conserved amino-terminal variant src homology (SH) 2 domain of Cbl-b did not affect its ligase activity. A distal carboxyl-terminal proline-rich region in Cbl-b was mapped to contain the primary binding sequences for the SH3 domain of p85. Deletion of either the distal proline-rich region in Cbl-b or the SH3 domain of p85 severely reduced ubiquitin conjugation to p85. The data suggest a molecular link for Cbl-b-mediated negative regulation of Vav, with phosphatidylinositol 3-kinase as a direct target for Cbl-b E3 ubiquitin ligase.
      Ub
      ubiquitin
      PI3-K
      phosphatidylinositol 3-kinase
      SH
      src homology
      mAb
      monoclonal antibody
      HA
      hemagglutinin
      a.a.
      amino acid(s)
      PV
      pervanadate
      Cbl-b and its close mammalian homologue, Cbl, consist of an amino-terminal variant SH21domain, a RING finger, and a carboxyl-terminal proline-rich domain with potential tyrosine phosphorylation sites (
      • Langdon W.Y.
      • Hartley J.W.
      • Klinken S.P.
      • Ruscetti S.K.
      • Morse H.C.d.
      ,
      • Keane M.M.
      • Rivero-Lezcano O.M.
      • Mitchell J.A.
      • Robbins K.C.
      • Lipkowitz S.
      ). Previous studies have shown that Cbl-b and Cbl function as adaptor proteins by interacting with other critical signal molecules, including the variant SH2 domain-dependent interaction with cell surface receptor tyrosine kinases or intracellular protein tyrosine kinases such as Syk and Zap-70 and the carboxyl-terminal region-dependent interaction with Grb2, 14-3-3, phosphatidylinositol 3-kinase (PI3-K), Vav, and Crk-L (
      • Lupher Jr., M.L.
      • Rao N.
      • Eck M.J.
      • Band H.
      ,
      • van Leeuwen J.E.
      • Samelson L.E.
      ). Genetic and biochemical studies have shown that Cbl family proteins including those from Drosophila andCaenorhabditis elegans attenuate intracellular signaling induced by the engagement of cell surface receptors. One mechanism for this negative role is Cbl-mediated ubiquitination of receptor tyrosine kinases (
      • Miyake S.
      • Lupher Jr., M.L.
      • Druker B.
      • Band H.
      ,
      • Lee P.S.
      • Wang Y.
      • Dominguez M.G.
      • Yeung Y.G.
      • Murphy M.A.
      • Bowtell D.D.
      • Stanley E.R.
      ,
      • Levkowitz G.
      • Waterman H.
      • Ettenberg S.A.
      • Katz M.
      • Tsygankov A.Y.
      • Alroy I.
      • Lavi S.
      • Iwai K.
      • Reiss Y.
      • Ciechanover A.
      • Lipkowitz S.
      • Yarden Y.
      ). It is now understood that Cbl functions as an E3 ubiquitin (Ub) ligase whose RING finger recruits a Ub-conjugating enzyme, E2, and whose SH2 domain recognizes activated receptor tyrosine kinases for Ub conjugation (
      • Levkowitz G.
      • Waterman H.
      • Ettenberg S.A.
      • Katz M.
      • Tsygankov A.Y.
      • Alroy I.
      • Lavi S.
      • Iwai K.
      • Reiss Y.
      • Ciechanover A.
      • Lipkowitz S.
      • Yarden Y.
      ,
      • Joazeiro C.A.
      • Wing S.S.
      • Huang H.
      • Leverson J.D.
      • Hunter T.
      • Liu Y.C.
      ,
      • Yokouchi M.
      • Kondo T.
      • Houghton A.
      • Bartkiewicz M.
      • Horne W.C.
      • Zhang H.
      • Yoshimura A.
      • Baron R.
      ,
      • Zheng N.
      • Wang P.
      • Jeffrey P.D.
      • Pavletich N.P.
      ).
      Ubiquitination is an important cellular process that involves ligation of a protein substrate with Ub, thereby marking it for degradation by the 26S proteasome (
      • Hochstrasser M.
      ,
      • Hershko A.
      • Ciechanover A.
      ,
      • Hicke L.
      ), and it involves a cascade of reactions including E1, E2, and E3 enzymes. Ub is first activated by an activating enzyme (E1) to form a high energy thiolester bond between Ub and E1 and is then transferred to a conjugating enzyme (E2). The E3s or Ub protein ligases are the components responsible for specific substrate recognition and for promoting Ub ligation to the target protein. Therefore, the E3s can provide specificity to the Ub system.
      Two recent genetic studies using Cbl-b gene-targeted mice showed that Cbl-b deficiency can change the signaling thresholds: Cbl-b deficiency uncouples T-cell proliferation and interleukin 2 production from the costimulation of CD28, and the gene-targeted mice develop spontaneous autoimmunity or become highly susceptible to exogenous antigen-induced autoimmune diseases (
      • Bachmaier K.
      • Krawczyk C.
      • Kozieradzki I.
      • Kong Y.Y.
      • Sasaki T.
      • Oliveira dos Santos A.
      • Mariathasan S.
      • Bouchard D.
      • Wakeham A.
      • Itie A.
      • Le J.
      • Ohashi P.S.
      • Sarosi I.
      • Nishina H.
      • Lipkowitz S.
      • Penninger J.M.
      ,
      • Chiang Y.J.
      • Kole H.K.
      • Brown K.
      • Naramura M.
      • Fukuhara S.
      • Hu R.J.
      • Jang I.K.
      • Gutkind J.S.
      • Shevach E.
      • Gu H.
      ). These studies suggest a critical role of Cbl-b in the regulation of T-cell activation thresholds and hence in the maintenance of a balance between immunity and tolerance. In Cbl-b-deficient T cells, the tyrosine phosphorylation and/or activation of Vav, a GDP/GTP exchange factor, are significantly enhanced (
      • Bachmaier K.
      • Krawczyk C.
      • Kozieradzki I.
      • Kong Y.Y.
      • Sasaki T.
      • Oliveira dos Santos A.
      • Mariathasan S.
      • Bouchard D.
      • Wakeham A.
      • Itie A.
      • Le J.
      • Ohashi P.S.
      • Sarosi I.
      • Nishina H.
      • Lipkowitz S.
      • Penninger J.M.
      ,
      • Chiang Y.J.
      • Kole H.K.
      • Brown K.
      • Naramura M.
      • Fukuhara S.
      • Hu R.J.
      • Jang I.K.
      • Gutkind J.S.
      • Shevach E.
      • Gu H.
      ), suggesting that Cbl-b negatively regulates T-cell signaling by inhibiting Vav activation. However, the molecular mechanism underlying Cbl-b-mediated negative regulation of Vav remains to be determined.
      Previous studies have shown that PI3-K, which phosphorylates PI lipid at the D3 position of the inositol ring to form active lipid second messengers, regulates the exchange activity of Vav through the binding of the active lipid products to the pleckstrin homology (PH) domain of Vav (
      • Han J.
      • Luby-Phelps K.
      • Das B.
      • Shu X.
      • Xia Y.
      • Mosteller R.D.
      • Krishna U.M.
      • Falck J.R.
      • White M.A.
      • Broek D.
      ,
      • Ma A.D.
      • Metjian A.
      • Bagrodia S.
      • Taylor S.
      • Abrams C.S.
      ). We have investigated whether Cbl-b acts as an E3 Ub ligase to promote ubiquitination of PI3-K. Here we show that Cbl-b binds p85, the regulatory subunit of PI3-K, and induces its ubiquitination. We propose a model by which Cbl-b indirectly regulates Vav activation by inducing ubiquitination of PI3-K.

      DISCUSSION

      The results in this report demonstrated that Cbl-b acts as an E3 Ub ligase by interacting with the p85 regulatory subunit of PI3-K and promoting its ubiquitination. A functional RING finger is essential for the Ub ligase activity toward p85, whereas the well-conserved amino-terminal variant SH2 domain is dispensable for this event. We further demonstrated that the distal proline-rich sequences in Cbl-b and the SH3 domain of p85 are required for the efficient interaction and subsequent Ub conjugation to p85. Our data suggest a novel role for Cbl-b in regulating T-cell activation by inducing p85 ubiquitination.
      Recent genetic studies on Cbl-b-deficient T cells showed that the activation of Vav is increased in these cells, which was proposed to be responsible for costimulation-independent cytokine production and cell proliferation (
      • Bachmaier K.
      • Krawczyk C.
      • Kozieradzki I.
      • Kong Y.Y.
      • Sasaki T.
      • Oliveira dos Santos A.
      • Mariathasan S.
      • Bouchard D.
      • Wakeham A.
      • Itie A.
      • Le J.
      • Ohashi P.S.
      • Sarosi I.
      • Nishina H.
      • Lipkowitz S.
      • Penninger J.M.
      ,
      • Chiang Y.J.
      • Kole H.K.
      • Brown K.
      • Naramura M.
      • Fukuhara S.
      • Hu R.J.
      • Jang I.K.
      • Gutkind J.S.
      • Shevach E.
      • Gu H.
      ). This finding is supported by a study showing that coexpression of Cbl-b and Vav inhibits the nucleotide exchange activity of Vav (
      • Bustelo X.R.
      • Crespo P.
      • Lopez-Barahona M.
      • Gutkind J.S.
      • Barbacid M.
      ). The recent findings, including our own, showing that Cbl functions as an E3 ubiquitin ligase (
      • Levkowitz G.
      • Waterman H.
      • Ettenberg S.A.
      • Katz M.
      • Tsygankov A.Y.
      • Alroy I.
      • Lavi S.
      • Iwai K.
      • Reiss Y.
      • Ciechanover A.
      • Lipkowitz S.
      • Yarden Y.
      ,
      • Joazeiro C.A.
      • Wing S.S.
      • Huang H.
      • Leverson J.D.
      • Hunter T.
      • Liu Y.C.
      ,
      • Yokouchi M.
      • Kondo T.
      • Houghton A.
      • Bartkiewicz M.
      • Horne W.C.
      • Zhang H.
      • Yoshimura A.
      • Baron R.
      ) prompted us to investigate a potential role of Cbl-b on Vav ubiquitination. Our efforts failed to prove that Vav is a direct target for Cbl-b E3 Ub ligase activity, even after repeated experiments (data not shown). A recent study showed that Vav could be ubiquitinated by the suppressor of cytokine signaling 1, a component of elongin Ub ligase complex. Suppressor of cytokine signaling 1 interacts with Vav and induces its ubiquitination and degradation (
      • De Sepulveda P.
      • Ilangumaran S.
      • Rottapel R.
      ). Although it cannot be ruled out that Vav is a target for Cbl-b E3 ligase activity, we favor a model in which suppressor of cytokine signaling 1 is the primary E3 ligase component for Vav, and Cbl-b may indirectly regulate Vav activation.
      We then investigated the possibility that Cbl-b regulates the upstream regulators for Vav activation. One of the well-established regulators is PI3-K, whose lipid product can bind to the pleckstrin homology domain of Vav and activates Vav nucleotide exchange activity (
      • Han J.
      • Luby-Phelps K.
      • Das B.
      • Shu X.
      • Xia Y.
      • Mosteller R.D.
      • Krishna U.M.
      • Falck J.R.
      • White M.A.
      • Broek D.
      ,
      • Ma A.D.
      • Metjian A.
      • Bagrodia S.
      • Taylor S.
      • Abrams C.S.
      ). We demonstrated that Cbl-b induces Ub conjugation to p85, the regulatory subunit of PI3-K, which is dependent on its RING finger domain and distal proline-rich sequences, suggesting that Cbl-b acts as an E3 Ub ligase for p85. Besides PI3-K, Src kinases can also regulate the tyrosine phosphorylation of Vav in synergy with PI3-K (
      • Han J.
      • Luby-Phelps K.
      • Das B.
      • Shu X.
      • Xia Y.
      • Mosteller R.D.
      • Krishna U.M.
      • Falck J.R.
      • White M.A.
      • Broek D.
      ). Although we did not address a potential role for Cbl-b on Src kinases in this study, our data support a model in which Cbl-b indirectly regulates Vav activation by promoting Ub conjugation to p85.
      Previous studies on Cbl have focused on the functional role of its amino-terminal variant SH2 domain in targeting the substrate proteins such as cell surface receptor tyrosine kinases (
      • Miyake S.
      • Lupher Jr., M.L.
      • Druker B.
      • Band H.
      ,
      • Lee P.S.
      • Wang Y.
      • Dominguez M.G.
      • Yeung Y.G.
      • Murphy M.A.
      • Bowtell D.D.
      • Stanley E.R.
      ,
      • Levkowitz G.
      • Waterman H.
      • Ettenberg S.A.
      • Katz M.
      • Tsygankov A.Y.
      • Alroy I.
      • Lavi S.
      • Iwai K.
      • Reiss Y.
      • Ciechanover A.
      • Lipkowitz S.
      • Yarden Y.
      ,
      • Joazeiro C.A.
      • Wing S.S.
      • Huang H.
      • Leverson J.D.
      • Hunter T.
      • Liu Y.C.
      ,
      • Yokouchi M.
      • Kondo T.
      • Houghton A.
      • Bartkiewicz M.
      • Horne W.C.
      • Zhang H.
      • Yoshimura A.
      • Baron R.
      ,
      • Lill N.L.
      • Douillard P.
      • Awwad R.A.
      • Ota S.
      • Lupher Jr., M.L.
      • Miyake S.
      • Meissner-Lula N.
      • Hsu V.W.
      • Band H.
      ). A loss of function mutation (G306E) at this domain of Cbl, which disrupts its interaction with receptor tyrosine kinases, can also abrogate its Ub ligase activity toward these kinases (
      • Levkowitz G.
      • Waterman H.
      • Ettenberg S.A.
      • Katz M.
      • Tsygankov A.Y.
      • Alroy I.
      • Lavi S.
      • Iwai K.
      • Reiss Y.
      • Ciechanover A.
      • Lipkowitz S.
      • Yarden Y.
      ,
      • Lill N.L.
      • Douillard P.
      • Awwad R.A.
      • Ota S.
      • Lupher Jr., M.L.
      • Miyake S.
      • Meissner-Lula N.
      • Hsu V.W.
      • Band H.
      ). However, a corresponding mutation at Cbl-b (G298E) did not affect its activity to promote p85 ubiquitination, suggesting that the amino-terminal variant SH2 domain of Cbl-b is dispensable for Ub conjugation to p85. Rather, we showed that the carboxyl-terminal proline-rich region and, more specifically, the distal proline-rich sequences of Cbl-b are required for efficient p85 ubiquitination. Thus, our results suggest a novel mechanism by which the Cbl-b carboxyl-terminal region can also recruit target proteins and, with the help of the RING finger, induce the transfer of Ub to them.
      Our finding that the Cbl-b carboxyl-terminal region can recruit protein substrates is consistent with the recent crystal structural study on Cbl RING finger and an E2, Ub-conjugating enzyme H7 (
      • Zheng N.
      • Wang P.
      • Jeffrey P.D.
      • Pavletich N.P.
      ). In this complex, the active cysteine of Ub-conjugating enzyme H7, which forms a thiol-ester bond with Ub, is on the opposite side of the complex relative to the binding site for tyrosine kinase binding domain (amino-terminal domain). Therefore, a substrate that associates with the carboxyl-terminal region would presumably allow an easy transfer of Ub to the substrate. To further support our model, we recently found that the Cbl carboxyl-terminal region could also recruit protein substrates such as Stat5 and that the RING finger and the carboxyl-terminal proline-rich region are sufficient for promoting Stat5 ubiquitination.
      H.-Y. Wang, D. Fang, L. Qiu, Y. Altman, C. Elly, and Y.-C. Liu, unpublished data.
      In fact, the Cbl family proteins including those from C.elegans or Drosophila are well conserved in the amino-terminal variant SH2 domain and the RING finger domain (
      • Lupher Jr., M.L.
      • Rao N.
      • Eck M.J.
      • Band H.
      ), whereas the carboxyl-terminal regions between Cbl and Cbl-b are more divergent, and some Cbl family members (Sli-1, D-Cbl, and Cbl-3) lack or contain only a short form of the carboxyl-terminal proline-rich region. Because both Cbl and Cbl-b carboxyl-terminal regions contain several protein interaction motifs, it is conceivable that differential recruitment of protein substrates by the carboxyl-terminal region would provide diversity and specificity to Cbl- and/or Cbl-b-mediated protein ubiquitination.
      The SH3 domain of p85 recognizes a consensus proline-rich motif: RXX RPLPPLPP (
      • Rickles R.J.
      • Botfield M.C.
      • Weng Z.
      • Taylor J.A.
      • Green O.M.
      • Brugge J.S.
      • Zoller M.J.
      ), which is also present in p85 itself. Inspection of the Cbl-b carboxyl-terminal region did not find a perfect match for this consensus sequence. We attempted but failed to map the exact binding motif in the distal proline-rich sequences, suggesting that more than one proline-rich motif is required for the interaction with the SH3 domain of p85. In support of this notion, it was previously shown that the proximal proline-rich stretches in Cbl, rather than one proline-rich motif, mediate the interaction with Grb2 (
      • Donovan J.A.
      • Ota Y.
      • Langdon W.Y.
      • Samelson L.E.
      ).
      The SH3 domain of p85 has been shown to mediate interaction with the proline-rich sequence of another p85 molecule to form a p85 heterodimer (
      • Harpur A.G.
      • Layton M.J.
      • Das P.
      • Bottomley M.J.
      • Panayotou G.
      • Driscoll P.C.
      • Waterfield M.D.
      ) or interaction with the proline-rich sequences of other molecules (
      • Li E.
      • Stupack D.G.
      • Brown S.L.
      • Klemke R.
      • Schlaepfer D.D.
      • Nemerow G.R.
      ,
      • Yudowski G.A.
      • Efendiev R.
      • Pedemonte C.H.
      • Katz A.I.
      • Berggren P.O.
      • Bertorello A.M.
      ). We have shown here that the p85 SH3 domain can also interact with Cbl-b proline-rich sequences, which results in p85 ubiquitination promoted by Cbl-b E3 Ub ligase. Thus, our study suggests a novel role for the SH3 domain of p85 in mediating its ubiquitination.
      Polyubiquitination of protein substrates by the Ub system targets them for degradation by the 26S proteasome (
      • Hochstrasser M.
      ,
      • Hershko A.
      • Ciechanover A.
      ). However, modification of biological functions through ubiquitination of protein substrates without proteolysis has been reported recently in several systems, including regulation of transcription by transcription factor (
      • Kaiser P.
      • Flick K.
      • Wittenberg C.
      • Reed S.I.
      ) or ubiquitination-dependent processing of precursor proteins (
      • Hoppe T.
      • Matuschewski K.
      • Rape M.
      • Schlenker S.
      • Ulrich H.D.
      • Jentsch S.
      ). In consideration of the fact that the protein levels of Cbl-b-binding proteins do not change in Cbl-b-deficient T cells such as Zap-70, Lck, or even Vav (
      • Bachmaier K.
      • Krawczyk C.
      • Kozieradzki I.
      • Kong Y.Y.
      • Sasaki T.
      • Oliveira dos Santos A.
      • Mariathasan S.
      • Bouchard D.
      • Wakeham A.
      • Itie A.
      • Le J.
      • Ohashi P.S.
      • Sarosi I.
      • Nishina H.
      • Lipkowitz S.
      • Penninger J.M.
      ,
      • Chiang Y.J.
      • Kole H.K.
      • Brown K.
      • Naramura M.
      • Fukuhara S.
      • Hu R.J.
      • Jang I.K.
      • Gutkind J.S.
      • Shevach E.
      • Gu H.
      ), it can be postulated that Cbl-b, as an E3 Ub ligase, may play a general role in functional regulation of its target proteins through ubiquitination in a protein degradation-independent manner. The present work provides us with a molecular basis for our ongoing study on how Cbl-b-promoted ubiquitination of p85 could affect its biological function.

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