Patrice Douillard

Cbl-mediated Negative Regulation of Platelet-derived Growth Factor Receptor-dependent Cell Proliferation A CRITICAL ROLE FOR Cbl TYROSINE KINASE-BINDING DOMAIN

The Cbl proto-oncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases. Our previous observations that Cbl overexpression in NIH3T3 cells enhanced the ubiquitination and degradation of the platelet-derived growth factor receptor-α (PDGFRα) and that the expression of oncogenic Cbl mutants up-regulated the PDGFRα signaling machinery strongly suggested that Cbl negatively regulates PDGFRα signaling. Here, we show that, similar to PDGFRα, selective stimulation of PDGFRβ induces Cbl phosphorylation, and its physical association with the receptor. Overexpression of wild type Cbl in NIH3T3 cells led to an enhancement of the ligand-dependent ubiquitination and subsequent degradation of the PDGFRβ, as observed with PDGFRα. We show that Cbl-dependent negative regulation of PDGFRα and β results in a reduction of PDGF-induced cell proliferation and protection against apoptosis. A point mutation (G306E) that inactivates the tyrosine kinase binding domain in the N-terminal transforming region of Cbl compromised the PDGF-inducible tyrosine phosphorylation of Cbl although this mutant could still associate with the PDGFR. More importantly, the G306E mutation abrogated the ability of Cbl to enhance the ligand-induced ubiquitination and degradation of the PDGFR and to inhibit the PDGF-dependent cell proliferation and protection from apoptosis. These results demonstrate that Cbl can negatively regulate PDGFR-dependent biological responses and that this function requires the conserved tyrosine kinase binding domain of Cbl.

The Evolutionarily Conserved N-terminal Region of Cbl Is Sufficient to Enhance Down-regulation of the Epidermal Growth Factor Receptor*

The mammalian proto-oncoprotein Cbl and its homologues in Caenorhabditis elegans andDrosophila are evolutionarily conserved negative regulators of the epidermal growth factor receptor (EGF-R). Overexpression of wild-type Cbl enhances down-regulation of activated EGF-R from the cell surface. We report that the Cbl tyrosine kinase-binding (TKB) domain is essential for this activity. Whereas wild-type Cbl enhanced ligand-dependent EGF-R ubiquitination, down-regulation from the cell surface, accumulation in intracellular vesicles, and degradation, a Cbl TKB domain-inactivated mutant (G306E) did not. Furthermore, the transforming truncation mutant Cbl-N (residues 1–357), comprising only the Cbl TKB domain, functioned as a dominant negative protein. It colocalized with EGF-R in intracellular vesicular structures, yet it suppressed down-regulation of EGF-R from the surface of cells expressing endogenous wild-type Cbl. Therefore, Cbl-mediated down-regulation of EGF-R requires the integrity of both the N-terminal TKB domain and additional C-terminal sequences. A Cbl truncation mutant comprising amino acids 1–440 functioned like wild-type Cbl in down-regulation assays. This mutant includes the evolutionarily conserved TKB and RING finger domains but lacks the less conserved C-terminal sequences. We conclude that the evolutionarily conserved N terminus of Cbl is sufficient to effect enhancement of EGF-R ubiquitination and down-regulation from the cell surface.

Graft-infiltrating T helper cells, CD45RC phenotype, and Th1/Th2-related cytokines in donor-specific transfusion-induced tolerance in adult rats.

Specific tolerance to LEW.1W (RT1u) heart allografts can be induced in adult LEW.1A (RT1a) rats by donor-specific blood transfusion (DST). We have previously shown that both rejected and tolerated grafts are heavily infiltrated by T lymphocytes, and that in both cases these T cells are capable of developing similar cytotoxic responses against donor cells in vitro; tolerance is therefore not due to the deletion of alloreactive T cells. At the same time, we found that the accumulation of IL-2 and IFN-gamma mRNA was decreased in tolerated grafts compared with rejected grafts. These results suggested that the induction of allograft tolerance in DST-treated animals could be mediated by anergy or suppression of graft-infiltrating Th1 cells. Although Th1 and Th2 clones have not yet been characterized in the rat, peripheral CD4+ rat T cells can be divided into two populations, based on their expression of the isoform RC of the CD45 molecule. Upon activation, CD45RChigh CD4+ T cells produce IL-2 and IFN-gamma and responsible for the induction of the graft-versus-host reaction, whereas CD45RClow CD4+ T cells produce IL-4 in vitro and provide B cell help. In the present study, we show that heart allografts from both DST-treated and untreated rats were infiltrated by equivalent numbers of leukocytes, of which CD4+ T cells also made up similar percentages. Among these CD4+ T cells, we observed that in allografts from DST-treated recipients the CD45RChigh population on day 5 was very significantly smaller (P = 0.004) than in the untreated group, while CD45RClow populations remained comparable. Moreover, using a new quantitative RT-PCR method, we found a dramatic reduction in the accumulation of IL-2, IFN-gamma, IL-10, IL-4, and IL-13 mRNA in hearts from DST-treated recipients compared with those of untreated recipients during the week following transplantation. These results show that in heart allografts from DST-treated recipients, despite phenotypic changes suggesting Th1 inhibition by Th2 imbalance, T helper function was inhibited as a whole, and that in vivo the phenotype CD4+ CD45RClow does not always correlate with Th2-related cytokine-producing cells.

Fas ligand, tumor necrosis factor-alpha expression, and apoptosis during allograft rejection and tolerance.

BACKGROUND Cytotoxic T cells can induce target cell lysis and apoptosis by different pathways. The interactions of CD95 antigen (Fas) with its ligand (CD95L) and of tumor necrosis factor (TNF)-alpha with its receptor (TNF-R1) lead to apoptotic cell death. Recently, conflicting studies have been published concerning the expression and the role of CD95L in allograft rejection and tolerance. METHODS In this study, the intragraft expression of CD95/CD95L and TNF-alpha and the frequency and distribution of apoptotic cells were compared in a model of heterotopic cardiac allograft in the rat in which recipients were either not treated (acute rejection) or pretreated with donor-specific blood transfusion (tolerant). RESULTS In the acutely rejected allografts, a peak in the expression of CD95L and TNF-alpha and in the number of apoptotic cells was observed during the first week after transplantation; apoptotic cells were confined to graft-infiltrating cells. In the tolerated allografts, however, levels of graft-infiltrating cell apoptosis and CD95L and TNF-alpha expression during the same period of time were dramatically lower. The expression of Fas was constitutive and was not modulated during acute rejection or tolerance. CONCLUSION This down-regulation of CD95L and TNF-alpha in allografts rendered tolerant by donor-specific transfusion suggests a role for apoptosis-inducing pathways in acute allograft rejection.

Binding of Cbl to a Phospholipase Cγ1-docking Site on Platelet-derived Growth Factor Receptor β Provides a Dual Mechanism of Negative Regulation

Ubiquitin conjugation to receptor tyrosine kinases is a critical biochemical step in attenuating their signaling through lysosomal degradation. Our previous studies have established Cbl as an E3 ubiquitin ligase for ubiquitinylation and degradation of platelet-derived growth factor receptor (PDGFR) α and PDGFRβ. However, the role of endogenous Cbl in PDGFR regulation and the molecular mechanisms of this regulation remain unclear. Here, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and degradation of PDGFRβ; this involves the Cbl TKB domain binding to PDGFRβ phosphotyrosine 1021, a known phospholipase C (PLC) γ1 SH2 domain-binding site. Lack of Cbl or ablation of the Cbl-binding site on PDGFRβ impedes receptor sorting to the lysosome. Cbl-deficient cells also show more PDGF-induced PLCγ1 association with PDGFRβ and enhanced PLC-mediated cell migration. Thus, Cbl-dependent negative regulation of PDGFRβ involves a dual mechanism that concurrently promotes ubiquitin-dependent lysosomal sorting of the receptor and competitively reduces the recruitment of a positive mediator of receptor signaling.

An essential role of ubiquitination in Cbl-mediated negative regulation of the Src-family kinase Fyn

The Cbl family of ubiquitin ligases function as negative regulators of activated receptor tyrosine kinases by facilitating their ubiquitination and subsequent lysosomal targeting. Here, we have investigated the role of Cbl ubiquitin ligase activity in the negative regulation of a non-receptor tyrosine kinase, the Src-family kinase Fyn. Using primary embryonic fibroblasts from Cbl(+/+) and Cbl(-/-) mice, we demonstrate that endogenous Cbl mediates the ubiquitination of Fyn and dictates the rate of Fyn turnover. By analyzing CHO-TS20 cells with a temperature-sensitive ubiquitin activating enzyme, we demonstrate that intact cellular ubiquitin machinery is required for Cbl-induced degradation of Fyn. Analyses of Cbl mutants, with mutations in or near the RING finger domain, in 293T cells revealed that the ubiquitin ligase activity of Cbl is essential for Cbl-induced degradation of Fyn by the proteasome pathway. Finally, use of a SRE-luciferase reporter demonstrated that Cbl-dependent negative regulation of Fyn function requires the region of Cbl that mediates the ubiquitin ligase activity. Given the conservation of structure between various Src-family kinases and the ability of Cbl to interact with multiple members of this family, Cbl-dependent ubiquitination could serve a general role to negatively regulate activated Src-family kinases.