E3 Ubiquitin Ligase c-Cbl Research Articles

Vinexin β regulates the phosphorylation of epidermal growth factor receptor on the cell surface

Epidermal growth factor (EGF) regulates various cellular events, including proliferation, differentiation, migration and oncogenesis. In this study, we found that exogenous expression of vinexin beta enhanced the phosphorylation of 180-kDa proteins in an EGF-dependent manner in Cos-7 cells. Western blot analysis using phospho-specific antibodies against EGFR identified EGFR as a phosphorylated 180-kDa protein. Vinexin beta did not stimulate the phosphorylation of EGFR but suppressed the dephosphorylation, resulting in a sustained phosphorylation. Mutational analyses revealed that both the first and third SH3 domains were required for a sustained phosphorylation of EGFR. Small interfering RNA-mediated knockdown of vinexin beta reduced the phosphorylation of EGFR on the cell surface in HeLa cells. The sustained phosphorylation of EGFR induced by vinexin beta was completely abolished by adding the EGFR-specific inhibitor AG1478 even after EGF stimulation, suggesting that the kinase activity of EGFR is required for the sustained phosphorylation induced by vinexin beta. We also found that E3 ubiquitin ligase c-Cbl is a binding partner of vinexin beta through the third SH3 domain. Expression of wild-type vinexin beta but not a mutant containing a mutation in the third SH3 domain decreased the cytosolic pool of c-Cbl and increased the amount of membrane-associated c-Cbl. Furthermore, over-expression of c-Cbl suppressed the sustained phosphorylation of EGFR induced by vinexin beta. These results suggest that vinexin beta plays a role in maintaining the phosphorylation of EGFR on the plasma membrane through the regulation of c-Cbl.

Src-like adaptor protein regulates TCR expression on thymocytes by linking the ubiquitin ligase c-Cbl to the TCR complex

The adaptor molecule SLAP and E3 ubiquitin ligase c-Cbl each regulate expression of T cell receptor (TCR)-CD3 on thymocytes. Here we provide genetic and biochemical evidence that both molecules function in the same pathway. TCR-CD3 expression was similar in the absence of SLAP and/or c-Cbl. SLAP and c-Cbl were found to interact, and their expression together downregulated CD3epsilon. This required multiple domains in SLAP and the ring finger of c-Cbl. Furthermore, expression of SLAP and c-Cbl together induced TCRzeta ubiquitination and degradation, preventing the accumulation of fully assembled recycling TCR complexes. These studies indicate that SLAP links the E3 ligase activity of c-Cbl to the TCR, allowing for stage-specific regulation of TCR expression.

Cortactin Overexpression Inhibits Ligand-Induced Down-regulation of the Epidermal Growth Factor Receptor

Ligand-induced receptor down-regulation by endocytosis is a critical process regulating the intensity and duration of receptor tyrosine kinase signaling. Ubiquitylation of specific receptor tyrosine kinases, for example, the epidermal growth factor receptor (EGFR) by the E3 ubiquitin ligase c-Cbl, provides a sorting signal for lysosomal degradation and leads to termination of receptor signaling. Cortactin, which couples the endocytic machinery to dynamic actin networks, is encoded by EMS1, a gene commonly amplified in breast and head and neck cancers. One mechanism whereby cortactin overexpression contributes to tumor progression is by enhancing tumor cell invasion and metastasis. However, in this study, we show that overexpression of cortactin in HeLa cells markedly inhibits ligand-induced down-regulation of the EGFR. This is independent of alterations in receptor autophosphorylation and correlates with impaired c-Cbl phosphorylation and association with the EGFR, reduced EGFR ubiquitylation, and sustained EGF-induced extracellular signal-regulated kinase activation. Furthermore, analysis of a panel of head and neck squamous cell carcinoma (HNSCC) cell lines revealed that cortactin overexpression is associated with attenuated ligand-induced EGFR down-regulation. Importantly, RNAi-mediated reduction of cortactin expression in an 11q13-amplified HNSCC cell line accelerates EGFR degradation. This represents the first demonstration of modulation of growth factor receptor signaling by cortactin. Moreover, enhanced EGFR signaling due to cortactin overexpression may provide an alternative explanation for EMS1 gene amplification in human cancers.

Negative regulation of receptor tyrosine kinases: unexpected links to c-Cbl and receptor ubiquitylation

Intracellular signals mediated by the family of receptor tyrosine kinases play pivotal roles in morphogenesis, cell fate determination and pathogenesis. Precise control of signal amplitude and duration is critical for the fidelity and robustness of these processes. Activation of receptor tyrosine kinases by their cognate growth factors not only leads to propagation of the signal through various biochemical cascades, but also sets in motion multiple attenuation mechanisms that ultimately terminate the active state. Early attenuators pre-exist prior to receptor activation and they act to limit signal propagation. Subsequently, late attenuators, such as Lrig and Sprouty, are transcriptionally induced and further act to dampen the signal. Central to the process of signaling attenuation is the role of the E3 ubiquitin ligase c-Cbl. While Cbl-mediated processes of receptor ubiquitylation and endocytosis are relatively well understood, the links of Cbl to other negative regulators are just now beginning to be appreciated. Here we review some emerging interfaces between Cbl and the transcriptionally induced negative regulators Lrig and Sprouty.

Human Sprouty Potentiates EGFR Signaling

Tight regulation of growth factor signaling through receptor tyrosine kinases (RTK) is critical to cell differentiation, growth, and survival. Sprouty (Spry) proteins are generally considered inhibitors of RTK signaling whose effects are mediated by interference with the mitogen-activated protein kinase (MAPK) cascade. Studies by Wong et al. and Egan et al. challenge this paradigm by demonstrating that human Sprouty2 (hSpry2) can potentiate epidermal growth factor (EGF) signaling by inhibiting ubiquitylation of the epidermal growth factor receptor (EGFR) by the E3 ubiquitin ligase c-Cbl. Wong et al. used confocal microscopy to show that overexpressed hSpry2, but not truncated constructs or isoforms that don't bind c-Cbl, inhibited endocytosis of immunofluorescently labeled EGFR in COS-1 cells stimulated with EGF. Transfected c-Cbl accelerated EGF-stimulated receptor down-regulation, whereas hSpry2 enhanced cell surface retention of EGFRs as demonstrated by radiolabeled EGF to EGFR binding. Using Western analysis on immunoprecipitates of intact and mutant proteins from transfected COS-1 cells, as well as an in vitro ubiquitylation assay, Wong et al. demonstrated that hSpry2 inhibited c-Cbl-mediated ubiquitylation of EGFR. Analysis of immunoprecipitates of cells transfected with hSpry2 and UbcH7 (human E2 ubiquitin ligase) indicated that hSpry competed with UbcH7 for binding to the c-Cbl RING finger domain. Decreased EGFR ubiquitylation in response to hSpry correlated with sustained EGF-induced ERK phosphorylation, indicating potentiation of the MAPK pathway. In PC12 cells, transfected hSpry2 induced morphological differentiation--normally associated with nerve growth factor (NGF) or fibroblast growth factor (FGF) stimulation--presumably secondary to sustained EGF-induced extracellular signal-regulated kinase (ERK) phosphorylation. In contrast, hSpry2 inhibited the response to FGF and NGF. These results indicate that, rather than being general inhibitors of RTK signaling, Sprys may inhibit certain specific pathways and potentiate others to fine tune the responses to RTK activation.E. S. M. Wong, M. Wong, C.W. Fong, J. Lin, P. Yusoff, B. C. Low, W. Y. Langdon, G. R. Guy, Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signalling. EMBO J. 21, 4796-4808 (2002). [Abstract] [Full Text] J. E. Egan, A. B. Hall, B. A. Yatsula, D. Bar-Sagi, The bimodal regulation of epidermal growth factor signaling by human Sprouty proteins, Proc. Natl. Acad. Sci. U.S.A. 99, 6041-6046 (2002). [Abstract] [Full Text]

Human Sprouty Potentiates EGFR Signaling

Tight regulation of growth factor signaling through receptor tyrosine kinases (RTK) is critical to cell differentiation, growth, and survival. Sprouty (Spry) proteins are generally considered inhibitors of RTK signaling whose effects are mediated by interference with the mitogen-activated protein kinase (MAPK) cascade. Studies by Wong et al. and Egan et al. challenge this paradigm by demonstrating that human Sprouty2 (hSpry2) can potentiate epidermal growth factor (EGF) signaling by inhibiting ubiquitylation of the epidermal growth factor receptor (EGFR) by the E3 ubiquitin ligase c-Cbl. Wong et al. used confocal microscopy to show that overexpressed hSpry2, but not truncated constructs or isoforms that don't bind c-Cbl, inhibited endocytosis of immunofluorescently labeled EGFR in COS-1 cells stimulated with EGF. Transfected c-Cbl accelerated EGF-stimulated receptor down-regulation, whereas hSpry2 enhanced cell surface retention of EGFRs as demonstrated by radiolabeled EGF to EGFR binding. Using Western analysis on immunoprecipitates of intact and mutant proteins from transfected COS-1 cells, as well as an in vitro ubiquitylation assay, Wong et al. demonstrated that hSpry2 inhibited c-Cbl-mediated ubiquitylation of EGFR. Analysis of immunoprecipitates of cells transfected with hSpry2 and UbcH7 (human E2 ubiquitin ligase) indicated that hSpry competed with UbcH7 for binding to the c-Cbl RING finger domain. Decreased EGFR ubiquitylation in response to hSpry correlated with sustained EGF-induced ERK phosphorylation, indicating potentiation of the MAPK pathway. In PC12 cells, transfected hSpry2 induced morphological differentiation--normally associated with nerve growth factor (NGF) or fibroblast growth factor (FGF) stimulation--presumably secondary to sustained EGF-induced extracellular signal-regulated kinase (ERK) phosphorylation. In contrast, hSpry2 inhibited the response to FGF and NGF. These results indicate that, rather than being general inhibitors of RTK signaling, Sprys may inhibit certain specific pathways and potentiate others to fine tune the responses to RTK activation. E. S. M. Wong, M. Wong, C.W. Fong, J. Lin, P. Yusoff, B. C. Low, W. Y. Langdon, G. R. Guy, Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signalling. EMBO J. 21 , 4796-4808 (2002). [Abstract] [Full Text] J. E. Egan, A. B. Hall, B. A. Yatsula, D. Bar-Sagi, The bimodal regulation of epidermal growth factor signaling by human Sprouty proteins, Proc. Natl. Acad. Sci. U.S.A. 99 , 6041-6046 (2002). [Abstract] [Full Text]