Hadassa Waterman

Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1.

Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that Cbl adaptor proteins directly control downregulation of the receptor for the epidermal growth factor (EGFR) by recruiting ubiquitin-activating and -conjugating enzymes. We infer a sequential process initiated by autophosphorylation of EGFR at a previously identified lysosome-targeting motif that subsequently recruits Cbl. This is followed by tyrosine phosphorylation of c-Cbl at a site flanking its RING finger, which enables receptor ubiquitination and degradation. Whereas all three members of the Cbl family can enhance ubiquitination, two oncogenic Cbl variants, whose RING fingers are defective and phosphorylation sites are missing, are unable to desensitize EGFR. Our study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways.

A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor.

The ErbB family includes four homologous transmembrane tyrosine kinases. Whereas ErbB-1 binds to the epidermal growth factor (EGF), both ErbB-3 and ErbB-4 bind to the Neu differentiation factors (NDFs, or neuregulins), and ErbB-2, the most oncogenic family member, is an orphan receptor whose function is still unknown. Because previous lines of evidence indicated the existence of interreceptor interactions, we used ectopic expression of individual ErbB proteins and their combinations to analyze the details of receptor cross talks. We show that 8 of 10 possible homo-and heterodimeric complexes of ErbB proteins can be hierarchically induced by ligand binding. Although ErbB-2 binds neither ligand, even in a heterodimeric receptor complex, it is the preferred heterodimer partner of the three other members, and it favors interaction with ErbB-3. Selective receptor overexpression in human tumor cells appears to bias the hierarchical relationships. The ordered network is reflected in receptor transphosphorylation, ErbB-2-mediated enhancement of ligand affinities, and remarkable potentiation of mitogenesis by a coexpressed ErbB-2. The observed superior ability of ErbB-2 to form heterodimers, in conjunction with its uniquely high basal tyrosine kinase activity, may explain why ErbB-2 overexpression is associated with poor prognosis.

Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions.

The ErbB family includes two receptors, ErbB‐1 and ErbB‐3, that respectively bind to epidermal growth factor and Neu differentiation factor, and an orphan receptor, ErbB‐2. Unlike ErbB‐1 and ErbB‐2, the intrinsic tyrosine kinase of ErbB‐3 is catalytically impaired. By using interleukin‐3‐dependent cells that ectopically express the three ErbB proteins or their combinations, we found that ErbB‐3 is devoid of any biological activity but both ErbB‐1 and ErbB‐2 can reconstitute its extremely potent mitogenic activity. Transactivation of ErbB‐3 correlates with heterodimer formation and is reflected in receptor phosphorylation and the transregulation of ligand affinity. Inter‐receptor interactions enable graded proliferative and survival signals: heterodimers are more potent than homodimers, and ErbB‐3‐containing complexes, especially the ErbB‐2/ErbB‐3 heterodimer, are more active than ErbB‐1 complexes. Nevertheless, ErbB‐1 signaling displays dominance over ErbB‐3 when the two receptors are coexpressed. Although all receptor combinations activate the mitogen‐activated protein kinases ERK and c‐Jun kinase, they differ in their rate of endocytosis and in coupling to intervening signaling proteins. It is conceivable that combinatorial receptor interactions diversify signal transduction and confer double regulation, in cis and in trans, of the superior mitogenic activity of the kinase‐defective ErbB‐3.

Differential endocytic routing of homo- and hetero-dimeric ErbB tyrosine kinases confers signaling superiority to receptor heterodimers

Both homo‐ and hetero‐dimers of ErbB receptor tyrosine kinases mediate signaling by a large group of epidermal growth factor (EGF)‐like ligands. However, some ligands are more potent than others, although they bind to the same direct receptor. In addition, signaling by receptor heterodimers is superior to homodimers. We addressed the mechanism underlying these two features of signal tuning by using three ligands: EGF; transforming growth factor α (TGFα); and their chimera, denoted E4T, which act on cells singly expressing ErbB‐1 as a weak, a strong, and a very strong agonist, respectively. Co‐expression of ErbB‐2, a developmentally important co‐receptor whose expression is frequently elevated in human cancers, specifically potentiated EGF signaling to the level achieved by TGFα, an effect that was partially mimicked by ErbB‐3. Analysis of the mechanism underlying this trans‐potentiation implied that EGF‐driven homodimers of ErbB‐1 are destined for intracellular degradation, whereas the corresponding heterodimers with ErbB‐2 or with ErbB‐3, dissociate in the early endosome. As a consequence, in the presence of either co‐receptor, ErbB‐1 is recycled to the cell surface and its signaling is enhanced. This latter route is followed by TGFα‐driven homodimers of ErbB‐1, and also by E4T‐bound receptors, whose signaling is further enhanced by repeated cycles of binding and dissociation from the receptors. We conclude that alternative endocytic routes of homo‐ and hetero‐dimeric receptor complexes may contribute to tuning and diversification of signal transduction. In addition, the ability of ErbB‐2 to shunt ligand‐activated receptors to recycling may explain, in part, its oncogenic potential.

The RING Finger of c-Cbl Mediates Desensitization of the Epidermal Growth Factor Receptor

Ligand-induced activation of surface receptors, including the epidermal growth factor receptor (EGFR), is followed by a desensitization process involving endocytosis and receptor degradation. c-Cbl, a tyrosine phosphorylation substrate shared by several signaling pathways, accelerates desensitization by recruiting EGFR and increasing receptor polyubiquitination. Here we demonstrate that the RING type zinc finger of c-Cbl is essential for ubiquitination and subsequent desensitization of EGFR. Mutagenesis of a single cysteine residue impaired the ability of c-Cbl to enhance both down-regulation and ubiquitination of EGFR in living cells, although the mutant retained binding to the activated receptor. Consequently, the mutant form of c-Cbl acquired a dominant inhibitory function and lost the ability to inhibit signaling downstream to EGFR. In vitroreconstitution of EGFR ubiquitination implies that the RING finger plays an essential direct role in ubiquitin ligation. Our results attribute to the RING finger of c-Cbl a causative role in endocytic sorting of EGFR and desensitization of signal transduction.

Molecular mechanisms underlying endocytosis and sorting of ErbB receptor tyrosine kinases.

The major process that regulates the amplitude and kinetics of signal transduction by tyrosine kinase receptors is endocytic removal of active ligand–receptor complexes from the cell surface, and their subsequent sorting to degradation or to recycling. Using the ErbB family of receptor tyrosine kinases we exemplify the diversity of the down regulation process, and concentrate on two sorting steps whose molecular details are emerging. These are the Eps15‐mediated sorting to clathrin‐coated regions of the plasma membrane and the c‐Cbl‐mediated targeting of receptors to lysosomal degradation. Like in yeast cells, sorting involves not only protein phosphorylation but also conjugation of ubiquitin molecules. The involvement of other molecules is reviewed and recent observations that challenge the negative regulatory role of endocytosis are described. Finally, we discuss the relevance of receptor down regulation to cancer therapy.

A mutant EGF-receptor defective in ubiquitylation and endocytosis unveils a role for Grb2 in negative signaling

Ligand‐induced desensitization of the epidermal growth factor receptor (EGFR) is controlled by c‐Cbl, a ubiquitin ligase that binds multiple signaling proteins, including the Grb2 adaptor. Consistent with a negative role for c‐Cbl, here we report that defective Tyr1045 of EGFR, an inducible c‐Cbl docking site, enhances the mitogenic response to EGF. Signaling potentiation is due to accelerated recycling of the mutant receptor and a concomitant defect in ligand‐induced ubiquitylation and endocytosis of EGFR. Kinetic as well as morphological analyses of the internalization‐defective mutant receptor imply that c‐Cbl‐mediated ubiquitylation sorts EGFR to endocytosis and to subsequent degradation in lysosomes. Unexpectedly, however, the mutant receptor displayed significant residual ligand‐induced ubiquitylation, especially in the presence of an overexpressed c‐Cbl. The underlying mechanism seems to involve recruitment of a Grb2 c‐Cbl complex to Grb2‐specific docking sites of EGFR, and concurrent acceleration of receptor ubiquitylation and desensitization. Thus, in addition to its well‐characterized role in mediating positive signals, Grb2 can terminate signal transduction by accelerating c‐Cbl‐dependent sorting of active tyrosine kinases to destruction.

Alternative Intracellular Routing of ErbB Receptors May Determine Signaling Potency

The ErbB signaling module consists of four receptor tyrosine kinases and several dozen ligands that activate specific homo- and heterodimeric complexes of ErbB proteins. Combinatorial ligand/receptor/effector interactions allow large potential for signal diversification. Here we addressed the possibility that turn-off mechanisms enhance the diversification potential. Concentrating on ErbB-1 and two of its ligands, epidermal growth factor (EGF) and transforming growth factor α (TGF-α), and the Neu differentiation factor (NDF/neuregulin) and one of its receptors, ErbB-3, we show that ligand binding variably accelerates endocytosis of the respective ligand-receptor complex. However, unlike the EGF-activated ErbB-1, which is destined primarily to degradation in lysosomes, NDF and TGF-α direct their receptors to recycling, probably because these ligands dissociate from their receptors earlier along the endocytic pathway. In the case of NDF, structural, as well as biochemical, analyses imply that ligand degradation occurs at a relatively late endosomal stage. Attenuation of receptor down-regulation by this mechanism apparently confers to both NDF and TGF-α more potent and prolonged signaling activity. In conclusion, alternative endocytic trafficking of ligand-ErbB complexes may tune and diversify signal transduction by EGF family ligands.

Epiregulin is a potent pan-ErbB ligand that preferentially activates heterodimeric receptor complexes

The ErbB signaling network consists of four transmembrane receptor tyrosine kinases and more than a dozen ligands sharing an epidermal growth factor (EGF) motif. The multiplicity of ErbB-specific ligands is incompletely understood in terms of signal specificity because all ErbB molecules signal through partially overlapping pathways. Here we addressed the action of epiregulin, a recently isolated ligand of ErbB-1. By employing a set of factor-dependent cell lines engineered to express individual ErbBs or their combinations, we found that epiregulin is the broadest specificity EGF-like ligand so far characterized: not only does it stimulate homodimers of both ErbB-1 and ErbB-4, it also activates all possible heterodimeric ErbB complexes. Consistent with its relaxed selectivity, epiregulin binds the various receptor combinations with an affinity that is approximately 100-fold lower than the affinity of ligands with more stringent selectivity, including EGF. Nevertheless, epiregulin’s action upon most receptor combinations transmits a more potent mitogenic signal than does EGF. This remarkable discrepancy between binding affinity and bioactivity is permitted by a mechanism that prevents receptor down-regulation, and results in a weak, but prolonged, state of receptor activation.

Pathogenic poxviruses reveal viral strategies to exploit the ErbB signaling network

Virulence of poxviruses, the causative agents of smallpox, depends on virus‐encoded growth factors related to the mammalian epidermal growth factor (EGF). Here we report that the growth factors of Shope fibroma virus, Myxoma virus and vaccinia virus (SFGF, MGF and VGF) display unique patterns of specificity to ErbB receptor tyrosine kinases; whereas SFGF is a broad‐specificity ligand, VGF binds primarily to ErbB‐1 homodimers, and the exclusive receptor for MGF is a heterodimer comprised of ErbB‐2 and ErbB‐3. In spite of 10‐ to 1000‐fold lower binding affinity to their respective receptors, the viral ligands are mitogenically equivalent or even more potent than their mammalian counterparts. This remarkable enhancement of cell growth is due to attenuation of receptor degradation and ubiquitination, which leads to sustained signal transduction. Our results imply that signal potentiation and precise targeting to specific receptor combinations contribute to cell transformation at sites of poxvirus infection, and they underscore the importance of the often ignored low‐affinity ligand–receptor interactions.