Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identifiedCbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl ؊/؊ mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization. Growth factor receptor tyrosine kinases (RTKs)1 play crucial roles in cellular proliferation, survival, migration, and differentiation. Epidermal growth factor receptor (EGFR/ErbB1) is a member of the ErbB family (ErbB1-4) of RTKs, which play crucial homeostatic roles and are implicated in oncogenesis. Ligand-induced activation of RTKs leads to the assembly of signaling protein complexes and subsequent activation of downstream signaling pathways. The ligand-activated RTKs also undergo rapid endocytosis (1). The endocytosed receptors then undergo a sorting process, which determines receptor fate and signal intensity. The receptors can be targeted to the lysosome for degradation, which terminates receptor signals. Alternatively, the internalized receptors can be recycled back to the cell surface for continued ligand binding and signaling (2-5). The relative efficiency of lysosomal sorting versus recycling is a key determinant of the signaling potency of RTKs (6). For example, EGFR is predominantly delivered to lysosomes when activated by EGF. In contrast, heregulin-activated ErbB2 is primarily recycled. The greater efficiency of the recycling process is thought to be a major determinant of the signaling superiority of ErbB2 over EGFR (7-9).Despite a critical role of endocytic sorting as a determinant of ErbB receptor down-regulation, the biochemical mechanisms that regulate this process have only recently begun to be elucidated. We, and others, have identified Cbl as one such regulator (10 -12). Cbl is recruited to the activated EGFR through both direct and indirect binding. Direct Cbl-EGFR interaction is mediated through the N-terminal tyrosine kinase-binding domain of Cbl, which binds to phosphorylated Tyr-1045 on EGFR (13). Indirect Cbl-E...

ErbB2 overexpression contributes to the evolution of a substantial group of human cancers and signifies a poor clinical prognosis. Thus, down-regulation of ErbB2 signaling has emerged as a new anti-cancer strategy. Ubiquitinylation, mediated by the Cbl family of ubiquitin ligases, has emerged as a physiological mechanism of ErbB receptor down-regulation, and this mechanism appears to contribute to ErbB2 down-regulation induced by therapeutic anti-ErbB2 antibodies. Hsp90 inhibitory ansamycin antibiotics such as geldanamycin (GA) induce rapid ubiquitinylation and down-regulation of ErbB2. However, the ubiquitin ligase(s) involved has not been identified. Here, we show that ErbB2 serves as an in vitro substrate for the Hsp70/Hsp90-associated U-box ubiquitin ligase CHIP. Overexpression of wild type CHIP, but not its U-box mutant H260Q, induced ubiquitinylation and reduction in both cell surface and total levels of ectopically expressed or endogenous ErbB2 in vivo, and this effect was additive with that of 17-allylamino-geldanamycin (17-AAG). The CHIP U-box mutant H260Q reduced 17-AAG-induced ErbB2 ubiquitinylation. Wild type ErbB2 and a mutant incapable of association with Cbl (ErbB2 Y1112F) were equally sensitive to CHIP and 17-AAG, implying that Cbl does not play a major role in geldanamycin-induced ErbB2 down-regulation. Both endogenous and ectopically expressed CHIP and ErbB2 coimmunoprecipitated with each other, and this association was enhanced by 17-AAG. Notably, CHIP H260Q induced a dramatic elevation of ErbB2 association with Hsp70 and prevented the 17-AAG-induced dissociation of Hsp90. Our results demonstrate that ErbB2 is a target of CHIP ubiquitin ligase activity and suggest a role for CHIP E3 activity in controlling both the association of Hsp70/Hsp90 chaperones with ErbB2 and the down-regulation of ErbB2 induced by inhibitors of Hsp90.

Mucosal tissues, such as the lung, are continually exposed to both foreign and environmental Ags. To counter the potential inflammatory tissue injury of chronic Th1-mediated responses against these Ags, mucosal sites may skew toward Th2 immune responses. However, the mechanism by which this occurs is unknown. Dendritic cells (DC), as orchestrators of the immune response, skew Th1/Th2 differentiation by cytokine secretion and expression of specific cell surface markers. We compared DC from mucosal and systemic locations. In this study, we show that the lung lacks a CD8α+ DC subpopulation and contains DC that appear less mature than splenic DC. Furthermore, we demonstrate that pulmonary DC produce significant levels of IL-6 and fail to produce the Th1-polarizing cytokine IL-12. Importantly, we demonstrate that IL-6 negatively regulates IL-12 production, as pulmonary DC from IL-6−/− mice produce significant levels of IL-12 and induce Th1 polarization of naive CD4+ T cells. Furthermore, we demonstrate that IL-6 is sufficient to explain the differential polarizing abilities of pulmonary and splenic DC, as splenic DC cocultures supplemented with IL-6 polarize naive T cells toward Th2, and pulmonary DC cultures in which IL-6 was removed with neutralizing Ab resulted in more Th1 polarization, pointing to IL-6 as the mechanism of Th2 polarization in the lung. We propose that the Th2 response seen in the lung is due to DC-mediated inhibition of Th1 responses via IL-6 production, rather than enhanced Th2 responses, and that this regulation decreases the likelihood of chronic inflammatory pathology in the lung.