In vitro endosome‐lysosome transfer of dephosphorylated EGF receptor and Shc in rat liver

Abstract

We have studied the endosome-lysosome transfer of internalized epidermal growth factor receptor (EGFR) complexes in a cell-free system from rat liver. Analytical subfractionation of a postmitochondrial supernatant fraction showed that a pulse of internalized 125 I]EGF to the cytosol was detected. To assess the fate of the internalized EGFR protein over the time course of the endo-lysosomal transfer of the ligand, the effect of a saturating dose of native EGF on subsequent lysosomal targeting of the EGFR was evaluated by immunoblotting. A massive translocation of the EGFR to the endosomal compartment was observed in response to ligand injection coincident with its tyrosine phosphorylation and receptor recruitment of the tyrosine-phosphorylated adaptor protein Shc. During cell-free endosome-lysosome fusion, a time-dependent increase in the content of the EGFR and the two 55-and 46-kDa Shc isoforms was observed in lysosomal fractions with a time course superimposable with the lysosomal transfer of the ligand; no transfer of the 66-kDa Shc isoform was detected. The relationship between EGFR tyrosine kinase activity and EGFR sorting in endosomes investigated by immunoblot studies with anti-phosphotyrosine antibodies revealed that endosomal dephosphorylation of EGFR and Shc preceded lysosomal transfer. These results support the view that a lysosomal targeting machinery distinct from the endosomal receptor kinase activity, such as the recruitment of the signaling molecule Shc, may regulate this sorting event in the endosome.z 1999 Federation of European Biochemical Societies.

Smap1 deficiency perturbs receptor trafficking and predisposes mice to myelodysplasia

“…SCF induces the phosphorylation and monoubiquitination of c-KIT, which are necessary for Grb2 association and endocytosis, respectively. The dephosphorylation of internalized RTKs occurs on early endosomes (7,35). We observed that the kinetics of c-KIT in the early phases, such as phosphorylation, dephosphorylation, endocytosis from the cell surface, and exit from early endosomes, were not affected by the loss of SMAP1.…”

Adaptor protein Shc undergoes translocation and mediates up‐regulation of the tyrosine kinase c‐Src in EGF‐stimulated A431 cells

“…Recently, Authier & Chauvet (1999) demonstrated that Shc is associated with the endocytosed EGFR. In addition, c-Src is reported to be enriched in the endosomal membranes in a ®broblast cell line that is derived from Rat-1 cells (Kaplan et al 1992).…”

Inhibition of Endosomal Insulin-like Growth Factor-I Processing by Cysteine Proteinase Inhibitors Blocks Receptor-mediated Functions

“…Alternatively, receptor retention in the endosomes may trigger an alternate signal transduction cascade that leads to inhibition rather than activation of DNA synthesis and MMP-2 production. Receptor phosphorylation and signaling within the endosomal compartment have been documented for the insulin receptor kinase (15,25), and epidermal growth factor receptor activation and Shc recruitment within endosomes have also been observed (26,27). Reports have linked receptor internalization to the activation of the Shc/mitogen-activated protein kinase pathway (28,29).…”

“…It has been clearly shown that ligand degradation is a key event in receptor recycling (11,15) and signaling (27). Recently it was reported that anti-p185/HER2 antibody-mediated targeting of a cysteine proteinase inhibitor to a cathepsin Bcontaining intracellular compartment resulted in growth inhibition of two breast carcinoma cell lines including MCF-7 (32).…”