Internalized Epidermal Growth Factor Receptors Participate in the Activation of p21 in Fibroblasts

Abstract

Regulated activation of the highly conserved RasGTPase is a central event in the stimulation of cell proliferation, motility, and differentiation elicited by receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR). In fibroblasts, this involves formation and membrane localization of Shc⅐Grb2⅐Sos complexes, which increases the rate of Ras guanine nucleotide exchange. In order to control Ras-mediated cell responses, this activity is regulated by receptor downregulation and a feedback loop involving the dual specificity kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK). We investigated the role of EGFR endocytosis in the regulation of Ras activation. Of fundamental interest is whether activated receptors in endosomes can participate in the stimulation of Ras guanine nucleotide exchange, because the constitutive membrane localization of Ras may affect its compartmentalization. By exploiting the differences in postendocytic signaling of two EGFR ligands, epidermal growth factor and transforming growth factor-␣, we found that activated EGFR located at the cell surface and in internal compartments contribute equally to the membrane recruitment and tyrosine phosphorylation of Shc in NR6 fibroblasts expressing wild-type EGFR. Importantly, both the rate of Ras-specific guanine nucleotide exchange and the level of Ras-GTP were depressed to near basal values on the time scale of receptor trafficking. Using the selective MEK inhibitor PD098059, we were able to block the feedback desensitization pathway and maintain activation of Ras. Under these conditions, the generation of Ras-GTP was not significantly affected by the subcellular location of activated EGFR. In conjunction with our previous analysis of the phospholipase C pathway in the same cell line, this suggests a selective continuation of specific signaling activities and cessation of others upon receptor endocytosis.The 170-kDa epidermal growth factor receptor (EGFR) 1 exerts its biological effects in response to binding of specific polypeptide ligands, including epidermal growth factor (EGF) and transforming growth factor-␣ (TGF␣). This leads to activation of the EGFR catalytic tyrosine kinase domain, autophosphorylation of specific residues in its carboxyl terminus, and recruitment and phosphorylation of heterologous signaling proteins (1). The EGFR can also transactivate other members of the erbB receptor family via heterodimerization, enhancing the diversity of potential signaling interactions (2). Overexpression and activating mutations of EGFR and other erbB family members, in conjunction with other permissive mutations, have been widely implicated in transformation and tumorigenesis.Increased ligand secretion and autocrine signaling through the EGFR can also contribute to uncontrolled cell proliferation. Secretion of TGF␣ in particular is potently mitogenic, because its dissociation from EGFR after endocytosis promotes receptor recycling; the sparing of receptors from proteolysis allows unabated si...

Rheb is in a high activation state and inhibits B-Raf kinase in mammalian cells

“…of at least three independent experiments performed in duplicate). These Rheb activation levels, particularly for serum-starved cells, are considerably higher than we and others have found for other wild type Ras-related proteins, including Ras and Rap 1: activation levels for serum-starved cells are generally in the range of 0.5 -8%, depending on the cell type, and activation levels for logarithmically growing or serum-stimulated cells are generally between 10 -18% (Scheele et al, 1995;Tian et al, 2000;von Lintig et al, 2000;Sharma et al, 1998;Prigent et al, 1996;Haugh et al, 1999;Satoh et al, 1990a,b). To perform experiments under identical conditions, we measured Rheb, Ras, and Rap 1 activation in serum-starved PC-12 cells, and found activation levels of 23+4%, 3+0.5%, and 5+0.7%, respectively.…”

Membrane Proximal ERK Signaling Is Required for M-calpain Activation Downstream of Epidermal Growth Factor Receptor Signaling

“…EGFR Signaling Compartmentation Protocol-Haugh et al (28,29) previously have shown that internalized EGFR can retain activity dependent on the binding properties of its ligand (Fig. 1A).…”

“…Images were false colored to grayscale; therefore, brightness correlates with increased fluorescence intensity and calpain activity. activated both from plasma membrane EGFR and endosomal EGFR (29). Obviously, the accessibility of focal adhesion components would differ greatly from these two sites.…”

Cellular Internalization of Insulin-like Growth Factor Binding Protein-3

“…The observation that the activated epidermal growth factor receptor can be detected on endosomes where it can activate Ras indicates that it is the receptor itself that needs to be internalized to initiate signaling (49,50). Furthermore, nerve growth factormediated signaling illustrates how one molecule can mediate dichotomous functions; the activated Trk receptor induces survival when at the plasma membrane, whereas it elicits differentiation of neurite outgrowth if internalized (51).…”