Enhanced Tumorigenic Behavior of Glioblastoma Cells Expressing a Truncated Epidermal Growth Factor Receptor Is Mediated through the Ras-Shc-Grb2 Pathway

Abstract

A mutant epidermal growth factor receptor (⌬EGFR) containing a deletion of 267 amino acids from the extracellular domain is common in human glioblastomas. We have previously shown that the mutant receptor fails to bind EGF, is constitutively phosphorylated, and confers upon U87MG glioblastoma cells expressing it (U87MG.⌬EGFR), an increased ability to form tumors in mice. Here we demonstrate that the constitutively phosphorylated ⌬EGFR enhances growth of glioblastoma cells through increased activity of Ras: 1) there was an increase in the proportion of Ras present in the GTPbound form, and 2) introduction of neutralizing anti-Ras 259 antibodies into U87MG and U87MG.⌬EGFR cells by microinjection inhibited DNA synthesis to the same low level in both cell populations. We also show that the truncated EGF receptor constitutively associates with the adapter proteins Shc and Grb2 which are involved in the recruitment of Ras to activated receptors. Several derivatives of ⌬EGFR containing single, or multiple mutations at critical autophosphorylation sites were constructed and used to demonstrate that the major Shc binding site is Tyr-1148, and that Grb2 association occurs primarily through Tyr-1068. We conclude that the increased tumorigenic potential of glioblastoma cells expressing the truncated EGF receptor is due at least in part to Ras activation presumably involving the Shc and Grb2 adapter proteins.

Aberrant activation of ALK kinase by a novel truncated form ALK protein in neuroblastoma

“…21,24 For example, in addition to ligand-independent self-dimerization, epidermal growth factor receptor class III variant has been shown to constitutively interact with adaptor proteins SHC and GRB2, which are involved in the recruitment of the RAS pathway. 25 The recepteur d'origine nantais (RON) RTK variant with a deletion in the first immunoglobulinplexin transcription domain (ROND160) has also been considered as a constitutively activated kinase in several human cancers. 20,26 RON belongs to the MET proto-oncogene family, which plays a critical role in epithelial cell homeostasis and tumorigenic development.…”

Quantitative determination of Rap 1 activation in cyclic nucleotide-treated HL-60 leukemic cells: lack of Rap 1 activation in variant cells

“…To measure Rapbound GTP, we used the RBD peptide to precipitate Rap´GTP from cell extracts and we measured GTP eluted from Rap using an enzymebased method (Scheele et al, 1995;Pilz et al, 1997;Guha et al, 1996Guha et al, , 1997Prigent et al, 1996;Liu et al, 1998;Egawa et al, 1999;Sharma et al, 1998). To determine the sum of Rap-bound GTP and GDP, we converted Rap´GDP to Rap´GTP by incubating half of the cell extract with 10 mM GTP and then measured Rap´GTP as described above.…”

“…GTP was measured by conversion to ATP using the enzyme nucleoside diphosphate kinase and the resulting ATP was measured by the ®re¯y luciferase system; this assay is sensitive to 1 fmol of GTP and has been described previously (Scheele et al, 1995;Pilz et al, 1997;Guha et al, 1996Guha et al, , 1997Prigent et al, 1996). In experiments where BHK cells were transfected with GST-tagged Rap 1A, the sum of GTP plus GDP bound to Rap 1A was measured by converting GDP to GTP using the enzyme pyruvate kinase (Sharma et al, 1998;Liu et al, 1998;Egawa et al, 1999) and total GTP was then measured.…”

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

“…As described in Materials and methods, we used a modification of a method for measuring Ras activation (Scheele et al, 1995;Dupuy et al, 2001;Prigent et al, 1996;Sharma et al, 1998;Suhasini et al, 1998), and this assay measures the amount of GTP and the sum of GTP plus GDP bound to Rheb. All samples are split in half, with one-half of the sample receiving an anti-Rheb or anti-myc antibody (the experimental sample), and the other half of the sample receiving non-specific immunoglobulin (control sample).…”