A mutant epidermal growth factor receptor with defective protein tyrosine kinase is unable to stimulate proto-oncogene expression and DNA synthesis.

Abstract

Cultured NIH-3T3 cells devoid of endogenous epidermal growth factor (EGF) receptors were transfected with cDNA expression constructs encoding either normal human EGF receptor or a rgceptor mutated in vitro at Lys-721, a residue that is thought to function as part of the ATP-binding site of the kinase domain. Unlike the wild-type EGF-receptor expressed in these cells, which exhibited EGF-dependent protein tyrosine kinase activity, the mutant receptor lacked protein tyrosine kinase activity and was unable to undergo autophosphorylation and to phosphorylate exogenous substrates. Despite this deficiency, the mutant receptor was normally expressed on the cell surface, and it exhibited both high-and low-affinity binding sites. The addition of EGF to cells expressing wild-type receptors caused the stimulation of various responses, including enhanced expression of proto-oncogenes c-fos and c-myc, morphological changes, and stimulation of DNA synthesis. However, in cells expressing mutant receptors, EGF was unable to stimulate these responses, suggesting that the tyrosine kinase activity is essential for EGF receptor signal transduction.The protein tyrosine kinase gene family includes a group of proteins which act as membrane receptors for growth factors (for a review, see reference 6). The enzymatic activity of the cytoplasmic kinase domain of these receptors is regulated by the binding of specific growth factors to the extracellular-ligand-binding domain (for a review, see references 6 and 13). This leads to receptor self-phosphorylation and to the phosphorylation of specific substrates which may play a role in the pleiotropic response leading to mitogenesis. All protein tyrosine kinases contain a consensus lysine residue in the kinase domain which is thought to function as part of the ATP-binding site (3,4,16,17) (i.e., in epidermal growth factor receptor [EGF-R] or Lys-1018 in insulin receptor). It has been shown that mutations at these consensus lysine residues abolish the protein tyrosine kinase activity of insulin receptor (4) and of other members of the src gene family (7,15,19). To examine the role of the kinase activity of EGF-R, we have prepared an Ala-721 EGF-R cDNA construct by in vitro site-directed mutagenesis (5). The Ala-721 EGF-R cDNA construct was cloned into a mammalian expression vector which utilizes the simian virus 40 early promoter for gene expression and also contains the dihydrofolate reductase and neomycin resistance genes as selectable markers (12). The plasmid was transfected into NIH-3T3 cells, and Geneticin G418 (GIBCO) was used for selection of cells expressing receptor molecules. Several cloned cell lines expressing the wild-type receptor on the Ala-721 receptor mutant were developed. The NIH-3T3 cells used for transfection were obtained from C. Fryling. We compared various clones of NIH-3T3 cells for the expression of EGF-R by using immunoprecipitation and 125I-labeled

The Linoleic Acid Metabolite, (13S)-Hydroperoxyoctadecadienoic Acid, Augments the Epidermal Growth Factor Receptor Signaling Pathway by Attenuation of Receptor Dephosphorylation

“…Ligand-induced activation of the EGFR tyrosine kinase appears to be the essential biochemical event for further EGF mitogenic signal transduction (17)(18)(19). In the work reported here, we demonstrate that the linoleic acid metabolite (13S)-HPODE modulates the tyrosine phosphorylation of the EGFR signaling pathway.…”

“…Occupation of the EGFR by EGF and other ligands causes dimerization of the receptor and stimulates the receptor's intrinsic tyrosine kinase activity (14). The intrinsic tyrosine kinase activity of the EGFR is the critical biochemical signal involved in cellular mitogenic responses to EGF (17)(18)(19). The kinase activity autophosphorylates the EGFR, phosphorylates key tyrosine residues present in several signaling proteins, and initiates a series of tyrosine, serine and threonine protein phosphorylations which link the cell surface with the nucleus (15).…”

Signal transduction through atypical PKCs, but not the EGF receptor, is necessary for UVC-induced AP-1 activation in immortal murine cells

“…The receptor has intrinsic protein tyrosine kinase activity, which is responsible for many of the pleotropic intracellular e ects (Honegger et al, 1987;Chen et al, 1987). Murine ®broblast B82 is a mouse L cell line that contains no EGF receptors, as assessed by EGF binding as well as by RNA blot analysis (Lin et al, 1986).…”

“…Ligand-induced activation of the receptor results in phosphorylation and activation of a number of substrates, including mitogen-activated protein (MAP) kinase (Carpenter and Cohen, 1991;Eldredge et al, 1994). The intrinsic kinase activity of the EGFR is responsible for many of the pleotropic intracellular e ects (Honegger et al, 1987;Chen et al, 1987). Reports from a number of laboratories have established that UVC irradiation activates signal transduction pathways that extend from the cell membrane (Sachsenmaier et al, 1994;Devary, 1991Devary, , 1993 and feed into common signal transduction pathways whose components are mostly shared among TPA and growth factors (Pelech and Songhere, 1992;Karin and Smeal, 1992).…”

Analogs of human epidermal growth factor which partially inhibit the growth factor‐dependent protein‐tyrosine kinase activity of the epidermal growth factor receptor

“…A third model invokes a ligand-induced intramolecular conformational change in the receptor's extracellular domain leading to the activation of the kinase activity of the receptor [lo, 111. While this issue is still being resolved, it is quite clear that the tyrosine kinase activity of the receptor is essential for the mitogenic response of the cells to EGF [12,13].…”