guanosine-diphosphate and Carcinoma--Squamous-Cell

Grb2-associated binder 1 (GAB1) is a scaffold protein involved in numerous interactions that propagate signaling by growth factor and cytokine receptors. Here we explore in silico and validate in vivo the role of GAB1 in the control of mitogenic (Ras/MAPK) and survival (phosphatidylinositol 3-kinase (PI3K)/Akt) signaling stimulated by epidermal growth factor (EGF). We built a comprehensive mechanistic model that allows for reliable predictions of temporal patterns of cellular responses to EGF under diverse perturbations, including different EGF doses, GAB1 suppression, expression of mutant proteins, and pharmacological inhibitors. We show that the temporal dynamics of GAB1 tyrosine phosphorylation is significantly controlled by positive GAB1-PI3K feedback and negative MAPK-GAB1 feedback. Our experimental and computational results demonstrate that the essential function of GAB1 is to enhance PI3K/Akt activation and extend the duration of Ras/MAPK signaling. By amplifying positive interactions between survival and mitogenic pathways, GAB1 plays the critical role in cell proliferation and tumorigenesis.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Line, Tumor; Cell Survival; Epidermal Growth Factor; Feedback; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Models, Biological; Transfection

The growth-inhibiting peptide hormone somatostatin stimulates phosphotyrosine phosphatase activity in the human pancreatic cell line MIA PaCa-2. This hormonal activation was mediated by a pertussis toxin-sensitive guanosine 5'-triphosphate-binding protein (G protein) in the membranes of these cells. Activation of this G protein by somatostatin stimulated the dephosphorylation of exogenous epidermal growth factor receptor prepared from A-431 cells in vitro. This pathway may mediate the antineoplastic action of somatostatin in these cells and in human tumors and could represent a general mechanism of G protein coupling that is utilized by normal cells in the hormonal control of cell growth.

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Dose-Response Relationship, Drug; Enzyme Activation; ErbB Receptors; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Kinetics; Pancreatic Neoplasms; Peptides; Pertussis Toxin; Phosphorylation; Protein Kinases; Protein Tyrosine Phosphatases; Somatostatin; Thionucleotides; Virulence Factors, Bordetella

Activation of epidermal growth factor (EGF) receptors stimulates inositol phosphate production in rat hepatocytes via a pertussis toxin-sensitive mechanism, suggesting the involvement of a G protein in the process. Since the first event after receptor-G protein interaction is exchange of GTP for GDP on the G protein, the effect of EGF was measured on the initial rates of guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) association and [alpha-32P]GDP dissociation in rat hepatocyte membranes. The initial rate of [35S]GTP gamma S binding was stimulated by EGF, with a maximal effect observed at 8 nM EGF. EGF also increased the initial rate of [alpha-32P]GDP dissociation. The effect of EGF on [35S]GTP gamma S association was blocked by boiling the peptide for 5 min in 5 mM dithiothreitol or by incubation of the membranes with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). EGF-stimulated [35S]GTP gamma S binding was completely abolished in hepatocyte membranes prepared from pertussis toxin-treated rats and was inhibited in hepatocyte membranes that were treated directly with the resolved A-subunit of pertussis toxin. The amount of guanine nucleotide binding affected by occupation of the EGF receptor was approximately 6 pmol/mg of membrane protein. Occupation of angiotensin II receptors, which are known to couple to G proteins in hepatic membranes, also stimulated [35S]GTP gamma S association with and [alpha-32P]GDP dissociation from the membranes. The effect of angiotensin II on [alpha-32P]GDP dissociation was blocked by the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II, demonstrating that the guanine nucleotide binding was receptor-mediated. In A431 human epidermoid carcinoma cells, EGF stimulates inositol lipid breakdown, but the effect is not blocked by treatment of the cells with pertussis toxin. In these cells, EGF had no effect on [35S]GTP gamma S binding. Occupation of the beta-adrenergic receptor in A431 cell membranes with isoproterenol did stimulate [35S] GTP gamma S binding, and the effect could be completely blocked by l-propranolol. These results support the concept that in hepatocyte membranes, EGF receptors interact with a pertussis toxin-sensitive G protein via a mechanism similar to other hormone receptor-G protein interactions, but that in A431 human epidermoid carcinoma cells, EGF may activate phospholipase C via different mechanisms.

Topics: Animals; Binding Sites; Calcium; Carcinoma, Squamous Cell; Cell Membrane; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Isoproterenol; Kinetics; Liver; Male; Pertussis Toxin; Phosphorylation; Protein-Tyrosine Kinases; Rats; Rats, Inbred Strains; Virulence Factors, Bordetella

Topics: Alkaline Phosphatase; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; Female; Giant Cells; Glycolipids; Glycosylphosphatidylinositols; Growth Substances; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Insulin; Isoenzymes; Kinetics; Phosphatidylinositols; Placenta; Pregnancy; Thionucleotides; Trophoblasts