Sir Peter MacCallum Department of Oncology - Research Publications

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Start date: 1990 × End date: 1999 × Author: Bowtell, David ×

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    A DOMINANT-NEGATIVE MUTANT OF MSOS1 INHIBITS INSULIN-INDUCED RAS ACTIVATION AND REVEALS RAS-DEPENDENT AND RAS-INDEPENDENT INSULIN SIGNALING PATHWAYS
    SAKAUE, M ; BOWTELL, D ; KASUGA, M (AMER SOC MICROBIOLOGY, 1995-01)
    The role of the Grb2-SOS complex in insulin signal transduction was investigated with a deletion mutant of mSOS1 that lacks the guanine nucleotide exchange domain of the wild-type protein. Cells over-expressing either wild-type (CHO-IR/SOS cells) or mutant (CHO-IR/delta SOS cells) mSOS1 were established by transfection of Chinese hamster ovary cells that express human insulin receptors (CHO-IR cells) with the appropriate expression plasmid. The mutant mSOS1 protein did not contain the guanine nucleotide exchange activity in vitro and associated with Grb2 both in vivo and in vitro. In both CHO-IR and CHO-IR/SOS cells, insulin rapidly stimulated the formation of GTP-bound Ras and the phosphorylation of mitogen-activated protein (MAP) kinase; both these effects of insulin were markedly inhibited in CHO-IR/delta SOS cells. Insulin-induced glycogen synthase and 70-kDa S6 kinase activities were not affected by expression of either wild-type or mutant mSOS1. These results show that the mutant mSOS1 acts in a dominant-negative manner and suggest that the Grb2-SOS complex mediates, at least in part, insulin-induced activation of Ras in intact cells. The data also indicate that Ras activation is not required for insulin-induced stimulation of glycogen synthase and 70-kDa S6 kinase.
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    Tissue hyperplasia and enhanced T-cell signalling via ZAP-70 in c-Cbl-deficient mice
    Murphy, MA ; Schnall, RG ; Venter, DJ ; Barnett, L ; Bertoncello, I ; Thien, CBF ; Langdon, WY ; Bowtell, DDL (AMER SOC MICROBIOLOGY, 1998-08)
    The c-Cbl protein is tyrosine phosphorylated and forms complexes with a wide range of signalling partners in response to various growth factors. How c-Cbl interacts with proteins, such as Grb2, phosphatidylinositol 3-kinase, and phosphorylated receptors, is well understood, but its role in these complexes is unclear. Recently, the Caenorhabditis elegans Cbl homolog, Sli-1, was shown to act as a negative regulator of epidermal growth factor receptor signalling. This finding forced a reassessment of the role of Cbl proteins and highlighted the desirability of testing genetically whether c-Cbl acts as a negative regulator of mammalian signalling. Here we investigate the role of c-Cbl in development and homeostasis in mice by targeted disruption of the c-Cbl locus. c-Cbl-deficient mice were viable, fertile, and outwardly normal in appearance. Bone development and remodelling also appeared normal in c-Cbl mutants, despite a previously reported requirement for c-Cbl in osteoclast function. However, consistent with a high level of expression of c-Cbl in the hemopoietic compartment, c-Cbl-deficient mice displayed marked changes in their hemopoietic profiles, including altered T-cell receptor expression, lymphoid hyperplasia, and primary splenic extramedullary hemopoiesis. The mammary fat pads of mutant female mice also showed increased ductal density and branching compared to those of their wild-type littermates, indicating an unanticipated role for c-Cbl in regulating mammary growth. Collectively, the hyperplastic histological changes seen in c-Cbl mutant mice are indicative of a normal role for c-Cbl in negatively regulating signalling events that control cell growth. Consistent with this view, we observed greatly increased intracellular protein tyrosine phosphorylation in thymocytes following CD3epsilon cross-linking. In particular, phosphorylation of ZAP-70 kinase in thymocytes was uncoupled from a requirement for CD4-mediated Lck activation. This study provides the first biochemical characterization of any organism that is deficient in a member of this unique protein family. Our findings demonstrate critical roles for c-Cbl in hemopoiesis and in controlling cellular proliferation and signalling by the Syk/ZAP-70 family of protein kinases.