The overall goal of this research program is to elucidate mechanisms by which the src oncogene product, p60v-src, alters cellular signal transduction systems. The model studied will focus on a recently discovered src-induced elevation of inositol tetrakisphosphate (InsP4) and stimulation of InsP3-3 kinase in cells. The modulation of these two novel components of the inositol lipid cascade identify a new site of potential control over cellular regulation by the src oncogene. The objectives of the research proposed here are to determine: (a) the role of InsP4 in cellular transformation by src and (b) the cellular and biochemical basis underlying a src-induced build-up of InsP4 in cells.
The specific aims to be pursued are: 1. Relationship of InsP4 and InsP3-3 kinase to src transformation. These studies will use a panel of various src mutants to establish the kinetics of src-induced changes in InsP4 levels and InsP3-3 kinase activity, determine the need for InsP4 in src transformation, and elucidate the mechanistic properties of src required for elevations of InsP4. These studies will also use immune complexes to investigate whether InsP3-3 kinase becomes activated by association with p60v-src or other activated tyrosine kinases. Finally, the generality and specificity of control over InsP4 by various signaling pathways will be explored in different types of normal cells. 2. Mechanisms of InsP4 elevation in normal and src-transformed cells. These studies will determine whether changes in other types of inositol polyphosphates or novel membrane phosphoinositides contribute to InsP4 elevation in src transformed cells. Studies are also proposed to determine if src alters the activities of InsP4-related enzymes and the metabolism of several inositol polyphosphates in cellular homogenates or permeabilized cells. Also studied will be the effects of src on the binding sites for InsP3 and InsP4 in cellular membranes. Together, these results will aid in identifying the sites of InsP4 metabolism affected by p60v-src. 3. Control a calcium dynamics. Since at least one isomer of InsP4 has been linked to calcium control, a series of pilot studies will be undertaken using laser-imaging to determine whether changes in cellular calcium homeostasis relate to InsP4 build-up in src-transformed cells. Overall, these studies will further our understanding of interactions between p60v-src and elements of cellular signaling pathways, and provide insight into how such interactions can lead to alterations in the control of normal cellular proliferation.
| Glazier, A T; Blackmore, P F; Nolan, R D et al. (1998) Attenuation of LPA-mediated calcium signaling and inositol polyphosphate production in rat-1 fibroblasts transformed by the v-src oncogene. Biochem Biophys Res Commun 245:607-12 |
| Mattingly, R R; Wasilenko, W J; Woodring, P J et al. (1992) Selective amplification of endothelin-stimulated inositol 1,4,5-trisphosphate and calcium signaling by v-src transformation of rat-1 fibroblasts. J Biol Chem 267:7470-77 |
