The search for the molecular mechanisms underlying neoplastic transformation has focused on the signal transduction pathways within cells. Several tyrosine kinase containing receptors have been implicated in this process but critical targets have remained elusive. Recently, it has been discovered that the EGF receptor can phosphorylate the gamma form of phospholipase C (PLC), a class of enzymes that catalyzes the formation of inositol triphosphate (IP3) and diacylglycerol, two potent mediators of intracellular signalling. We have cloned the human form of PLC-gamma from a glial tumor and have found that when it is overexpressed in NIH-3T3 cells it can cause transformation. The goals of this proposal are to clarify the mechanisms by which PLC-gamma is involved in transformation and to examine its potential role in glial tumors. Preliminary experiments have shown that transformation by PLC-gamma was not associated with an increase in enzymatic activity, but an increase in tyrosine phosphorylation was always found. To evaluate these two processes, PLC-gamma will be placed under the control of an inducible promoter. By obtaining graded amounts of protein, it will be possible to determine the relationship between PLC-gamma levels, IP3 production, phosphotyrosine content, and the properties associated with transformation including effects on cell growth, cloning efficiency in soft agar, and tumorigenicity in athymic mice. To study the relationship between the EGF receptor and PLC-gamma in transformation, the cell lines described above will be transfected with the EGF receptor to see if this augments any of these parameters. Coordinate changes in the levels of PLC-gamma during transformation by the EGF receptor will also be examined. Functional domains of PLC-gamma will be mapped with respect to transforming ability. Two constructs will be focused upon. The first construct will contain the catalytic domain but delete the presumptive regulatory domain. The second will contain the region that is homologous to the v-crk oncogene, which has been found to induce tyrosine phosphorylation despite having no intrinsic kinase activity. These cell lines will be used to define the proteins that interact with PLC-gamma and potentially effect transformation. The relevance of these finding to glial tumors will be explored. The possibility that a mutation may have occurred in our tumor-derived clone will be examined. If the mutation does confer transforming ability, then its prevalence in other tumors will be determined. To correlate previous results with primary tumors, the levels of PLC-gamma, IP3 and phosphotyrosine will be studied in glial tumors. Since preliminary evidence shows that PLC-gamma is elevated in tumors, antibodies against this protein will be used to stain glial tumors to examine their utility in diagnosis or evaluating prognosis. It is anticipated that these studies will add to our knowledge of the basic mechanisms underlying transformation as well as providing potentially useful clinical information.
| Lawe, D C; Hahn, C; Wong, A J (1997) The Nck SH2/SH3 adaptor protein is present in the nucleus and associates with the nuclear protein SAM68. Oncogene 14:223-31 |
| Emlet, D R; Moscatello, D K; Ludlow, L B et al. (1997) Subsets of epidermal growth factor receptors during activation and endocytosis. J Biol Chem 272:4079-86 |
| Moscatello, D K; Montgomery, R B; Sundareshan, P et al. (1996) Transformational and altered signal transduction by a naturally occurring mutant EGF receptor. Oncogene 13:85-96 |
| Wong, A J; Zoltick, P W; Moscatello, D K (1994) The molecular biology and molecular genetics of astrocytic neoplasms. Semin Oncol 21:139-48 |
