Growth control represents a balance of positive and negative growth stimuli, and is dependent on the precis relay of intracellular signals. The broad goal of this research proposal is to understand the process of signa transduction, as it relates to growth control. This information will provide the basis for designing strategies for the effective treatment of pathological conditions such as cancer, which arise from unmoderated proliferation. To study signal transduction we are using the platelet-derived growth factor (PDGF) beta receptor (betaPDGFR) model system. In response to activation by PDGF, the betaPDGFR undergoes tyrosine phosphorylation and tightly associates with numerous cellular signal transduction enzymes including phospholipase C-gamma1 (PLCgamma), the GTPase activating protein of ras (GAP), phosphatidylinositol 3 kinase (PI3K), three src family members, several adapter proteins such as Nck and Shc, and the phosphotyrosine phosphatase Syp. Since these receptor-associated proteins are signal relay enzymes, it seemed likely that they are the receptor's intracellular effectors. We tested this hypothesis by developing and characterizing the add-back betaPDGFR mutants, which revealed that signaling by the betaPDGFR requires stable association with the receptor- associated proteins. In addition, our studies strongly suggested that PI3K and PLCgamma are the betaPDGFR's intracellular effectors. This add-back system has proven to be a unique and powerful approach to study betaPDGFR signal transduction. We will continue to use this system to further investigated signal relay by the betaPDGFR as outlined in the following 4 specific aims. 1. Determine the contribution of PI3K and PLCgamma to the DNA synthesis response of the PI3K and PLCgamma add-back mutants. The hypothesis to be tested is that PLCgamma and PI3K, (as opposed to other proteins that may also associate with the PDGFR via these same sites), are the betaPDGFR's effectors. 2. Test the ability of the various add-back betaPDGFR mutants to drive cellular transformation. The hypothesis to be tested is that the receptor- associated proteins which are required for a DNA synthesis response are the same ones that mediate signals responsible for PDGF-driven transformation of cells. 3. Determine whether the PI3K- and PLCgamma-directed pathways are independent signaling cascades by looking at the immediate early genes induced by the appropriate add-back mutants. The hypothesis to be tested is that PI3K and PLCgamma initiate distinct signal relay pathways. 4. Construct a panel of double add-back mutants (two of the binding sites are restored) and use then to determine if GAP and Syp modulate the PI3K or PLCgamma signaling pathway. The hypothesis to be tested is that GAP and/or Syp function to suppress the positive signals sent by the betaPDGFR. The outcome of these studies will dramatically increase our understanding of how the betaPDGFR sends a mitogenic signal, and how such a signal is regulated. Importantly, the members of the betaPDGFR signal relay cascades are common to many signaling systems, and as a result our findings will provide novel and useful information for a broad spectrum of the signal transduction field.
Showing the most recent 10 out of 28 publications
