Andrius Kazlauskas, Ph.D. - US grants

Growth control represents a balance of positive and negative growth stimuli, and is dependent on the precise relay of intracellular signals . The broad goal of this research proposal is to understand the process of signal transduction, as it relates to growth control. This information will provide the foundation for designing strategies for the effective treatment of pathological conditions such as cancer, which arise from unmoderated proliferation. My long-term objectives are to define the mechanism of signal transduction by the platelet-derived growth factor receptor (PDGFR). The intracellular domain of the PDGFR is a tyrosine kinase which is activated by the binding of PDGF. This results in the tyrosine phosphorylation of numerous intracellular proteins including the receptor itself. Receptor autophosphorylation permits the stable association of several signal transduction enzymes. Our studies with receptor mutants have shown that those receptors that fail to associate with any of the signal transduction molecules also fail to mediate a biological signal, indicating that the receptor associated proteins are the likely intracellular mediators of PDGFs mitogenic signal. The projects in this proposal focus on the PDGFR-associated proteins. The binding sites for two of the receptor-associated proteins (GAP and PI3K) have been identified. The First Specific Aim of this proposal is to identify the binding sites on the PDGFR for the other receptor-associated proteins. GAP and PI3K bind to a region of the PDGFR that include a phosphorylated tyrosine residue. The binding of the other receptor-associated proteins also requires that the receptor be tyrosine phosphorylated, and binding can be blocked by antiphosphotyrosine antibodies. We will test the hypothesis that, like the binding sites for GAP and P13K, the binding sites for the other receptor-associated proteins include a phosphotyrosine. This will be accomplished by site-directed mutagenesis of tyrosine residues that are within candidate binding sites. This approach will identify the important tyrosine residues of each of the binding sites, and also provide mutants that selectively fail to bind one (or more) of the receptor-associated proteins. Specific Aim 2: Once the binding sites for each of the receptor-associated proteins have been identified a panel of PDGFR mutants will be made that bind none, or only one of the receptor-associated proteins. Specific Aim 3: The ability of the various PDGFR mutants to trigger mitogenesis will be tested. Given that the mutant repertoire will include receptors that do not associate with any of the signal transduction molecules, as well as PDGFRs that bind only one of the receptor-associated proteins, it will be possible to define the relative contribution of each of the receptor-associated proteins to PDGFR signal transduction. Importantly, the identification of the receptor-associated proteins that are able to mediate PDGF's biological signal will focus future studies, designed to identify all the components of a signal transduction cascade, on these signal transduction enzymes.

Growth control represents a balance of positive and negative growth stimuli, and is dependent on the precise relay of intracellular signals. The broad goals of this research proposal are to understand the process of signal transduction, as it relates to growth control. This information will provide the foundation for designing strategies for the effective treatment of pathological conditions such ad cancer, which arise from unmoderated proliferation. The proposed research examines the mechanism of signal transduction of the platelet-derived growth factor receptor (PDGFR). The intracellular domain of the PDGFR is a tyrosine kinase which is activated by the binding of PDGF. This results in the tyrosine phosphorylation of numerous intracellular proteins including the receptor itself. Receptor autophosphorylation permits the stable association of several signal transduction enzymes. Our studies with receptor mutants have shown that those receptors that fail to associate with any of the signal transduction molecules also fail to mediate a biological signal which strongly suggests that the receptor associated proteins are the most likely intracellular mediators of PDGF's mitogenic signal. I propose to define the relative contribution of each of the receptor- associated proteins to PDGF-mediated signal transduction. This will be accomplished by generating random receptor mutants, screening for those that constitutively mediate a biological signal, and then identifying the proteins that associate. By focusing on the constitutively activated receptor mutants that bind a reduced set of signal relay molecules, we will define the minimal number of proteins needed to bind to the PDGFR in order to relay PDGF's biological signal. In addition, by determining the precise location of the activating mutations we will identify regions of the receptor that regulate the binding of these signal transduction molecules. The proteins that associate with the PDGFR have in large part been identified, and this information has provided valuable insight into how the receptor transmits a biological signal. Two of the receptor-associated proteins, a 120 and 65 kd species, have not yet been identified. As the second aim of this application, I propose to isolate the cDNAs corresponding to these two proteins, so that they could be identified and studied further. This will be accomplished by screening a cDNA expression library with the phosphorylated receptor, to which we have demonstrated that the 120 and 65 kd proteins bind in vitro. These two approaches are both the most likely to succeed and the most direct way to answering the question of how PDGF's mitogenic signal is relayed intracellularly. In addition, given that the proteins that associate with the PDGFR also associate with a number of other growth factor receptors and activated tyrosine kinases, what we learn of the mechanism of signal relay by these associated proteins for the PDGFR system will be widely received in the field of signal transduction.