The colony stimulating factor-1 receptor (CSF-1R), encoded by the proto-oncogene c-fms, is a tyrosine kinase receptor which modulates macrophage growth and metabolism. This investigator is interested in the mechanisms that regulate receptor function. This project focuses on the regulation of this growth factor receptor by phosphorylation/dephosphorylation of serines and threonines. The goal of the planning period to be supported by this grant is to set up an experimental system for the identification of sites of serine phosphorylation in the C-terminus of this receptor. The first objective is to construct a C-terminal truncation mutant by site- directed mutagenesis, express the mutant receptor in myeloid 32D cells and then confirm the presence or absence of C-terminal phosphorylation by 2D thin layer tryptic phosphopeptide mapping of immunoprecipitated CSF-1R. A complementary strategy will be to synthesize the C-terminal domain as a bacterial fusion protein and to utilize the fragment in an in vitro kinase assay that contains preparations of either candidate serine/threonine kinases or unfractionated cellular extracts. Following, serine to alanine mutants that cover the entire C-terminus will be constructed to pinpoint the actual sites of phosphorylation. The phenotypes of these serine mutants will be studied in terms of effects on receptor tyrosine kinase activity, down-regulation and desensitization, interaction with intracellular substrates and growth potential. Work will also be initiated to identify the responsible serine/threonine kinase. The second objective of the planning period is to begin studies that explore reciprocal mechanisms involving serine/threonine phosphorylation which may regulate the interactions between CSF-1R and the platelet-derived growth factor receptor in 3T3 fibroblasts and between CSF-1R and the interleukin-3 receptor in 32D cells. %%% The phosphorylation of the amino acids serine and threonine is likely to represent an important regulatory mechanism for certain classes of growth factor receptors. This award will provide start- up support for a beginning investigator undertaking research in this important area.
