Regulation of Signal Transduction in the Colony Stimulating Factor-1 Receptor by Serine/Threonine Phosphorylation

原癌基因c-fms编码的集落刺激因子-1受体(CSF-1R)是一种酪氨酸激酶受体，调节巨噬细胞的生长和代谢。这位研究者对调节受体功能的机制很感兴趣。这个项目的重点是通过丝氨酸和苏氨酸的磷酸化/去磷酸化来调节这种生长因子受体。由这笔赠款支持的规划期的目标是建立一个实验系统，用于识别该受体C-末端的丝氨酸磷酸化位点。第一个目的是通过定点突变构建C端截短突变体，在髓系32D细胞中表达突变体受体，然后通过免疫沉淀的CSF-1R的二维薄层胰酶磷酸肽图来证实C端磷酸化的存在或缺失。一种补充策略是将C-末端结构域合成为细菌融合蛋白，并将该片段用于包含候选丝氨酸/苏氨酸激酶或普通细胞提取物的体外激酶试验。接下来，将构建覆盖整个C末端的丝氨酸到丙氨酸的突变体，以精确定位磷酸化的实际位置。这些丝氨酸突变体的表型将从对受体酪氨酸激酶活性的影响、下调和脱敏、与细胞内底物的相互作用和生长潜力等方面进行研究。还将开始工作，以确定负责的丝氨酸/苏氨酸激酶。规划期的第二个目标是开始研究涉及丝氨酸/苏氨酸磷酸化的相互作用机制，该机制可能调节3T3成纤维细胞中CSF-1R和血小板衍生生长因子受体之间的相互作用，以及32D细胞中CSF-1R和白细胞介素3受体之间的相互作用。氨基酸丝氨酸和苏氨酸的磷酸化可能代表着某些类别的生长因子受体的重要调节机制。该奖项将为在这一重要领域开展研究的初学者提供启动支持。

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.