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

丝氨酸/苏氨酸磷酸化对集落刺激因子-1 受体信号转导的调节

• 批准号: 9306519
• 负责人: Angel Lee
• 金额: $ 1.8万
• 依托单位: Washington University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9306519&HistoricalAwards=false
• 关键词: Regulation; Signal; Transduction; Colony; Stimulating

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.

集落刺激因子 1 受体 (CSF-1R) 由原癌基因 c-fms 编码，是一种调节巨噬细胞生长和代谢的酪氨酸激酶受体。 该研究者对调节受体功能的机制感兴趣。 该项目的重点是通过丝氨酸和苏氨酸的磷酸化/去磷酸化来调节该生长因子受体。这笔资金支持的规划期的目标是建立一个实验系统来识别该受体 C 末端的丝氨酸磷酸化位点。 第一个目标是通过定点诱变构建 C 端截短突变体，在骨髓 32D 细胞中表达突变体受体，然后通过免疫沉淀 CSF-1R 的 2D 薄层胰蛋白酶磷酸肽图谱确认 C 端磷酸化的存在或不存在。 一种补充策略是将 C 端结构域合成为细菌融合蛋白，并在体外激酶测定中利用该片段，其中包含候选丝氨酸/苏氨酸激酶的制剂或未分级的细胞提取物。 接下来，将构建覆盖整个 C 末端的丝氨酸到丙氨酸突变体，以查明磷酸化的实际位点。 这些丝氨酸突变体的表型将在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方面进行研究。 还将启动鉴定相关丝氨酸/苏氨酸激酶的工作。 计划期的第二个目标是开始研究，探索涉及丝氨酸/苏氨酸磷酸化的相互机制，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进行研究的新研究者提供启动支持。