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

集落刺激因子殖民地（CSF-1 R），由 原癌基因c-fms是一种酪氨酸激酶受体， 巨噬细胞的生长和代谢。 这个调查员对 调节受体功能的机制。 这个项目 着重于这种生长因子受体的调节， 丝氨酸和苏氨酸的磷酸化/去磷酸化。的 计划期间的目标将由这笔赠款支持， 建立了一个丝氨酸位点鉴定的实验系统， 在该受体的C-末端磷酸化。 第一 目的是构建一个C端截短突变体， 定向诱变，在髓系32 D中表达突变型受体 细胞，然后确认是否存在C-末端 通过2D薄层胰蛋白酶磷酸肽图谱的磷酸化 免疫沉淀的CSF-1 R。 一项补充战略将是 将C-末端结构域合成为细菌融合蛋白， 为了在体外激酶测定中利用该片段， 候选丝氨酸/苏氨酸激酶或 未分级的细胞提取物。 接着，丝氨酸变成丙氨酸 将构建覆盖整个C末端的突变体， 精确定位磷酸化的实际位置。 的表型 将研究这些丝氨酸突变体对 受体酪氨酸激酶活性，下调和 脱敏，与细胞内底物的相互作用， 增长潜力 还将着手确定 负责丝氨酸/苏氨酸激酶。 的第二个目标 规划期是开始研究，探索互惠 涉及丝氨酸/苏氨酸磷酸化的机制， 调节CSF-1 R和血小板衍生的 3 T3成纤维细胞中的生长因子受体以及CSF-1 R和 32 D细胞中的白细胞介素-3受体。 %%% 氨基酸丝氨酸和苏氨酸的磷酸化是 这可能是一种重要的监管机制， 生长因子受体的种类。 该奖项将提供启动- 支持一个开始的研究人员进行研究， 这一重要领域。

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.