MOLECULAR MECHANISMS FOR REGULATION OF NA+/H+ EXCHANGE

该提案描述了我们继续研究的实验， 在有丝分裂原刺激的，培养的细胞中调节Na+/H+交换活性 人成纤维细胞。 本项目的长期目标是了解 一种分子和结构基础， 激活Na+/H+交换系统。 我们具体建议：（1） 确定参与调节的激酶和磷酸酶， Na+/H+交换对促分裂原刺激的反应。 这将涉及 利用融合蛋白和完整细胞膜作为底物的研究 用于用来自细胞提取物的激酶进行体外磷酸化测定， 用纯化的激酶如PKC、CaM激酶II和MAP激酶/MAP样 激酶。 磷酸酶参与的研究将使用32 P标记的 交换蛋白作为细胞提取物中磷酸酶的底物。 冈田酸将用于区分磷酸酶的类型 （2）确定Na+/H+磷酸化位点， 有丝分裂原活化细胞中的交换分子。 这将涉及 利用融合蛋白作为底物进行体外磷酸化研究 蛋白质和合成肽，代表不同部分的 Na+/H+交换器的胞质尾部，以及体内磷酸化 实验后，用免疫沉淀法对交换剂进行免疫沉淀， 我们实验室生产的抗体 融合蛋白上的磷酸化位点 蛋白质和完整的Na+/H+交换器将通过胰蛋白酶 运输蛋白的片段化，胰蛋白酶的纯化 碎片的TLEC或反相FPLC和气相序列分析， 胰蛋白酶片段;（3）确定是否差异调节 在HSWP和WI-38细胞中的Na+/H+交换（作为表现出两种Na +/H+交换的模型细胞） Na+/H+交换剂的不同类型调节）是以下因素的结果 不同的调节途径（激酶/磷酸酶途径）或结果 不同的交换分子存在于两种细胞类型中。 这 将涉及比较磷酸化位点和 参与调节Na+/H+交换的激酶/磷酸酶途径 这两种细胞类型。 由于Na+/H+交换剂是促分裂剂的最佳实例之一， 对生长因子活性增加的活化的膜靶 刺激，阐明调节的分子机制， 这一途径的研究对于我们理解正常的 细胞生长，并最终在了解异常细胞生长， 癌症

This proposal describes experiments to continue our research on the regulation of Na+/H+ exchange activity in mitogen-stimulated, cultured human fibroblasts. The long-term goal of this project is to understand on a molecular and structural basis the mechanism by which growth factors activate the Na+/H+ exchange system. We propose specifically to: (1) Determine the kinases and phosphatases involved in the regulation of the Na+/H+ exchanger in response to mitogen stimulation. This will involve studies utilizing fusion proteins and intact cell membranes as substrates for in vitro phosphorylation assays with kinases from cell extracts and with purified kinases such as PKC, CaM kinase II and MAP kinase/MAP-like kinases. Investigation of phosphatase involvement will utilize 32P-labeled exchanger protein as a substrate for phosphatases in cell extracts. Okadaic acid will be utilized to distinguish the type of phosphatase involved; (2) Identify the site(s) of phosphorylation on the Na+/H+ exchanger molecule in mitogen-activated cells. This will involve performing in vitro phosphorylation studies utilizing as substrates fusion proteins and synthetic peptides that represent different portions of the cytosolic tail of the Na+/H+ exchanger, as well as in vivo phosphorylation experiments followed by immunoprecipitation of the exchanger with antibodies produced in our laboratory. Phosphorylation sites on the fusion proteins and the intact Na+/H+ exchanger will be identified by tryptic fragmentation of the transport protein, purification of the tryptic fragments on TLEC or reverse phase FPLC and gas phase sequence analysis of the tryptic fragments; (3) Determine whether the differential regulation of Na+/H+ exchange in HSWP and WI-38 cells (as model cells exhibiting two different types of regulation of the Na+/H+ exchanger) is the result of different regulatory pathways (kinase/phosphatase pathways) or the result of different exchanger molecules being present in the two cell types. This will involve a comparison of the phosphorylation sites and the kinase/phosphatase pathways involved in regulation of Na+/H+ exchange in these two cell types. Since the Na+/H+ exchanger is one of the best examples of a mitogen- activated membrane target whose activity is increased on growth factor stimulation, the elucidation of the molecular mechanism for the regulation of this pathway is important for our understanding of the process of normal cell growth and ultimately in the understanding of abnormal cell growth in the disease of cancer.