Role of KOS in Regulating Cell Growth

KOS 在调节细胞生长中的作用

• 批准号: 7237309
• 负责人: William Stratford MAY
• 金额: $ 28.28万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-16 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7237309
• 关键词: Affect; Agar; Apoptosis; Arts; Binding; Biochemical; Cell Line; Cells; Clathrin; Cloning; Data; Development; Embryo; Embryonic Development; Future; Goals; Growth; Growth Factor; Growth and Development function; Hematopoietic; Human Cell Line; IL3 gene; In Vitro; Knockout Mice; Knowledge; Liquid substance; MEKs; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Methodology; Modeling; Molecular; Mus; NIH 3T3 Cells; Oncogenic; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Property; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Receptor Protein-Tyrosine Kinases; Regulation; Role; Serum; Signal Pathway; Signal Transduction; Staging; Stress; T47D; Testing; Time; Tumor Suppressor Proteins; Tyrosine; Tyrosine Phosphorylation; Tyrosine Phosphorylation Site; United States National Institutes of Health; Up-Regulation; Withdrawal; anti-cancer therapeutic; cell growth; day; embryonic stem cell; genetic regulatory protein; homologous recombination; in vivo; leukemia inhibitory factor; mutant; novel; paxillin; receptor; stem

DESCRIPTION (provided by applicant): We recently discovered Kos1 (Kinase of the embryonic stem cells), a novel, nonreceptor protein tyrosine kinase (NRPTK) in differentiating murine embryonic stem (ES) cells following withdrawal of LIF. Kosl is a ubiquitously expressed 47kDa protein, whose forced expression in either mouse or human cell lines, including NIH 3T3, T47D and NCI-H82 cells, is associated with inhibition of growth and apoptosis. Kos1 was shown to autophosphorylate and its catalytic activity is required for its growth inhibitory function. Thus, expression of a kinase null version of Kos1 [Kos1 (K148A)CN] promotes NIH 3T3 cell growth even in liquid and soft agar cultures, indicating Kosl may function as a negative regulator of growth. Preliminary studies also indicate that Kos1 but not Kos1 (K148A)CN can indirectly inhibit Ras and the downstream Raf-MAPK growth signal pathway. Therefore, we hypothesize that Kos1 functions as a negative regulator of cell growth in a mechanism involving inhibition of Ras and may have tumor suppressor properties. Thus, Kos1 may play a role in cancer. To test this hypothesis two specific aims have been identified: 1) To determine how Kos1 is regulated and how it regulates Ras activity and cell growth. How is Kos1 catalytically activated and what are the required Kos1 tyrosine-phosphorylation site(s)? Is tyrosine phosphorylation of Kos1 required for its growth and Ras inhibitory functions? 2) To determine whether Kos1 is required for growth suppression and whether Kos1 can affect growth and development in vivo. Is Kos1 a tumor suppressor? State of the art molecular and biochemical methodologies will be employed to facilitate the studies outlined including mutational analysis of Kos1, in vivo and in vitro expression studies of Kos1 mutants, interactive cloning strategies using Kos1 as "bait" to identify binding/regulatory proteins, and the creation of a Kos1 null mouse through targeted homologous recombination to assess its role in negative growth regulation in vivo. Results are expected to fill in fundamental gaps in our knowledge regarding how NRPTKs and Kos1 specifically may negatively regulate the Ras-Rafl-MAPK growth pathway and thereby potentially aid in the future development of novel anti-cancer therapeutic strategies targeting Kos1.

描述（由申请人提供）：我们最近发现了Kos1（胚胎干细胞激酶），一种新型非受体蛋白酪氨酸激酶（NRPTK），可在LIF撤除后分化小鼠胚胎干（ES）细胞。 Kosl 是一种普遍表达的 47kDa 蛋白，其在小鼠或人类细胞系（包括 NIH 3T3、T47D 和 NCI-H82 细胞）中强制表达，与生长和细胞凋亡的抑制相关。 Kos1 被证明可以自磷酸化，其催化活性是其生长抑制功能所必需的。因此，Kos1 [Kos1 (K148A)CN] 的激酶无效版本的表达即使在液体和软琼脂培养物中也能促进 NIH 3T3 细胞生长，表明 Kosl 可能充当生长的负调节因子。初步研究还表明，Kos1而非Kos1 (K148A)CN可以间接抑制Ras及其下游Raf-MAPK生长信号通路。因此，我们假设 Kos1 在涉及抑制 Ras 的机制中充当细胞生长的负调节因子，并且可能具有肿瘤抑制特性。因此，Kos1 可能在癌症中发挥作用。为了检验这一假设，我们确定了两个具体目标：1) 确定 Kos1 的调节方式以及它如何调节 Ras 活性和细胞生长。 Kos1 是如何催化激活的以及所需的 Kos1 酪氨酸磷酸化位点是什么？ Kos1 的酪氨酸磷酸化是其生长和 Ras 抑制功能所必需的吗？ 2)确定Kos1是否是生长抑制所必需的以及Kos1是否能影响体内生长发育。 Kos1是抑癌基因吗？将采用最先进的分子和生化方法来促进概述的研究，包括 Kos1 的突变分析、Kos1 突变体的体内和体外表达研究、使用 Kos1 作为“诱饵”来识别结合/调节蛋白的交互式克隆策略，以及通过靶向同源重组创建 Kos1 无效小鼠以评估其在体内负生长调节中的作用。预计结果将填补我们关于 NRPTK 和 Kos1 如何具体负向调节 Ras-Rafl-MAPK 生长途径的知识空白，从而可能有助于未来开发针对 Kos1 的新型抗癌治疗策略。