Role of KOS in Regulating Cell Growth

描述（由申请人提供）：我们最近发现Kos 1（胚胎干细胞激酶），一种新的非受体蛋白酪氨酸激酶（NRPTK），在LIF撤除后分化小鼠胚胎干（ES）细胞。Kosl是一种广泛表达的47 kDa蛋白，其在小鼠或人细胞系（包括NIH 3 T3、T47 D和NCI-H82细胞）中的强制表达与生长抑制和凋亡相关。Kos 1显示出自磷酸化，其催化活性是其生长抑制功能所必需的。因此，Kos 1 [Kos 1（K148 A）CN]的激酶无效版本的表达促进NIH 3 T3细胞生长，即使在液体和软琼脂培养物中，表明Kos 1可能作为生长的负调节剂起作用。初步研究还表明，Kos 1而不是Kos 1（K148 A）CN可以间接抑制Ras和下游的Raf-MAPK生长信号通路。因此，我们假设Kos 1在涉及Ras抑制的机制中作为细胞生长的负调节剂发挥作用，并且可能具有肿瘤抑制特性。因此，Kos 1可能在癌症中发挥作用。为了验证这一假设，已经确定了两个具体的目标：1）确定Kos 1是如何调节的，以及它如何调节Ras活性和细胞生长。Kos 1是如何被催化激活的，所需的Kos 1酪氨酸磷酸化位点是什么？Kos 1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Kos 1是否是生长抑制所必需的，以及Kos 1是否可以影响体内生长和发育。Kos 1是肿瘤抑制因子吗？将采用最先进的分子和生物化学方法来促进所概述的研究，包括Kos 1的突变分析，Kos 1突变体的体内和体外表达研究，使用Kos 1作为“诱饵”以鉴定结合/调节蛋白的交互式克隆策略，以及通过靶向同源重组创建Kos 1 null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NRPTKs和Kos 1如何特异性地负调节Ras-Rafl-MAPK生长途径的知识的根本空白，从而可能有助于未来开发针对Kos 1的新型抗癌治疗策略。

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.