Role of KOS in Regulating Cell Growth

描述（由申请人提供）：我们最近发现了Kos1（胚胎干细胞激酶），这是一种新的非受体蛋白酪氨酸激酶（NRPTK），在小鼠胚胎干细胞（ES）细胞退出LIF后分化。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）中的强制表达与抑制生长和凋亡有关。研究显示，Kos1具有自磷酸化功能，其催化活性是其生长抑制功能所必需的。因此，即使在液体和软琼脂培养物中，激酶缺失版本Kos1 [Kos1 (K148A)CN]的表达也能促进NIH 3T3细胞的生长，这表明Kosl可能作为生长的负调节因子。初步研究还表明，Kos1而非Kos1 (K148A)CN可以间接抑制Ras和下游Raf-MAPK生长信号通路。因此，我们假设Kos1在抑制Ras的机制中作为细胞生长的负调节因子，并可能具有肿瘤抑制特性。因此，Kos1可能在癌症中发挥作用。为了验证这一假设，已经确定了两个特定的目标：1)确定如何调节Kos1以及它如何调节Ras活性和细胞生长。Kos1是如何被催化激活的？需要哪些Kos1酪氨酸磷酸化位点？Kos1的生长和Ras抑制功能是否需要酪氨酸磷酸化？2)确定是否需要Kos1来抑制生长，以及Kos1在体内是否能影响生长发育。Kos1是肿瘤抑制因子吗？将采用最先进的分子和生化方法来促进研究概述，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”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。研究结果有望填补我们关于NRPTKs和Kos1如何特异性负调控Ras-Rafl-MAPK生长途径的基本知识空白，从而可能有助于未来开发针对Kos1的新型抗癌治疗策略。

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.