Role of KOS in Regulating Cell Growth

描述(申请人提供)：我们最近发现了KOS1(胚胎干细胞激酶)，一种新的，非受体蛋白酪氨酸激酶(NRPTK)，在LIF停用后分化小鼠胚胎干细胞(ES细胞)。KOSL是一种普遍表达的47 kDa蛋白，在NIH3T3、T47D和NCI-H82等人和小鼠细胞系中强制表达与抑制细胞生长和诱导细胞凋亡有关。KOS1被证明是自磷酸化的，它的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中，Kos1[Kos1(K148A)CN]的表达也能促进NIH 3T3细胞的生长，表明KOSL可能作为生长的负调控因子发挥作用。初步研究还表明，KOS1而不是KOS1(K148A)CN可以间接抑制RAS及其下游的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控因子发挥作用，并可能具有肿瘤抑制特性。因此，KOS1可能在癌症中起作用。为了验证这一假说，已经确定了两个具体的目标：1)确定KOS1是如何被调控的，以及它是如何调控RAS活性和细胞生长的。KOS1是如何被催化激活的？所需的KOS1酪氨酸磷酸化位点是什么(S)？KOS1的酪氨酸磷酸化是否是其生长和RAS抑制功能所必需的？2)确定KOS1是否是生长抑制所必需的，以及KOS1是否可以影响体内的生长和发育。KOS1是肿瘤抑制因子吗？将采用最先进的分子和生物化学方法促进所概述的研究，包括KOS1的突变分析，KOS1突变体的体内和体外表达研究，以KOS1为“诱饵”识别结合/调节蛋白的交互克隆策略，以及通过定向同源重组创建KOS1缺失小鼠，以评估其在体内负生长调控中的作用。这些结果有望填补我们关于NRPTKs和KOS1如何特异性地负调控Ras-raf1-MAPK生长途径的知识的根本空白，从而可能有助于未来针对KOS1的新型抗癌治疗策略的开发。

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.