GENETIC ANALYSIS OF RAS TRANSFORMATION AND JUN FUNCTION

描述：(改编自申请者摘要)：关键环节 Ras原癌基因的激活和转录激活是 C-jun和AP-1转录因子的反式激活。申请人 已经在小鼠体内产生了c-jun零突变。衍生的原代细胞 这样的胚胎在培养过程中立即停止生长。增长停滞可能 通过与大T抗原和激活的RAS共转化克服； 然而，这些细胞不同于由相同试剂转化的野生型细胞， 保持非致瘤性。这些数据表明c-jun在RAS中起着关键作用 诱导肿瘤形成。申请人建议进一步澄清这一点。 使用生物化学和遗传学相结合的方法发挥作用。首先，他会 使用S63/S73突变体进行c-jun的靶向替换，以评估其作用 体内JNK介导的磷酸化对c-Jun功能的影响。类似 将使用各种方法来确定 三角洲地区，该地区被认为在JNK招聘和在 C-jun的泛素化/不稳定。可能存在的冗余 不同的c-jun基因将通过c-jun被取代的研究进行评估 在6月B或6月D之前，申请人将评估Jund的角色 直接通过执行一次Jund击倒。最后，申请者将使用 一种新的体内致瘤性建立和候选筛选方法 Ras/AP-1相关靶基因参与肿瘤的发生。

DESCRIPTION: (adapted from applicant's abstract): A critical link between the activation of the ras proto-oncogene and transcriptional activation is the transactivation of c-Jun and AP-1 transcription factors. The applicant has created a null mutation of c-jun in the mouse. Primary cells derived from such embryos growth arrest immediately in culture. Growth arrest can be overcome by co-transformation with large T antigen and activated Ras; however, these cells, unlike wild type cells transformed by the same agents, remain non-tumorigenic. These data indicate a critical role of c-jun in Ras induced tumor formation. The applicant proposes to further elucidate this role using a combined biochemical and genetic approach. First, he will perform targeted replacement of c-jun with a S63/S73 mutant, to assess role of JNK-mediated phosphorylation on c-jun function in vivo. Similar approaches will be used to determine the role of and the requirement for the delta region, which is thought to play a role both in JNK recruitment and in ubiquitination/destabilization of c-jun. The possible redundancy of different c-jun genes will be assessed by studies in which c-jun is replaced by jun B or jun D. In addition, the applicant will assess the role of junD directly by performing a junD knockout. Finally, the applicant will use a novel in vivo assay of tumorigenic establishment and screen for candidate target genes involved in ras/AP-1 mediate tumorigenesis.