Mirk Kinase in Colon Cancer Development

描述：(改编自调查人员的摘要)米尔克是一个 一种可介导细胞存活的结构性活性蛋白激酶 缺乏有丝分裂原。瞬时转染的MERK激活ERK亚家族 在没有生长因子或血清的情况下，MAP激酶的活性。稳定污垢 转染体，在无血清条件下生长和存活，以及 载体控制细胞不表现出ERKS的低结构性激活， 是载体对照细胞的5倍，对有丝分裂原有更多的反应。这些 观察很可能与生理相关，而不仅仅是一种人工制品 过度表达或使转基因细胞适应培养，因为米尔克是 已知的生存因子之一--胰岛素样生长因子1所必需的 (IGF-1)作为有丝分裂原发挥作用。当NIH3T3细胞中MERK蛋白水平 被硫代化的反义寡核苷酸降低，IGF-1不再 这些细胞的有丝分裂原。改变反义寡核苷酸的剂量会导致 剂量依赖性地降低IGF-1的反应，因此MERK减少得越多， 促有丝分裂的IGF-1较少。因此，内源性的MERK水平是必要的。 用于体内的有丝分裂反应。此外，还存在着相互制约的关系 ERK和MERK：过度表达的MERK激活ERK，而持续激活 的人压低了米尔克。MERK与癌症有关。稳定过表达 MERK在体内存在于表现为MERK的结肠癌的一大亚群中 水平是配对正常结肠的5-40倍。如果野生型MERK稳定 在2个结肠癌细胞系中过表达，细胞在 在无血清条件下，以一种依赖于MERK的方式，不影响 激酶失活的MERK。目的1：分析MERK在ERK激活中的作用。 瞬时转染中激活ERKs所需的MERK区域 实验将通过突变/缺失分析来确定。MERK可能会激活 ERK通过磷酸化ERK信号级联的某些组成部分。此入站 在酵母双杂交中，利用MERK作为“诱饵”可以找到活体底物。 互补实验筛选人骨骼肌cdna文库。米克可能会 调节ERK信号，使MERK激活启动子元件的能力 与报告基因相关联的基因将通过瞬时共转染进行检测。 目的2：分析ERKS对MERK的调控。ERK监管是否 转录或转录后将使用以下条件确定 其中(A)ERK激活被阻断并且mik蛋白水平增加数倍， (B)ERK持续激活，MERK水平下降数倍。 MERK是一种带有ERK磷酸化位点的MAP激酶底物 终点站。ERK的作用，如果有的话，在C‘末端的产生中被删除57 将确定KDA核沉淀物种类，以及生物特性 这个C‘终端删除了MERK。目的3：检测MERK蛋白的表达 通过免疫组织化学方法在人类癌组织中检测。

DESCRIPTION: (Adapted from the investigator's abstract) Mirk is a constitutively active protein kinase which can mediate cell survival in the absence of mitogens. Transiently transfected mirk activates the erk subfamily of MAP kinases in the absence of growth factors or serum. Stable mirk transfectants, under serum-free conditions in which they grow and survive, and vector control cells do not, exhibit a low constitutive activation of erks, 5-fold over that of vector control cells, and more response to mitogens. These observations are likely to be physiologically relevant, not simply an artifact of overexpression or an adaptation of transfectants to culture, because mirk is required for one of the known survival factors, insulin-like growth factor 1 (IGF-1) to function as a mitogen. When mirk protein levels in NIH3T3 cells were decreased by phosphorothiolated antisense oligonucleotides, IGF-1 no longer was a mitogen for these cells. Varying the dosage of the antisense oligos caused a dose-dependent decrease in IGF-1 response, so the more mirk was reduced, the less mitogenic IGF-1 was. Therefore, endogenous levels of mirk are necessary for mitogenic response in vivo. There is also reciprocal regulation between erks and mirk: overexpressed mirk activates erks, whereas sustained activation of erks downregulate mirk. Mirk is relevant to cancer. Stable overexpression of mirk occurs in vivo in a large subset of colon cancers which exhibit mirk levels 5-40-fold those in paired normal colon. If wild-type mirk is stably overexpressed in 2 colon cancer cell lines, cells grow and survive in serum-free conditions in a mirk-dependent manner, with no effect of kinase-inactive mirk. Aim 1 :analysis of the role of mirk in erk activation. The regions of mirk necessary to activate erks in transient transfection experiments will be determined by mutation/deletion analysis. Mirk may activate erks by phosphorylating some component of the erk signaling cascade. This in vivo substrate will be found by using mirk as "bait" in a yeast two-hybrid complementation assay to screen a human skeletal muscle cDNA library. Mirk may modulate erk signaling so the capacity of mirk to activate promoter elements linked to reporter genes will be assayed by transient co-transfections. Aim 2 : analysis of mirk regulation by erks. Whether erk regulation is transcriptional or post-transcriptional will be determined using conditions in which (a) erk activation is blocked and mirk protein levels rise several fold, (b) erks undergo a sustained activation and mirk levels decrease several fold. Mirk is a MAP kinase substrate with erk phosphorylation sites in its C'terminus. The role of erks, if any, in generation of a C'terminal deleted 57 kDa nuclear mirk species will be determined, as will the biological properties of this C'terminal deleted mirk. Aim 3 : measurement of mirk protein expression by immunohistochemistry in human cancer tissues.