Mirk Kinase in Colon Cancer Development

描述：（改编自研究者的摘要）Mirk是一个 组成型活性蛋白激酶，可以介导细胞存活， 没有丝裂原。瞬时转染的mirk激活erk亚家族 MAP激酶在生长因子或血清的情况下。稳定的泥泞 转染子在无血清条件下生长和存活，和 载体对照细胞没有表现出ERK的低组成性激活， 5-倍于载体对照细胞，并且对有丝分裂原有更多的反应。这些 观察结果可能与生理相关，而不仅仅是人为因素 过度表达或适应的转染培养，因为mirk是 胰岛素样生长因子1是一种已知的生存因子， （IGF-1）作为有丝分裂原发挥作用。当NIH 3 T3细胞中mirk蛋白水平降低时， 通过硫代磷酸化反义寡核苷酸降低，IGF-1不再被 这些细胞的促分裂原改变反义寡核苷酸的剂量， IGF-1反应呈剂量依赖性降低，因此mirk减少越多， IGF-1促有丝分裂作用较弱。因此，内源性mirk水平是必要的， 用于体内促有丝分裂反应。也有相互调节之间 erks和mirk：过表达的mirk激活erks，而持续激活 下调了mirk。Mirk与癌症有关。稳定高表达 MIRK在体内发生在表现MIRK的结肠癌的大部分中 水平是配对正常结肠的5-40倍。如果野生型mirk稳定地 在2种结肠癌细胞系中过表达，细胞在 在无血清条件下，以依赖于mirk的方式， 激酶失活的mirk目的1：分析mirk在erk激活中的作用。 在瞬时转染中激活erks所必需的mirk区域 实验将通过突变/缺失分析来确定。迈尔克可能会激活 通过磷酸化ERK信号级联的一些组分来调节ERK。这 在酵母双杂交系统中，用mirk作为“诱饵”， 互补分析来筛选人骨骼肌cDNA文库。米尔克·梅 调节erk信号传导，使mirk激活启动子元件的能力 通过瞬时共转染来检测与报道基因相连的细胞。 目的2：分析erks对mirk的调控作用。无论erk法规是 转录或转录后将使用以下条件来确定： 其中（a）ERK激活被阻断并且MIRK蛋白水平升高数倍， (b)ERK经历持续的激活，并且MILK水平降低数倍。 Mirk是一种MAP激酶底物，在其 C末端。erks的作用，如果有的话，在产生C '末端删除57 kDa核镜种类将被确定，生物学特性也将被确定 C '端删除的病毒目的3：测量mirk蛋白表达 通过免疫组织化学的方法。

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.