Intestinal Cell Growth Control: Role of Tyrosine Kinases

描述(由申请人提供)：我们有兴趣了解正常的肠道细胞如何调节它们的生长，以及这种调节的丧失如何导致恶变。我们的研究集中在Src酪氨酸激酶及其抑制剂参与调控的分子机制上。我们发现了两种调节Src活性的基本机制。一种是通过在C末端尾部高度保守的酪氨酸上添加磷酸。这个位点的突变将正常细胞中的Src转化为一种转化蛋白。另一种是通过与Rack1相互作用，Rack1是一种内源性底物，也是Src激酶和结肠细胞生长的抑制因子。我们最近的体外研究表明 Rack1通过抑制关键细胞周期检查点和细胞凋亡过程中的Src活性来调节结肠细胞的生长。我们假设Rack1也在体内调节细胞生长，并在线粒体凋亡中发挥关键作用。为了验证这些假说，我们将：目标1：分析Rack1在体内调节肠上皮细胞生长的机制。为此，将建立一种肠道上皮细胞中Rack1缺失的小鼠模型。将评估Rack1‘S对隐窝和干细胞增殖、上皮细胞迁移、分化和凋亡基因表达的影响以及肠肿瘤的发生发展。我们将确定Src的缺失是否可以挽救肠上皮细胞中的Rack1缺乏症。目的2：进一步分析Rack1在线粒体细胞死亡中的作用。研究的重点是Rack1参与和激活促凋亡的Bax和Bim，诱导它们移位到线粒体，介导它们之间的相互作用和与存活的Bcl2家族成员的相互作用，调节Bax在线粒体的滞留，促进Bax的寡聚。在细胞周期的关键检查点、在隐窝和干细胞的增殖以及在细胞凋亡过程中起作用的内源性致癌蛋白抑制物将对细胞的生长起到强大和普遍的控制作用。对这些功能的开发可以用来开发新的、更强大的、有选择性的治疗人类结肠癌的策略。

DESCRIPTION (provided by applicant): We are interested in understanding how normal intestinal cells regulate their growth and how loss of that regulation results in malignant transformation. Our research focuses on molecular mechanisms by which the Src tyrosine kinases and their inhibitors contribute to the regulation. We discovered two fundamental mechanisms whereby Src activity is regulated. One is by addition of a phosphate to a highly conserved tyrosine in the C-terminal tail. Mutation of this site converts the normal cellular Src into a transforming protein. The other is by interaction with Rack1, an endogenous substrate and inhibitor of Src kinases and colonic cell growth. Our recent in vitro studies demonstrate that Rack1 regulates colonic cell growth by suppressing Src activity at critical cell cycle checkpoints and during apoptosis. We hypothesize that Rack1 also regulates cell growth in vivo and plays a key role in mitochondrial apoptosis. To test these hypotheses we will: Aim 1: Analyze mechanisms by which Rack1 regulates growth of intestinal epithelia in vivo. A mouse model with Rack1 deleted in intestinal epithelia will be generated to do so. Rack1's influence on proliferation of crypt and stem cells, migration, differentiation and apoptosis of epithelial cells gene expression and development of intestinal neoplasia will be assessed. We will determine whether loss of Src rescues Rack1 deficiency in intestinal epithelia. Aim 2: Further analyze Rack1 function in mitochondrial cell death. Studies focus on mechanisms by which Rack1 engages and activates proapoptotic Bax and Bim, induces their translocation to mitochondria, mediates their interaction with each other and with prosurvival Bcl-2 family members, regulates Bax sequestration at mitochondria and facilitates Bax oligomerization. Endogenous inhibitors of oncogenic kinases that work at key checkpoints in the cell cycle, in proliferating crypt and stem cells and during apoptosis would exert powerful and pervasive control over cell growth. Exploitation of these multiple functions could be used to develop new and more powerful and selective strategies for treatment of human colon cancer.