The Fnk Kinase and Vascular Cell Growth Control

说明(申请人摘要)：本研究所研究的长期目标 实验室是为了了解调节生长的分子机制 因子刺激的细胞周期进程。成体的大多数细胞，高等生物体 通常处于静止的非增殖状态，称为GO 细胞周期的阶段。如果被适当的细胞外有丝分裂原激活， 这些细胞可以退出休眠状态，重新启动细胞增殖。 在血管生物学的背景下，内皮细胞(EC)的发病 增殖与许多“血管新生”的发病机制有关 疾病“(如癌症、糖尿病视网膜病变)，而平滑肌细胞(SMC) 积聚发生在动脉粥样硬化的发展过程中， 移植相关动脉硬化和血管壁损伤后的再狭窄。 成纤维细胞生长因子-1和成纤维细胞生长因子-2可能在 在血管细胞生长控制中的作用。这些因素通过绑定起作用，因此 激活特定的跨膜受体酪氨酸激酶。这会触发 下游细胞内事件，包括蛋白质的刺激 磷酸化的级联和特定基因的转录激活。 我们已经使用差异显示方法来鉴定成纤维细胞生长因子诱导的基因 NIH3T3细胞和血管细胞。其中一个名为Fnk的基因是一种 编码POLO家族成员的即刻-早期反应基因 结构上相关的丝氨酸/苏氨酸激酶。我们假设Fnk 表达和酶活性是生长因子刺激细胞的关键 循环递增。该提案的具体目的是：1)确定 在静止的小鼠NIH3T3细胞中加入成纤维细胞生长因子-2是否促进 FNK合成、磷酸氨基酸含量、酶活性和 亚细胞定位，2)确定候选Fnk调节蛋白和 通过筛选Fnk结合蛋白的底物，3)确定Fnk 高表达或低表达改变NIH 3T3细胞、人微血管内皮细胞、 或人主动脉SMC体外增殖，4)测定表型 体内Fnk缺乏的后果。预计这些研究 将提供有关Fnk生物功能的重要信息 蛋白质及其在体内血管细胞生长控制中的潜在作用。

DESCRIPTION (Applicant's abstract): The long-term goal of the research in our laboratory is to understand the molecular mechanisms that regulate growth factor-stimulated cell cycle progression. Most cells in adult, higher organisms are generally in a quiescent non-proliferative state, referred to as the GO phase of the cell cycle. If activated by an appropriate extracellular mitogen, these cells can exit from the resting state and reinitiate cell proliferation. In the context of vascular biology, the onset of endothelial cell (EC) proliferation is associated with the pathogenesis of numerous "angiogenic diseases" (e.g., cancer, diabetic retinopathy) while smooth muscle cell (SMC) accumulation occurs during the development of atherosclerosis, transplant-associated arteriosclerosis and restenosis after vessel wall injury. It is likely that fibroblast growth factor (FGF)- 1 and FGF-2 play an important role in vascular cell growth control. These factors act by binding and thereby activating specific transmembrane receptor tyrosine kinases. This triggers downstream intracellular events, including the stimulation of protein phosphorylation cascades and the transcriptional activation of specific genes. We have used a differential display approach to identify FGF-inducible genes in NIH 3T3 cells and vascular cells. One of these genes, named Fnk, is an immediate-early response gene encoding a member of the polo family of structurally-related serine/threonine kinases. We hypothesize that Fnk expression and enzymatic activity is critical for growth factor-stimulated cell cycle progression. The specific aims of this proposal are: 1) To determine whether the addition of FGF-2 to quiescent murine NIH 3T3 cells promotes changes in Fnk synthesis, phosphoamino acid content, enzymatic activity and subcellular localization, 2) To identify candidate Fnk regulatory proteins and substrates by screening for Fnkbinding proteins, 3) To determine whether Fnk overexpression or underexpression alters NIH 3T3 cell, human microvascular EC, or human aortic SMC proliferation in vitro, and 4) To determine the phenotypic consequences of Fnk deficiency in vivo. It is anticipated that these studies will provide important information on the biological functions of the Fnk protein and its potential role in vascular cell growth control in vivo.