NOVEL PUTATIVE PROTEIN KINASE ENCODED BY A MITOGEN INDUCIBLE GENE

成纤维细胞生长因子-1和成纤维细胞生长因子-2蛋白可能 体内血管细胞生长的重要介质，因为它们(1)是 有效的血管生成因子和平滑肌细胞有丝分裂原和(2)是 由内皮细胞、平滑肌细胞和单核细胞表达- 衍生的巨噬细胞。成纤维细胞生长因子参与血管病变的可能性 疾病已经促使许多研究确定有效的治疗策略 抑制成纤维细胞生长因子的生物活性。一种这样的策略是打断 成纤维细胞生长因子促有丝分裂信号通路。当成纤维细胞生长因子被激活时激活的基因 添加到静止细胞中可能编码参与这一过程的蛋白质 因此，采用了mRNA差异显示技术 小鼠表达成纤维细胞生长因子-1反应基因的cDNA克隆 NIH3T3成纤维细胞。其中一个基因，命名为Fnk，意思是成纤维细胞生长因子诱导的 是一种受成纤维细胞生长因子-1、成纤维细胞生长因子-2、血小板衍生生长因子-2高度诱导的即刻早期基因。 BB、血清和佛波酯。该基因也在体内瞬时表达。 小鼠肝部分切除后和大鼠颈动脉切除后的肝脏 气球受伤。核苷酸序列分析表明，Fnk基因的c DNA 编码一种新的丝氨酸/苏氨酸蛋白激酶，其催化结构域 占据了分子的氨基末端的一半。预测的Fnk 蛋白质与小鼠的氨基酸序列同源性约为49% SNK蛋白，它也是一种可能的丝氨酸/苏氨酸激酶，由 一种中早熟基因。此外，FNK在结构上与 哺乳动物PLK和果蝇Polo，细胞周期所需的蛋白激酶 进步。本文件中描述的研究的主要目标 应用是确定Fnk是否是丝氨酸/苏氨酸激酶 参与了成纤维细胞生长因子的细胞内信号通路。因此， 这项建议的具体目的是：1)确定FNK蛋白， 与Fnk一样，在成纤维细胞生长因子-1刺激的细胞中瞬时表达，并 确定其亚蜂窝位置；2)确定FNK是否真的 丝氨酸/苏氨酸特异的蛋白激酶；3)确定FNK是否 在成纤维细胞生长因子-1刺激的细胞中磷酸化，如果是这样的话，鉴定 磷酸化位点(S)，并确定磷酸化是否改变 酶活性；以及4)确定是否需要FNK功能 对于成纤维细胞生长因子-1刺激的细胞增殖以及FNK本身是否 有丝分裂。预计这些研究将提供新颖的 成纤维细胞生长因子-1和成纤维细胞生长因子-2促分裂信号机制的研究进展 转导，并可能确定FNK作为治疗的靶点 抗增殖治疗。

The fibroblast growth factor (FGF)-1 and FGF-2 proteins are likely to be important mediators of vascular cell growth in vivo since they (1) are potent angiogenic factors and smooth muscle cell mitogens and (2) are expressed by endothelial cells, smooth muscle cells and blood monocyte- derived macrophages. The possibility that FGFs may be involved in vascular disease has prompted numerous studies to identify strategies for effective inhibition of FGF biological activity. One such strategy is to interrupt the FGF mitogenic signaling pathway. Genes that are activated when FGF is added to quiescent cells may encode proteins that participate in this pathway; therefore, a mRNA differential display technique was used to obtain cDNA clones representing FGF-1-responsive genes expressed in murine NIH 3T3 fibroblasts. One of these genes, named Fnk for FGF-inducible kinase, is an immediate-early gene highly inducible by FGF-1, FGF-2, PDGF- BB, serum and phorbol ester. This gene is also transiently expressed in mouse liver after partial hepatectomy and in rat carotid artery after balloon injury. Nucleotide sequence analysis indicated that the Fnk cDNA encoded a novel serine/threonine protein kinase, with the catalytic domain occupying the amino-terminal half of the molecule. The predicted Fnk protein has approximately 49% amino acid sequence identity to the mouse Snk protein, which is also a putative serine/threonine kinase encoded by an mediate-early gene. Furthermore, Fnk is structurally-related to mammalian Plk and Drosophila polo, protein kinases required for cell cycle progression. The major objective of the research described in this application is to determine whether Fnk is a serine/threonine kinase involved in the FGF intracellular signaling pathway. Accordingly, the specific aims of this proposal are: 1) to determine whether Fnk protein, like Fnk mRNA, is transiently expressed in FGF-1-stimulated cells and to identify its subcellular location; 2) to determine whether Fnk is in fact a serine/threonine-specific protein kinase; 3) to determine whether Fnk is phosphorylated in FGF-1-stimulated cells, and if so, to identify the phosphorylation site(s) and determine whether phosphorylation alters enzymatic activity; and 4) to determine whether Fnk function is required for FGF-1-stimulated cell proliferation and whether Fnk itself is mitogenic. It is anticipated that these studies will provide novel information on the mechanism of FGF-1 and FGF-2 mitogenic signal transduction and may identify Fnk as a therapeutic target for antiproliferative therapy.