NOVEL PUTATIVE PROTEIN KINASE ENCODED BY A MITOGEN INDUCIBLE GENE

成纤维细胞生长因子（FGF）-1和FGF-2蛋白可能是 体内血管细胞生长的重要介质，因为它们（1） 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、平滑肌细胞和血液单核细胞表达， 衍生的巨噬细胞。FGFs参与血管生成的可能性 疾病促使许多研究确定有效的策略， 抑制FGF生物活性。其中一个策略是打断 FGF促有丝分裂信号通路。当FGF被激活时， 添加到静止细胞可能编码蛋白质，参与这一过程， 因此，mRNA差异显示技术被用于 获得代表在小鼠中表达的FGF-1应答基因的cDNA克隆， NIH 3T3成纤维细胞。其中一个基因，命名为Fnk， 激酶，是一种可被FGF-1、FGF-2、PDGF-1、FGF-2、FGF-3、FGF-4、FGF-4、FGF-5、FGF-6、FGF-7、FGF-8、FGF-8、FGF-9、FGF-10、FGF-11、FGF-11、FGF-11、FGF-11、FGF-12、FGF-11、FGF-11、FGF-11、FGF-12、FGF-11、FGF-11、FGF-12、FGF-11、FGF-11、FGF-12、FGF-11、FGF-12、FGF-11、FGF-13、FGF-12、FGF-13、FGF-12、FGF-13、FGF-13、FGF-13、FGF-14、FGF-13、FGF-13、FGF-14、FGF-13、FGF-13、FGF-14、FGF-13、FGF-13、FGF-14、FGF-13、FGF-14、FGF BB，血清和佛波酯。该基因也瞬时表达于 小鼠肝部分切除后和大鼠颈动脉 气囊损伤核苷酸序列分析表明，Fnk cDNA 编码一种新的丝氨酸/苏氨酸蛋白激酶，具有催化结构域 占据了分子氨基末端的一半。预测的FNK 蛋白质与小鼠具有约49%的氨基酸序列同一性 snk蛋白，其也是由 中早期基因此外，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 进展本文所述研究的主要目的是 本申请的目的是确定Fnk是否是丝氨酸/苏氨酸激酶 参与FGF细胞内信号通路。因此 该建议的具体目的是：1）确定Fnk蛋白， 与Fnk mRNA一样，在FGF-1刺激的细胞中瞬时表达， 识别其亚细胞位置; 2）确定Fnk是否实际上 丝氨酸/苏氨酸特异性蛋白激酶; 3）确定Fnk是否是 在FGF-1刺激的细胞中磷酸化，如果是这样， 磷酸化位点，并确定磷酸化是否改变 酶活性;和4）确定是否需要Fnk功能 对于FGF-1刺激的细胞增殖以及Fnk本身是否 促有丝分裂预计这些研究将提供新的 FGF-1和FGF-2促有丝分裂信号机制的信息 转导，并可能确定Fnk作为治疗靶点， 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.