REGULATION OF P93C-FES PROTEIN-TYROSINE KINASE ACTIVITY

人c-fes基因座编码93 kDa胞质蛋白酪氨酸 在细胞终末分化过程中被激活的一种蛋白激酶(PTK) 体外培养髓系白血病细胞株。未成熟基因的转染法 携带c-fes基因的髓系白血病细胞株(K562)的诱导 提示p93(c-fes)在肿瘤细胞分化中起积极作用。 髓系生长控制。人们对c-fes的调控知之甚少。 PTK活性，或p93(c-)磷酸化底物的特性。 FES)，调节分化。这项提案将调查这些 通过关注已知的p93(c-fes)的独特结构特征来提问 作为src同源2(SH2)结构域。这个非催化结构域位于 P93(c-fes)和其他蛋白酪氨酸激酶的N-末端，包括 病毒的c-fes同源物，v-fps。已经显示了v-fps的SH2结构域 通过与自身磷酸化的酪氨酸结合来调节PTK活性 激酶结构域中的残基，并介导与 转化相关底物和调节蛋白。要测试 假设c-fes SH2结构域调节相邻的激酶 域，将在c-FE的SH2域中进行一系列删除 CDNA.由此产生的突变体家族将在大肠杆菌中表达，并且 用免疫复合激动法测定PTK活性。变化 IN活性将与改变的SH2-激酶结构域关联相关 使用了一种抗蛋白酶的检测方法。C-fes SH2结构域在细胞周期调控中的作用 通过将该基因导入K562细胞进行生物学功能的评估 Sh2缺失突变体，并对细胞进行功能标记分析 差异化。含磷酸酪氨酸的蛋白质模式将 野生型K562细胞与SH2细胞的BE比较 变种人。存在于细胞中的酪氨酸磷酸化蛋白质 野生型但不存在分化缺陷的SH2 突变体将是很好的c-fes底物候选者。直接测试 C-fes SH2结构域可识别c-fes底物的假设 和调控蛋白，重组c-fes SH2结构域蛋白将被 在细菌中合成，生物素化，用于探测蛋白质印迹 来自K562细胞的蛋白质。此外，重组SH2蛋白 将被连接到珠状琼脂糖凝胶上并用作亲和基质 纯化SH2结合蛋白。最后，c-fes自磷酸化位点。 将通过2-D胰酶磷酸肽图谱和位点- 定向诱变。这些位点的突变预计会影响 P93(c-fes)PTK活性、结构和生物学功能 中断了与SH2结构域的相互作用。成功完成 这些研究将提供新的信息，关于 C-Fes PTK调控中的Sh2结构域及其与底物的复合体形成 和/或调节蛋白。对这种增长的进一步理解- 调控途径可能为新基因的设计提供分子基础 通过刺激诱导终末分化的抗白血病药物 P93(c-fes)PTK活性。

The human c-fes locus encodes a 93 kDa cytoplasmic protein-tyrosine kinase (PTK) that is activated during the terminal differentiation of myeloid leukemia cell lines in vitro. Transfection of an immature myeloid leukemia cell line (K562) with the c-fes gene induced differentiation, suggesting that p93(c-fes) plays an active role in myeloid growth control. Little is known about the regulation of c-fes PTK activity, or the identity of the substrates phosphorylated by p93(c- fes) that mediate differentiation. This proposal will investigate these questions by focusing on a unique structural feature of p93(c-fes) known as the src homology 2 (SH2) domain. This non-catalytic domain is located N-terminal to the kinase domain in p93(c-fes) and other PTKs, including the viral c-fes homolog, v-fps. The SH2 domain of v-fps has been shown to regulate PTK activity by binding to autophosphorylated tyrosine residues in the kinase domain, and to mediate interactions with transformation-related substrates and regulatory proteins. To test the hypothesis that the c-fes SH2 domain regulates the adjacent kinase domain, a series of deletions will be made in the SH2 domain of the c-fes cDNA. The resulting family of mutants will be expressed in E. coli, and assayed for PTK activity using an immune-complex kinase assay. Changes in activity will be correlated with altered SH2-kinase domain association using a protease-resistance assay. The role of the c-fes SH2 domain in biological function will be assessed by transfecting K562 cells with the SH2 deletion mutants, and assaying the cells for functional markers of differentiation. Patterns of phosphotyrosine-containing proteins will be compared between K562 cells transfected with the wild-type and SH2 mutants. Proteins phosphorylated on tyrosine that are present in cells transfected with the wild-type but not in differentiation-defective SH2 mutants will be good candidates for c-fes substrates. To directly test the hypothesis that the c-fes SH2 domain can recognize c-fes substrates and regulatory proteins, recombinant c-fes SH2 domain protein will be synthesized in bacteria, biotinylated, and used to probe western blots of proteins from K562 cells. Additionally, the recombinant SH2 protein will be attached to beaded agarose and used as an affinity matrix to purify SH2-binding proteins. Finally, c-fes autophosphorylation sites will be identified using 2-D tryptic phosphopeptide mapping and site- directed mutagenesis. Mutagenesis of these sites is expected to affect p93(c-fes) PTK activity, structure and biological function due to disrupted interaction with the SH2 domain. Successful completion of these studies will provide novel information regarding the role of the SH2 domain in c-fes PTK regulation and complex formation with substrates and/or regulatory proteins. Further understanding of this growth- regulatory pathway may provide a molecular basis for the design of novel anti-leukemic agents that induce terminal differentiation by stimulating p93(c-fes) PTK activity.