REGULATION OF P93C-FES PROTEIN-TYROSINE KINASE ACTIVITY

人c-fes基因编码一个93 kDa的胞质蛋白酪氨酸 PTK是一种在细胞终末分化过程中被激活的蛋白激酶， 体外髓系白血病细胞系。 转染未成熟的 c-fes基因诱导的髓系白血病细胞系K562 提示p93（c-fes）在细胞分化中起着积极的作用， 骨髓生长控制。 对c-fes的监管知之甚少 PTK活性，或由p93（c-100）磷酸化的底物的身份， FES）介导分化。 本提案将调查这些 通过关注已知的p93（c-fes）的独特结构特征， src homology 2（SH 2）结构域。 该非催化结构域位于 p93（c-fes）和其他PTK中激酶结构域的N端，包括 病毒c-fes同系物v-fps 已显示v-fps的SH 2结构域 通过与自磷酸化酪氨酸结合来调节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。 测试 c-fes SH 2结构域调节邻近激酶的假设 结构域，将在c-fes的SH 2结构域中进行一系列缺失 cDNA。 所得突变体家族将在E.杆菌和 使用免疫复合物激酶测定法测定PTK活性。 变化 活性将与改变的SH 2-激酶结构域结合相关 使用蛋白酶抗性测定。 c-fes SH 2结构域在细胞凋亡中的作用 生物学功能将通过用 SH 2缺失突变体，并测定细胞的SH 2缺失突变体的功能标志物。 分化 含磷酸酪氨酸蛋白的模式将 在用野生型和SH 2转染的K562细胞之间进行比较 变种人 存在于细胞中的酪氨酸磷酸化蛋白质 用野生型转染但不转染分化缺陷型SH 2 突变体将是C-FES底物的良好候选物。 直接测试 c-fes SH 2结构域可以识别c-fes底物的假设 和调节蛋白，重组c-fes SH 2结构域蛋白将被 在细菌中合成，生物素化，并用于探测蛋白质印迹 K562细胞的蛋白质。 此外，重组SH 2蛋白 将附着在珠状琼脂糖上，并用作亲和基质， 纯化SH 2结合蛋白。 最后，c-fes自身磷酸化位点 将使用2-D胰蛋白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 破坏与SH 2结构域的相互作用。 成功完成 这些研究将提供关于 SH 2结构域在c-fes PTK调控及与底物复合物形成中的作用 和/或调节蛋白。 进一步了解这种增长- 调控途径可能为设计新的 通过刺激诱导终末分化的抗白血病剂 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.