REGULATION OF P93C FES PROTEIN TYROSINE KINASE ACTIVITY

描述：人类c-fes原癌基因编码一个93 kDa的细胞质。 酪氨酸激酶(Fes)参与造血、血管生成和胚胎发育 发展。未成熟髓系白血病细胞系(K-562)的转基因研究 FES导致生长抑制和末端分化， 确认FES是髓系细胞生长的关键调节因子和合理的 髓系白血病分化治疗的靶点。从结构上讲， FES由独特的N-末端区域、Src同源2(SH2)结构域、 和一个C-末端激活域。关于每个角色的四个假设 结构域在调节激酶活性，与下游的相互作用 效应器，并将进行生物功能测试：1)Fes N-末端 区域包含一个新的蛋白质-蛋白质相互作用结构域，对 BCR和其他底物的识别。FES的特定区域 N-末端结构域负责与Rho家族的调节因子BCR结合 小的GTP酶和Fes底物，将在体外定位。生物学的 BCR结合结构域的重要性将通过检测缺失来评估， 该区域在K-562细胞中的插入和替换突变 差异化模型。2)FES SH2结构域对于 分化信号中的蛋白质-蛋白质相互作用。嵌合体Fes 含有其他信号分子SH2结构域的蛋白质将是 在K-562分化模型中进行了检测。证明了嵌合体 在保持激酶活性的同时表现出生物活性减弱 正确的亚细胞定位将有力地支持FE的角色 Sh2结构域在特定的蛋白质-蛋白质相互作用中。3)含SH2的 Vav、STAT-3和PI3K蛋白是分化相关的FES底物。 初步数据表明，这些蛋白质是Fes下游的效应器。这 AIM将测试Fes在髓系细胞中是否与这些蛋白质相互作用 分化，并确定Fes激酶结构域Tyr 自磷酸化位点与这些分子的SH2结构域结合。 体外和体内。4)FeS的活化需要齐聚和 转磷酸化。初步数据表明，活性FeS为低聚物 并通过分子间机制进行自磷酸化。诱变 实验将测试由以下因素确定的N端螺旋线圈结构域 齐聚反应和生物活性需要计算机分析。 在互补实验中，Fes的自我磷酸化缺陷突变体 将接受显性负性活动测试。激酶失活Fes突变体 被预测为抑制依赖于内源性FE的生物反应 非生产性齐聚事件的激活。

DESCRIPTION: The human c-fes proto-oncogene encodes a 93 kDa cytoplasmic tyrosine kinase (Fes) involved in hematopoiesis, angiogenesis, and embryonic development. Transfection of an immature myeloid leukemia cell line (K-562) with Fes results in growth suppression and terminal differentiation, identifying Fes as a key regulator of myeloid cell growth and a rational target for the differentiation therapy of myeloid leukemia. Structurally, Fes consists of a unique N-terminal region, a Src homology 2 (SH2) domain, and a C-terminal kinase domain. Four hypotheses regarding the role of each domain in the regulation of kinase activity, interaction with downstream effectors, and biological function will be tested: 1) The Fes N-terminal region contains a novel protein-protein interaction domain essential for the recognition of BCR and other substrates. The specific region of the Fes N-terminal domain responsible for binding to BCR, a regulator of Rho-family small GTPases and Fes substrate, will be mapped in vitro. The biological significance of the BCR-binding domain will be assessed by testing deletion, insertion, and substitution mutants of this region in the K-562 cell differentiation model. 2) The Fes SH2 domain is essential for protein-protein interaction during differentiation signaling. Chimeric Fes proteins containing the SH2 domains of other signaling molecules will be tested in the K-562 differentiation model. Demonstration that the chimeras show diminished biological activity while maintaining kinase activity and correct subcellular localization will strongly support a role for the Fes SH2 domain in specific protein-protein interactions. 3) The SH2-containing proteins Vav, STAT-3, and PI3K are differentiation-related Fes substrates. Preliminary data implicate these proteins as downstream Fes effectors. This Aim will test whether Fes interacts with these proteins during myeloid differentiation, and determine whether Fes kinase domain Tyr autophosphorylation sites bind to the SH2 domains of these molecules in vitro and in vivo. 4) Activation of Fes requires oligomerization and transphosphorylation. Preliminary data show that active Fes is an oligomer and autophosphorylates via an intermolecular mechanism. Mutagenesis experiments will test whether an N-terminal coiled-coil domain identified by computer analysis is required for oligomerization and biological activity. In complementary experiments, autophosphorylation-defective mutants of Fes will be tested for dominant-negative activity. Kinase-inactive Fes mutants are predicted to suppress biological responses dependent upon endogenous Fes activation by non-productive oligomerization events.