Construction of a cDNA for the human c-fes protooncogene protein-tyrosine kinase and its expression in a baculovirus system.
Construction of a cDNA for the human c-fes protooncogene protein-tyrosine kinase and its expression in a baculovirus system.
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人 c-fes 原癌基因蛋白酪氨酸激酶 cDNA 的构建及其在杆状病毒系统中的表达。
DOI：
10.1021/bi00135a013
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Glazer,RI
中科院分区：
文献类型：
--
作者：
Smithgall,TE;Goswami,BB;Nagashfar,Z;Ahmad,S;Glazer,RI
乔治城大学医学中心药理学教研室，华盛顿特区，20007收到1991年11月21日；修订稿收到1992年3月5日摘要：以前的研究已经证实，由人c-fes原癌基因编码的93 kDa蛋白酪氨酸激酶(PTK)在诱导髓系终末分化中起积极作用。然而，这种酶在髓系细胞中的表达水平非常低，因此很难分离出足够的量来进行详细的生化分析。为了克服这一问题，我们利用聚合酶链式反应从重叠的5‘和3’部分cDNA序列中构建了全长c-fes基因。在杆状病毒系统中高效表达了c-fes基因，并用DEAE-Sepharose和酪氨酸-琼脂糖柱层析部分纯化了具有催化活性的重组c-fes基因产物p93c‘-/IL。重组p93c‘-/“在SDS-PAGE表观相对分子质量、催化活性、对聚(Glu，Tyr)4；1的Km、抗原性和金黄色葡萄球菌蛋白酶产生的磷酸肽图谱等方面与天然蛋白无明显区别。人类c-fes基因编码93 kDa的蛋白酪氨酸激酶(PTK)1(p93c‘af)，它只在粒细胞和单核细胞系的造血细胞中表达(Feldman等人，1985；Glazer等人，1991；MacDonald等人，1985；Smithgall等人，1988)。先前的研究表明，c-fes信号水平(Liebermann&Liebermann，1989)和酪氨酸激酶活性(Glazer等人，1986,1987；Yu等人，1988；Yu&Glazer，1987；Chapekar等人，1986)在髓样细胞的终末分化过程中增加，这表明这个原癌基因可能直接调控成熟过程。进一步支持这一假设的是使用人类髓系白血病细胞系K-562进行的基因转移研究(Lozzio等人，1981)，该细胞系不表达c-fes，并且对髓系分化诱导剂具有抵抗力(Koeffler等人，1981)。将人c-fes基因导入K-562细胞后，细胞生长速度和成熟粒细胞功能特性的表达显著降低(Yu等，1989)。这一结果表明，仅p93-fes酪氨酸激酶活性就足以触发适当细胞类型的终末分化，并表明它可能代表着一种重要的骨髓生成生理调节机制，如结肠癌刺激因子(Nicola，1989)。需要更详细地分析p93c‘-/“调控其酪氨酸激酶活性和与其他细胞蛋白相互作用的结构特征，以阐明这一原癌基因产物在髓系分化中的作用。P93<；：-/？js是由原癌基因编码的独特的细胞质PTK亚家族的成员，与以p60c‘i，’c为例的更大的PTK亚家族具有许多结构特征。同源的主要区域是催化结构域，它位于
Department of Pharmacology, Georgetown University Medical Center, Washington, DC 20007 Received November 21, 1991; Revised Manuscript Received March 5, 1992 abstract: Previous studies have established that the 93-kDa protein-tyrosine kinase (PTK) encoded by the human c-fes protooncogene plays an active role in the induction of terminalmyeloid differentiation. However, this enzyme is expressed at very low levels in myeloid cells, making isolation of sufficient quantities for detailed biochemical analysis difficult. To overcome this problem, we used the polymerase chain reaction to construct a full-length c-fes cDNA from overlapping 5'and 3'partial cDNA sequences. The c-fes cDNA was expressed at high levels in a baculovirus system, and the catalytically active recombinant c-fes gene product p93c'-/il was partially purified by DEAE-Sepharose and tyrosine-agarose chromatography. Re-combinant p93c'-/" was indistinguishable from the native protein in terms of its apparent molecular weight following SDS-PAGE, catalytic activity, Km for poly (Glu, Tyr) 4; 1, antigenicity, and phosphopeptide pattern generated with Staphylococcus aureus protease. e human c-fes locusencodes a protein-tyrosine kinase (PTK) 1 of 93 kDa (p93c'Af) that is expressed exclusively in hematopoietic cells of the granulocytic and monocytic lineages (Feldman et al., 1985; Glazer et al., 1991; MacDonald et al., 1985; Smithgall et al., 1988). Previous studies have shown that c-fes message levels (Liebermann & Liebermann, 1989) and tyrosine kinase activity (Glazer et al., 1986, 1987; Yu et al., 1988; Yu & Glazer, 1987; Chapekar et al., 1986) increase during the terminal differentiation of myeloidcells, suggesting that this protooncogene maydirectly regulate the maturation process. Further support for this hypothesis comes from gene-transfer studies using the human myeloid leukemia cell line K-562 (Lozzio et al., 1981), which does not express c-fes and is resistant to myeloid differentiation inducers (Koeffler et al., 1981). Transfection of K-562 cells with the human c-fes gene resulted in a significant reduction in the cellular growth rate and the expression of functionalproperties of mature granulocytes (Yu et al., 1989). This result indicates that p93-fes tyrosine kinase activity alone is sufficient to trigger terminal differentiation in an appropriate cell type and suggests that it may represent an important effectormechanism for physiological regulators of myelopoiesis such as the colonystimulating factors (Nicola, 1989). A more detailed analysis of the structural features of p93c'-/" that regulate its tyrosine kinase activity and interaction with other cellular proteins is required to clarify the role of this protooncogene product in myeloid differentiation. p93<:-/«js a member of a unique subfamily of cytoplasmic PTKs encoded by protooncogenes and shares a number of structural features with a larger subfamily of PTKs exemplified by p60c ‘I,'c. The primary region of homology is the catalytic domain, which is located in the C-terminal portion of both the