Phosphorylation of the ras GTPase-activating protein (GAP) by the p93c-fes protein-tyrosine kinase in vitro and formation of GAP-fes complexes via an SH2 domain-dependent mechanism.
Phosphorylation of the ras GTPase-activating protein (GAP) by the p93c-fes protein-tyrosine kinase in vitro and formation of GAP-fes complexes via an SH2 domain-dependent mechanism.
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p93c-fes 蛋白酪氨酸激酶体外磷酸化 ras GTP 酶激活蛋白 (GAP)，并通过 SH2 结构域依赖性机制形成 GAP-fes 复合物。
DOI：
10.1021/bi00090a031
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Smithgall,TE
中科院分区：
文献类型：
--
作者：
Hjermstad,SJ;Briggs,SD;Smithgall,TE
摘要：人c-fes原癌基因(p93c-f“)编码的蛋白酪氨酸激酶(p93c-f”)在髓系细胞分化中起直接作用，但其下游底物尚未确定。本文报道人ras GTP酶激活蛋白(GAP)是p93c‘^“的体外底物。经细菌表达的p93c’-/”可使重组蛋白在酪氨酸残基上磷酸化。二维胰酶图谱显示一个单一的GAP磷酸肽，与p93c‘7w在一个或几个紧密排列的酪氨酸残基上对GAP的特异性磷酸化一致。自磷酸化的p93？/？也与GAP形成稳定的络合物。复合体的形成可能涉及p93c‘^J的GAP和自磷酸化酪氨酸残基的src同源2(SH2)结构域，因为Fes SH2结构域的缺失并不取消复合体的形成。此外，含有其中一个或两个GAP SH2结构域的固定化重组融合蛋白能够以与抗重组FES蛋白的单抗相同的亲和力沉淀p93？/？含有GAP N末端、C末端催化结构域或SH3结构域的融合蛋白不与p93c‘7“结合。GAP SH2结构域与p93c‘-fo的相互作用依赖于磷酸化，因为重组的SH2结构域蛋白不能与激酶缺陷的c-fes突变体结合，并且显示出两个酪氨酸自动磷酸化位点之一被苯丙氨酸取代的突变体的结合减少。C-fes自身磷酸化的刺激可能导致与GAP的相互作用，导致p21ras功能改变。人c-fesgene编码一个93 kDa的PTK1(p93c‘^Cj)，主要表达于粒细胞和单核细胞系的髓系细胞(Feldman等人，1985；MacDonald等人，1985；Smithgall等人，1988)。P93c‘7“酪氨酸激酶活性在人髓系白血病细胞系体外分化过程中显著增强，
Revised Manuscript Received July 7, 1993® abstract: The protein-tyrosine kinaseencoded by the human c-fes protooncogene (p93c_-f") plays a direct role in myeloid differentiation, but downstream substrates for this kinase have not been identified. Here we report that the human ras GTPase-activating protein (GAP) is a substrate for p93c'^“invitro. Purified, recombinant GAP was readily phosphorylated on tyrosine residues by bacterially-expressed p93c'-/". Two-dimensional tryptic mapping revealed a single GAP phosphopeptide, consistent with specific phosphorylation of GAP by p93c'7w on one or several closely-spaced tyrosine residues. Autophosphorylated p93 «-/«also formed a stable complex with GAP. Complex formation is likely to involve the src homology 2 (SH2) domains of GAP and autophosphorylated tyrosine residues of p93c'^ J, as deletion of the fes SH2 domain did not abolish complex formation. Furthermore, immobilized recombinant fusion proteins containing either or both of the GAP SH2 domains were able to precipitate p93 «-/«with an affinity equal to that observed with a monoclonal antibody against the recombinant fes protein. Fusion proteins containing the GAP N-terminal, C-terminal catalytic, or SH3 domains did not bind to p93c'7". Interaction of the GAP SH2 domains with p93c ‘-fo is phosphorylation-dependent, as the recombinant SH2 domain proteins were unable to bind to a kinase-defective c-fes mutant and showed reduced binding of a mutant in which one of the two tyrosine autophosphorylation sites was replaced with phenylalanine. Stimulation of c-fes autophosphorylation in vivo may induce interaction with GAP, resulting in altered p21ras function.The human c-fesgene encodes a 93-kDa PTK1 (p93c'^ cj) that is expressed predominantly in myeloid cells of the granulocytic and monocytic lineages (Feldman et al., 1985; MacDonald et al., 1985; Smithgall et al., 1988). p93c'7 “tyrosine kinase activity is greatly enhanced during the differentiation of human myeloid leukemia cell lines in vitro,
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DOI：
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发表时间：
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期刊：
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DOI：
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发表时间：
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期刊：
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