Synergistic activation of PtdIns 3-kinase by tyrosine-phosphorylated peptide and beta gamma-subunits of GTP-binding proteins.
Synergistic activation of PtdIns 3-kinase by tyrosine-phosphorylated peptide and beta gamma-subunits of GTP-binding proteins.
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酪氨酸磷酸化肽和 GTP 结合蛋白的 β-γ 亚基协同激活 PtdIns 3-激酶。
DOI：
10.1042/bj3170475
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
T. Katada
中科院分区：
文献类型：
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作者：
Taro Okada;O. Hazeki;M. Ui;T. Katada
用胰岛素刺激分化的THP-1细胞导致PtdIns 3-激酶的产物PtdIns（3，4，5）P3的快速积累。用N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）刺激GTP结合蛋白连接的受体也诱导PtdIns（3，4，5）P3在细胞中的积累。胰岛素的作用是，而fMLP的作用不是，伴随着增加PtdIns 3-激酶活性的抗磷酸酪氨酸免疫沉淀。胰岛素和fMLP的组合诱导的PtdIns（3，4，5）P3的产生多于单独效应的总和。胰岛素诱导的PtdIns 3-激酶活性在抗磷酸酪氨酸免疫沉淀物的募集不受fMLP联合治疗的影响。为了研究PtdIns（3，4，5）P3协同积累的机制，我们在Mono Q柱上分离了THP-1细胞的胞浆蛋白。PtdIns 3-激酶活性在两个峰中洗脱，并且其中一个峰在添加GTP结合蛋白的β γ亚基（G β γ）时显著增加。另一个峰仅受G β γ轻微影响，但G β γ和酪氨酸磷酸化肽协同增加，酪氨酸磷酸化肽根据胰岛素受体底物-1的氨基酸序列合成。后一级分中的活性完全由针对PtdIns 3-激酶（p85）的调节亚基的抗体免疫沉淀。这些结果表明，传统的PtdIns 3-激酶（p85/p110），这已牵连在胰岛素诱导的细胞事件，或密切相关的同工酶是由酪氨酸磷酸化蛋白和GTP结合蛋白在完整细胞的组合控制。
Stimulation of differentiated THP-1 cells by insulin led to rapid accumulation of PtdIns(3,4,5)P3, a product of PtdIns 3-kinase. Stimulation of the GTP-binding-protein-linked receptor by N-formylmethionyl-leucyl-phenylalanine (fMLP) also induced the accumulation of PtdIns(3,4,5)P3 in the cells. The effect of insulin was, while that of fMLP was not, accompanied by increased PtdIns 3-kinase activity in the anti-phosphotyrosine immuno-precipitate. The combination of insulin and fMLP induced more PtdIns(3,4,5)P3 production than the sum of the individual effects. The insulin-induced recruitment of PtdIns 3-kinase activity in the anti-phosphotyrosine immunoprecipitate was unaffected by the combined treatment with fMLP. To investigate the mechanism underlying the synergistic accumulation of PtdIns(3,4,5)P3, we separated the cytosolic proteins of THP-1 cells on a Mono Q column. PtdIns 3-kinase activities were eluted in two peaks, and one of the peaks markedly increased on the addition of beta gamma-subunits of GTP-binding proteins (G beta gamma). The other peak was affected only slightly by G beta gamma, but was synergistically increased by G beta gamma and a tyrosine-phosphorylated peptide which was synthesized accordingly to the amino acid sequence of insulin receptor substrate-1. The activity in the latter fraction was completely immunoprecipitated by an antibody against the regulatory subunit of PtdIns 3-kinase (p85). These results suggest that the conventional PtdIns 3-kinase (p85/p110), which has been implicated in insulin-induced cellular events, or a closely related isoenzyme is controlled by a combination of a tyrosine-phosphorylated protein and a GTP-binding protein in intact cells.