Okada T, Hazeki O, Ui M, Katada T
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Biochem J. 1996 Jul 15;317 ( Pt 2)(Pt 2):475-80. doi: 10.1042/bj3170475.
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.
胰岛素刺激分化的THP-1细胞会导致磷脂酰肌醇-3,4,5-三磷酸（PtdIns(3,4,5)P3）迅速积累，PtdIns(3,4,5)P3是磷脂酰肌醇3激酶的产物。N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）刺激GTP结合蛋白偶联受体也会诱导细胞内PtdIns(3,4,5)P3积累。胰岛素的作用伴随着抗磷酸酪氨酸免疫沉淀物中PtdIns 3激酶活性增加，而fMLP的作用则不然。胰岛素和fMLP联合诱导产生的PtdIns(3,4,5)P3比各自单独作用之和更多。fMLP联合处理不影响胰岛素诱导的抗磷酸酪氨酸免疫沉淀物中PtdIns 3激酶活性募集。为了研究PtdIns(3,4,5)P3协同积累的潜在机制，我们在Mono Q柱上分离了THP-1细胞的胞质蛋白。PtdIns 3激酶活性以两个峰洗脱，加入GTP结合蛋白的βγ亚基（Gβγ）后，其中一个峰明显增加。另一个峰仅受到Gβγ的轻微影响，但Gβγ和根据胰岛素受体底物-1氨基酸序列合成的酪氨酸磷酸化肽可使其协同增加。后一组分中的活性可被抗磷脂酰肌醇3激酶调节亚基（p85）的抗体完全免疫沉淀。这些结果表明，参与胰岛素诱导细胞事件的传统磷脂酰肌醇3激酶（p85/p110）或密切相关的同工酶在完整细胞中受酪氨酸磷酸化蛋白和GTP结合蛋白组合的调控。
