Formisano P, Condorelli G, Condorelli G, Beguinot F
Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, II Facoltà di Medicina, Università degli studi di Napoli, Italy.
Endocrinology. 1991 Jun;128(6):2949-57. doi: 10.1210/endo-128-6-2949.
Phosphotyrosine-containing proteins (PYPs) from FRTL5 epithelial cells were immunoprecipitated with Sepharose-linked phosphotyrosine antibody and phosphorylated in vitro with insulin and insulin-like growth factor-I (IGF-I) receptor kinases. Insulin preactivation of the kinases resulted in increased phosphorylation of a single 175,000 mol wt PYP (p175) beside insulin and IGF-I receptors. Phosphorylation of both p175 and receptors exhibited almost identical time courses and insulin dose responses, suggesting that p175 may serve as a substrate for the insulin and IGF-I receptor kinases. Preincubation of autophosphorylated receptors with PYPs derived from insulin-stimulated cells decreased 32P-labeled receptor precipitation by antiphosphotyrosine-Sepharose by 50-70% compared to that obtained upon preincubation with PYPs from unstimulated cells. This effect was not the result of PYP-receptor competition, was observed on both in vivo and in vitro phosphorylated receptors, and was almost completely blocked by 100 microM sodium vanadate, suggesting PYP copurification of a phosphotyrosine phosphatase. No phosphatase was recovered in lysates from insulin-unstimulated cells. Maximum activity was detected after 2 min of insulin stimulation, decreasing by more than 50% after 30 min. Antiphosphotyrosine recovery of p175 from cell lysates exhibited almost identical insulin dependency. Complete recovery of both p175 and phosphatase activity was obtained after receptor immunodepletion of the extracts. Thus, a receptor phosphatase activity from insulin-stimulated FRTL5 cells is retained on an antiphosphotyrosine affinity matrix and correlates with phosphorylation of the p175 substrate for the insulin and IGF-I receptor kinases.
用琼脂糖偶联的磷酸酪氨酸抗体对来自FRTL5上皮细胞的含磷酸酪氨酸蛋白（PYP）进行免疫沉淀，并在体外与胰岛素和胰岛素样生长因子-I（IGF-I）受体激酶一起磷酸化。激酶的胰岛素预激活导致除胰岛素和IGF-I受体外，一种单一的175,000摩尔分子量的PYP（p175）磷酸化增加。p175和受体的磷酸化表现出几乎相同的时间进程和胰岛素剂量反应，表明p175可能是胰岛素和IGF-I受体激酶的底物。与用未刺激细胞的PYP预孵育相比，将自磷酸化受体与来自胰岛素刺激细胞的PYP预孵育后，抗磷酸酪氨酸琼脂糖对32P标记受体的沉淀减少了50 - 70%。这种效应不是PYP-受体竞争的结果，在体内和体外磷酸化的受体上均观察到，并且几乎完全被100 microM钒酸钠阻断，提示PYP共纯化了一种磷酸酪氨酸磷酸酶。在未用胰岛素刺激的细胞裂解物中未回收磷酸酶。胰岛素刺激2分钟后检测到最大活性，30分钟后下降超过50%。从细胞裂解物中回收p175的抗磷酸酪氨酸几乎表现出相同的胰岛素依赖性。提取物经受体免疫去除后，p175和磷酸酶活性均完全恢复。因此，来自胰岛素刺激的FRTL5细胞的受体磷酸酶活性保留在抗磷酸酪氨酸亲和基质上，并与胰岛素和IGF-I受体激酶的p175底物的磷酸化相关。
