OSMOTIC LOADING OF NEUTRALIZING ANTIBODIES DEMONSTRATES A ROLE FOR PROTEIN-TYROSINE-PHOSPHATASE 1B IN NEGATIVE REGULATION OF THE INSULIN ACTION PATHWAY
OSMOTIC LOADING OF NEUTRALIZING ANTIBODIES DEMONSTRATES A ROLE FOR PROTEIN-TYROSINE-PHOSPHATASE 1B IN NEGATIVE REGULATION OF THE INSULIN ACTION PATHWAY
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DOI：
10.1074/jbc.270.35.20503
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发表时间：
1995-09-01
影响因子：
4.8
通讯作者：
GOLDSTEIN, BJ
中科院分区：
文献类型：
--
作者：
AHMAD, F;LI, PM;GOLDSTEIN, BJ
蛋白酪氨酸磷酸酶（PTPases）被认为可以平衡胰岛素受体及其底物蛋白的稳态磷酸化和激活状态。PTP1B是一种广泛表达的非受体型PTPase，为了探究PTP1B是否调节胰岛素信号，我们采用渗透休克的方法，用亲和纯化的中和抗体负载大鼠KRC-7肝癌细胞，这些抗体在体外免疫沉淀并灭活重组大鼠PTP1B的酶活性。在携带PTP1B抗体的细胞中，与携带免疫前IgG的对照细胞相比，胰岛素刺激的DNA合成和磷脂酰肌醇3′-激酶活性分别增加了42%和38% （p < 0.005）。为了表征PTP1B在胰岛素信号传导中的潜在作用位点，我们还确定胰岛素刺激的受体自磷酸化和胰岛素受体底物1酪氨酸磷酸化分别增加了2.2倍和2.0倍，胰岛素刺激的受体激酶对外源性肽底物的活性在PTP1B抗体负载的细胞中增加了57%。渗透负载没有改变PTP1B蛋白的细胞含量，这表明该抗体通过立体阻断PTP1B与其生理底物之间的催化相互作用在细胞中起作用。这些研究表明，PTP1B在胰岛素信号的负向调控中起作用，至少部分直接作用于胰岛素受体水平。这些结果还表明，胰岛素信号可以通过抑制特定的PTPases而增强，这在胰岛素抵抗和II型糖尿病的治疗中具有潜在的临床意义。
Protein-tyrosine phosphatases (PTPases) have been postulated to balance the steady-state phosphorylation and the activation state of the insulin receptor and its substrate proteins. To explore whether PTP1B, a widely expressed, non-receptor type PTPase, regulates insulin signaling, we used osmotic shock to load rat KRC-7 hepatoma cells with affinity-purified neutralizing antibodies that immunoprecipitate and inactivate the enzymatic activity of recombinant rat PTP1B in vitro. In cells loaded with PTP1B antibody, insulin-stimulated DNA synthesis and phosphatidylinositol 3'-kinase activity were increased by 42% and 38%, respectively, compared with control cells loaded with preimmune IgG (p < 0.005). In order to characterize the potential site(s) of action of PTP1B in insulin signaling, we also determined that insulin-stimulated receptor autophosphorylation and insulin receptor substrate 1 tyrosine phosphorylation were increased 2.2- and 2.0-fold, respectively, and that insulin-stimulated receptor kinase activity toward an exogenous peptide substrate was increased by 57% in the PTP1B antibody loaded cells. Osmotic loading did not alter the cellular content of PTP1B protein, suggesting that the antibody acts in the cell by sterically blocking catalytic interactions between PTP1B and its physiological substrates. These studies demonstrate that PTP1B has a role in the negative regulation of insulin signaling and acts, at least in part, directly at the level of the insulin receptor. These results also show that insulin signaling can be enhanced by the inhibition of specific PTPases, a maneuver that has potential clinical relevance in the treatment of insulin resistance and Type II diabetes mellitus.