Requirement for PIKfyve enzymatic activity in acute and long-term insulin cellular effects
Requirement for PIKfyve enzymatic activity in acute and long-term insulin cellular effects
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DOI：
10.1210/en.2002-220615
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发表时间：
2002-12-01
期刊：
影响因子：
4.8
通讯作者：
Shisheva, A
中科院分区：
文献类型：
--
作者：
Ikonomov, OC;Sbrissa, D;Shisheva, A
PIKfyve 是一种磷酸肌醇 5-激酶，也可充当蛋白激酶。 PIKfyve 在急性胰岛素作用中的作用是基于其与胰岛素可刺激的磷脂酰肌醇-3-激酶的关联以及急性胰岛素在 3T3-L1 脂肪细胞的细胞内膜上招募和磷酸化 PIKfyve 的能力。在这里，我们检查了胰岛素敏感细胞中几种经典的胰岛素调节的长期和短期反应，这些细胞表达高水平的活性 PIKfyve 或具有显性负效应的激酶死亡突变体。在培养的 3T3-L1 成纤维细胞分化为脂肪细胞的早期阶段，记录了 PIKfyve 蛋白表达的上调，并且将激酶死亡突变体 PIKfyveDeltaK 引入前脂肪细胞阶段，显着延迟了激素诱导的脂肪生成。接下来，胰岛素诱导的有丝分裂在 HEK293 稳定细胞系中被显着抑制，诱导表达显性失活激酶死亡 PIKfyve(K1831E) 突变体，但在表达 PIKfyve(WT) 的细胞中则不然。同样，显性失活突变体 PIKfyve(K1831E) 或 PIKfyveDeltaK 的表达强烈抑制 3T3-L1 脂肪细胞中胰岛素刺激的 GLUT4 易位，或表达人胰岛素受体的中国仓鼠卵巢 T 细胞中 GLUT1 介导的葡萄糖摄取。中国仓鼠卵巢T细胞中PIKfyveDeltaK和PIKfyve(WT)的表达分别减少或增加，胰岛素刺激Akt在Ser473处磷酸化，但在Thr308处不磷酸化。此外，细胞渗透性激酶抑制剂姜黄素在极低浓度（ID50 = 6 mum）下对 PIKfyve 具有强大的抑制作用。当引入 3T3-L1 脂肪细胞时，姜黄素显着抑制胰岛素诱导的 GLUT4 易位和葡萄糖转运。这些数据共同表明 PIKfyve 酶活性在胰岛素急性和长期生物反应中充当正向调节中间体，并将 Akt 中的 Ser473 确定为一个潜在的 PIKfyve 下游靶标。
PIKfyve is a phosphoinositide 5-kinase that can also act as a protein kinase. PIKfyve's role in acute insulin action has been suggested on the basis of its association with the insulin stimulatable phosphatidylinositol-3-kinase and the ability of acute insulin to recruit and phosphorylate PIKfyve on intracellular membranes of 3T3-L1 adipocytes. Here we have examined several classical insulin-regulated long- and short-term responses in insulin-sensitive cells expressing high levels of either active PIKfyve or kinase-dead mutants with a dominant-negative effect. Up-regulation of PIKfyve protein expression was documented in the early stages of differentiation of cultured 3T3-L1 fibroblasts into adipocytes and a kinase-dead mutant, PIKfyveDeltaK, introduced into the preadipocyte stage profoundly delayed the hormone-induced adipogenesis. Next, insulin-induced mitogenesis was markedly inhibited in HEK293 stable cell lines, inducibly expressing the dominant-negative kinase-dead PIKfyve(K1831E) mutant but not in cells expressing PIKfyve(WT). Similarly, expression of the dominant negative mutants PIKfyve(K1831E) or PIKfyveDeltaK strongly inhibited insulin-stimulated translocation of GLUT4 in 3T3-L1 adipocytes, or GLUT1-mediated glucose uptake in Chinese hamster ovary T cells expressing the human insulin receptor. Expression of PIKfyveDeltaK and PIKfyve(WT) in Chinese hamster ovary T cells decreased or increased, respectively, insulin-stimulated Akt phosphorylation at Ser473 but not at Thr308. Furthermore, a powerful inhibition of PIKfyve was documented at a very low concentration (ID50 = 6 mum) of the cell-permeable kinase inhibitor curcumin. When introduced into 3T3-L1 adipocytes, curcumin markedly inhibited insulin-induced GLUT4 translocation and glucose transport. Together these data indicate that PIKfyve enzymatic activity functions as a positive regulatory intermediate in insulin acute and long-term biological responses and identify Ser473 in Akt as one potential PIKfyve downstream target.