Heart 6-phosphofructo-2-kinase activation by insulin results from Ser-466 and Ser-483 phosphorylation and requires 3-phosphoinositide-dependent kinase-1, but not protein kinase B
Heart 6-phosphofructo-2-kinase activation by insulin results from Ser-466 and Ser-483 phosphorylation and requires 3-phosphoinositide-dependent kinase-1, but not protein kinase B
复制标题
DOI：
10.1074/jbc.274.43.30927
复制
发表时间：
1999-10-22
影响因子：
4.8
通讯作者：
Hue, L
中科院分区：
文献类型：
--
作者：
Bertrand, L;Alessi, DR;Hue, L
既往研究表明：（1）胰岛素诱导的心脏6-磷酸果糖-2-激酶（PFK-2）激活对渥曼菌素敏感，而对雷帕霉素不敏感，提示有磷脂酰肌醇3-激酶参与;和（ii）蛋白激酶B（PKB）通过磷酸化Ser-466和Ser-483在体外活化PFK-S。在这项工作中，我们已经研究了通过用谷氨酸替换每个或两个残基，这些残基的磷酸化对PFK-2活性的影响。Ser-466突变增加PFK-2的V-max，而Ser-483突变降低柠檬酸盐抑制。这两个残基的突变需要降低6-磷酸果糖的Km。我们还研究了在人胚肾293细胞中进行的转染实验中胰岛素诱导的心脏PFK-2的活化。胰岛素激活转染PFK-S，磷酸化Ser-466和Ser-483。激酶死亡（KD）PKB和KD 3-磷酸肌醇依赖性激酶-1（PDK-1）共转染子作为显性负作用，因为两者都阻止了胰岛素诱导的PKB激活以及糖原合成酶激酶-3（PKB的既定底物）的失活。然而，胰岛素诱导的PFK-2活化仅被KD PDK-1阻止，而不是被KD PKB阻止。这些结果表明，胰岛素诱导的心脏PFK-S的激活是由PDK-1激活的蛋白激酶而不是PKB介导的。
Previous studies have shown that (i) the insulin-induced activation of heart 6-phosphofructo-2-kinase (PFK-2) is wortmannin-sensitive, but is insensitive to rapamycin, suggesting the involvement of phosphatidylinositol 3-kinase; and (ii) protein kinase B (PKB) activates PFK-S in vitro by phosphorylating Ser-466 and Ser-483, In this work, we have studied the effects of phosphorylation of these residues on PFK-2 activity by replacing each or both residues with glutamate. Mutation of Ser-466 increased the V-max of PFK-2, whereas mutation of Ser-483 decreased citrate inhibition. Mutation of both residues was required to decrease the K-m for fructose 6-phosphate. We also studied the insulin-induced activation of heart PFK-2 in transfection experiments performed in human embryonic kidney 293 cells. Insulin activated transfected PFK-S by phosphorylating Ser-466 and Ser-483. Kinase-dead (KD) PKB and KD 3-phosphoinositide-dependent kinase-1 (PDK-1) cotransfectants acted as dominant negatives because both prevented the insulin-induced activation of PKB as well as the inactivation of glycogen-synthase kinase-3, an established substrate of PKB. However, the insulin-induced activation of PFK-2 was prevented only by KD PDK-1, but not by KD PKB. These results indicate that the insulin-induced activation of heart PFK-S its mediated by a PDK-1-activated protein kinase other than PKB.