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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

1999; Elsevier BV; Volume: 274; Issue: 43 Linguagem: Inglês

10.1074/jbc.274.43.30927

1083-351X

Luc Bertrand, Dario R. Alessi, Johan Deprez, Mária Deák, Eric Viaene, Mark H. Rider, Louis Hue,

PI3K/AKT/mTOR signaling in cancer

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-2 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-2 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-2 is mediated by a PDK-1-activated protein kinase other than PKB.