PKD1 Inhibits AMPK2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells

Background: Diminished activity of the enzyme AMP-activated protein kinase (AMPK) is associated with impaired insulin signaling. Results: Protein Kinase (PK)C/D1 activation inhibits AMPK2 via Ser(491) phosphorylation; PKD1 inhibition prevents this in skeletal muscle cells. Conclusion: PKD1 is a novel upstream AMPK-kinase that phosphorylates AMPK on Ser(491) and regulates insulin signaling. Significance: PKD1 inhibition may be a novel strategy for improving insulin sensitivity. AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser(485/491) of its 1/2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser(485/491) phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser(485/491) phosphorylation, which was associated with a approximate to 60% decrease in AMPK2 activity. Expression of a phosphodefective AMPK2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Go6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser(485/491) phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPK2 at Ser(491) in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPK2 Ser(491) that plays a negative role in insulin signaling in muscle cells.