Protein kinase C phosphorylates AMP-activated protein kinase α1 Ser487

Abstract

The key metabolic regulator, AMP-activated protein kinase (AMPK), is reported to be down-regulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491, respectively, as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC-stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues.

Canagliflozin inhibits interleukin-1β-stimulated cytokine and chemokine secretion in vascular endothelial cells by AMP-activated protein kinase-dependent and -independent mechanisms

“…BrdU cell proliferation assay kits were obtained from Millipore (Watford, UK). All other reagents were from sources described previously 26 , 27 , 50 .…”

“…Human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs) were cultured in MV2 medium (Promocell, Heidelberg, Germany) and used for experiments between passages 3 and 6 as described previously 50 . HEK-293 cells were cultured in DMEM, supplemented with 10% (v/v) foetal calf serum, 1 mmol/l pyruvate and 2 mmol/l L-glutamine as described previously 19 .…”

“…HUVECs or HAECs were serum-starved in Medium 199 for 2 h prior to addition of SGLT2 inhibitors, AMPK activators or IL-1β. Cell lysates were prepared, proteins resolved by SDS-PAGE and subjected to quantitative immunoblotting with the antibodies indicated as described previously 27 , 50 . Immunolabelled proteins were visualized using infrared dye-labelled secondary antibodies and an Odyssey Sa infrared imaging system (LiCor Biosciences UK Ltd, Cambridge, UK).…”

AMPK: Mechanisms of Cellular Energy Sensing and Restoration of Metabolic Balance

“…Similarly, S6K (p70S6 kinase) has been reported to inhibit AMPK activity by phosphorylating Ser491 in AMPKα2, and this phosphorylation was proposed as the mechanism by which leptin inhibits AMPK in the hypothalamus (Dagon et al, 2012). Other kinases reported to inhibit AMPK by phosphorylating various residues in the ST-loop are GSK3 (glycogen synthesis kinase 3) (Suzuki et al, 2013), PKD1 (protein kinase D) (Coughlan et al, 2016) and PKC (protein kinase C) (Heathcote et al, 2016), though it remains to be determined which of these kinases are relevant in different tissues in vivo. Although the mechanism of AMPK inhibition is not entirely clear, it seems that phosphorylation of the ST-loop reduces net phosphorylation of Thr172, by either physically interfering with its phosphorylation or by promoting its dephosphorylation (Hawley et al, 2014).…”

Promise and challenges for direct small molecule AMPK activators

“…Phosphorylation of AMPK1 at Ser485 and AMPK2 at Ser491 by PKA [32,33], Akt [34,35] and S6K [36] antagonizes AMPK activation. Phosphorylation at other sites within the ST-loop by GSK3 [37], PKC [38] and PKD1 [39] has been also involved in the reduction of AMPK activity. In addition to phosphorylation, AMPK activity is also modulated via post-translational ubiquitination and degradation by ubiquitin ligases specifically targeting the 1-isoform [40] or the 2-isoform [41].…”