PF-4708671 Activates AMPK Independently of p70S6K1 Inhibition

<div><p>The P70 ribosomal protein S6 kinase 1 (P70S6K1) is activated by the mammalian target of rapamycin (mTORC1) and regulates proliferation, growth, and metabolism. PF-4708671 is a novel, cell-permeable, has been proposed to be a highly specific inhibitor of p70S6K1. It is used in micromolar concentration range to dissect signaling pathways downstream of mTORC1 and to study the function of p70S6K1. Here we show that PF-4708671 induces AMP-activated protein kinase (AMPK) phosphorylation and activation in immortalized mouse embryonic fibroblasts (MEF) independently of p70S6K1, due to specific inhibition of mitochondrial respiratory chain Complex I.</p></div

AMPK activation by PF-4708671.

<p>Wild type MEF (<b>A</b>) and p70S6K double knockout MEF (<b>B</b>) were treated with PF-4708671 as indicated. Phospho-ACC (Ser79), ACC, phospho-AMPK(Thr172), AMPK, phospho-S6 and (235/236 and 240/244 were determined by SDS-PAGE as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107364#s2" target="_blank">Methods</a>. Tubulin and beta-Catenin served as loading controls.</p

Inhibition of oxygen consumption by PF-4708671.

<p>(<b>A</b>) S6K double knockout MEF were treated with vehicle (red line); rapamycin 50 nM (turquoise); PF-4708671 2.5 µM (blue) and 5 µM (pink). Oxygen consumption rate (OCR) of was determined by the Seahorse platform as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107364#s2" target="_blank">Methods</a>. (<b>B</b>) ANOVA statistical analysis of the OCR area under the curve showed that after 2.5 uM or 5 uM PF-4708671 treatment the OCR was significantly decreased (p = 2.6*10⁻⁵ and 1.97*10⁻⁹, respectively; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107364#pone.0107364.s005" target="_blank">Table S1</a>).</p

Inhibition of mitochondrial complex I by PF-4708671.

<p>MEF mitochondria were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107364#s2" target="_blank">Methods</a> and incubated in the presence of PF-4708671, metformin or rotenone as indicated. (<b>A</b>) Activities of citrate synthase and mitochondrial respiratory chain complexes were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107364#s2" target="_blank">Methods</a>. Activities of mitochondrial respiratory chain complexes in the presence of added PF-4708671 are presented relative to respective activities in the presence of vehicle. (<b>B</b>) Relative activity of mitochondrial Complex I in the presence of increasing PF-4708671 concentrations as indicated. (<b>C</b>) Relative activity of mitochondrial Complex I in the presence of PF-4708671, metformin or rotenone as indicated. Activities of mitochondrial respiratory chain complexes are presented relative to (DMSO).</p