ChemBio Spotlight #4
Paper: PKD1 Inhibits AMPK2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells
Whodunit? – The Culprit of AMPK Phosphorylation in Insulin Resistance
It is no secret that millions of people worldwide are affected by type II diabetes. People with this disease are unable to properly store glucose in their tissues due to insulin resistance. Insulin resistance means that the insulin receptors and/or their pathways are not functioning properly. Normally, when glucose is available in the bloodstream, insulin receptors are told to activate the downstream enzymes in their pathway to cause for metabolic changes in the tissues of the body. One of these changes can be to store the glucose present in the blood. However, in insulin resistance, one of these downstream enzymes, called AMPK, is inactivated by a phosphorylation at one of its serine residues. As a result, the pathway is disrupted and glucose cannot be stored. This leads to high blood glucose levels, a symptom seen in untreated diabetic patients. So who is causing this trouble by directly phosphorylating AMPK? A recent study by Coughlan et al. investigates two possible enzymes responsible: PKC and PKD1. Only one of the two enzymes, however, is proven guilty.
The authors first demonstrated that PMA, a PKC activator, caused an increase in AMPK phosphorylation when incubated with PKC or PKD1. This experiment served to establish that both enzymes have some sort of contribution to AMPK phosphorylation when stimulated to do so. In order to eliminate other enzymes as responsible for the direct phosphorylation of AMPK, the authors conducted an experiment with enzymes popularly mentioned in the literature to be involved in this insulin-receptor pathway. Inhibition of all three enzymes did not prevent PMA-induced phosphorylation of AMPK. If they were phosphorylating AMPK, their inhibition should have resulted in a decreased phosphorylation in this PMA environment. This made the authors sure PKC and PKD1 were the only two left in the running. The authors proceeded to test whether inhibitors of PKC and PKD1 would have any effect on AMPK phosphorylation. Inhibition of both enzymes prevented PMA-induced phosphorylation of AMPK’s serine. This further solidified the two enzymes’ role in the pathway disruption. Coughlan et al. went on to see the effects a knockdown of PKD1 has on the phosphorylation of AMPK versus IRS-1 (another intermediate in the insulin signaling pathway). They found that the PKD1 knockdown prevented PMA-induced phosphorylation of AMPK but not of IRS-1. IRS-1 phosphorylation is also known to impair insulin signaling. Thus the authors realized that PKD1 activation must diminish insulin signaling through an unprecedented mechanism independent of IRS-1. In order to exhibit that PKD1 is directly phosphorylating AMPK, the authors tested whether recombinant PKD1 phosphorylates AMPK in a cell-free system. A positive control of AMPK with Akt was compared to AMPK with PKD1 as well as AMPK alone. PKD1 did indeed phosphorylate AMPK in these conditions, suggesting PKD1 does so directly. Establishing PKD1 is directly responsible for this phosphorylation opens doors for drug-targeting approaches to combat insulin resistance.
Coughlan, Kimberly A., Rudy J. Valentine, Bella S. Sudit, Katherine Allen, Yossi Dagon, Barbara B. Kahn, Neil B. Ruderman, and Asish K. Saha. 2016. “PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells.” Journal of Biological Chemistry 291 (11): 5664–75. doi:10.1074/jbc.M115.696849.
Mancini, Arturo D., and Vincent Poitout. 2013. “The Fatty Acid Receptor FFA1/GPR40 a Decade Later: How Much Do We Know?” Trends in Endocrinology & Metabolism 24 (8): 398–407. doi:10.1016/j.tem.2013.03.003.