Abstract 3724: Identification of Myosin Light Chain Kinase as a Novel Myocardial 5′-AMP Activated Protein Kinase Kinase
5′-adenosine monophosphate activated protein kinase (AMPK), a key regulator of energy metabolism, is activated by myocardial ischemia via phosphorylation of thr-172 within its catalytic α-subunit by upstream AMPK kinases (AMPKKs). In this study, we identified a novel myocardial AMPKK via its ability to phosphorylate a recombinant AMPK α1 catalytic subunit (α312). Myocardial AMPKK was purified from a 6.25% polyethylene glycol supernatant fraction from rat heart homogenates. Anion-exchange chromatography (DEAE sepharose) resolved two peaks of AMPKK activity. Subsequent resolution (blue sepharose) resolved a single peak free of the previously identified AMPKKs, LKB1 and CaMKKβ. Further resolution (Source 30Q) substantially purified myocardial AMPKK, that phosphorylated AMPK α312 in a concentration-and time-dependent manner, and was active over a pH range of 6.0 –9.0 (maximal activity -pH 6.8 –7.4). The activity of myocardial AMPKK was similar in the absence and presence of AMP (100 μM). SDS-PAGE analysis of myocardial AMPKK revealed several protein bands and further purification using calmodulin sepharose chromatography resulted in two distinct protein bands of 130 –150 kDa and 65 kDa. Size-exclusion chromatography indicated AMPKK activity was associated with the band migrating at 130 –150 kDa, and mass spectrometry analysis identified peptides associated with the catalytic domain of myosin light chain kinase (MLCK). Myocardial AMPKK phosphorylated myosin light chain (ser-19). The selective MLCK inhibitors, ML7 and ML9, inhibited the phosphorylation/activation of AMPKα312 by purified AMPKK. MLCK is sensitive to activation by increased [Ca2+]I, and the Ca2+ ionophore, A23187 (10 μM), increased AMPK phosphorylation and activity in neonatal rat cardiac myocytes. In cultured C2C12 myotubes, siRNA directed against mylk1, (the gene encoding MLCK) decreased MLCK expression, and abrogated the ability of the Ca2+ ionophore, ionomycin to increase AMPK phosphorylation. In working rat hearts, severe low flow ischemia increased MLCK activity, AMPK phosphorylation, and glycolysis, effects attenuated by ML-7. In conclusion, these data identify 130 kDa MLCK as a novel myocardial AMPKK that participates in the ischemia-induced activation of AMPK.