title: PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells.
abstract: 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 α1/α2 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 ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 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 Gö6983 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 AMPKα2 at Ser(491) in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser(491) that plays a negative role in insulin signaling in muscle cells.
authors.affiliation: From the Endocrinology, Diabetes, and Nutrition Section, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Coughlan
authors.affiliation: Department of Kinesiology, Iowa State University, Ames, Iowa 50011, and.
authors.forename: Rudy J
authors.initials: RJ
authors.lastname: Valentine
authors.affiliation: From the Endocrinology, Diabetes, and Nutrition Section, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Bella S
authors.initials: BS
authors.lastname: Sudit
authors.affiliation: From the Endocrinology, Diabetes, and Nutrition Section, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Katherine
authors.initials: K
authors.lastname: Allen
authors.affiliation: Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215.
authors.forename: Yossi
authors.initials: Y
authors.lastname: Dagon
authors.affiliation: Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215.
authors.forename: Barbara B
authors.initials: BB
authors.lastname: Kahn
authors.affiliation: From the Endocrinology, Diabetes, and Nutrition Section, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Neil B
authors.initials: NB
authors.lastname: Ruderman
authors.affiliation: From the Endocrinology, Diabetes, and Nutrition Section, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118,. Electronic address: aksaha@bu.edu.
authors.forename: Asish K
authors.initials: AK
authors.lastname: Saha
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D007328:Insulin; D012694:Serine; C089212:protein kinase D; C534978:Prkaa2 protein, mouse; D011493:Protein Kinase C; D055372:AMP-Activated Protein Kinases
keywords: AMP-activated kinase (AMPK); Akt PKB; Serine485/491; insulin resistance; protein kinase C (PKC); protein kinase D (PKD); skeletal muscle