New Candidate Targets of AMP-Activated Protein Kinase in Murine Brain Revealed by a Novel Multidimensional Substrate-Screen for Protein Kinases

Abstract

AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase that is involved in the maintenance of energy homeostasis and recovery from metabolic stresses both at the cellular and whole body level. AMPK is found in all tissues examined so far, and a number of downstream targets have been identified. Recent work suggests that AMPK has specialized functions in the brain, such as involvement in appetite control. Nevertheless, brain-specific substrates of AMPK are unknown. Here, we performed a proteomic in vitro screen to identify new putative AMPK targets in brain. Prefractionation of murine brain lysates by liquid chromatography, utilizing four different, serially connected columns with different chemistries was found to be superior to a single column method. A pilot screen involving incubation of small volumes of individual fractions with radiolabeled ATP in the presence or absence of active AMPK, followed by one-dimensional SDS-PAGE and autoradiography, revealed the presence of potential AMPK substrates in a number of different fractions. On the basis of these results, several kinase assays were repeated with selected fractions on a preparative scale. Following separation of the radiolabeled proteins by two-dimensional electrophoresis and comparison of samples with or without added AMPK by differential autoradiography, 53 AMPK-specific phospho-spots were detected and excised. Thereof, 26 unique proteins were identified by mass spectrometry and were considered as new potential downstream targets of AMPK. Kinase assays with 14 highly purified candidate substrate proteins confirmed that at least 12 were direct targets of AMPK in vitro. Although the physiological consequences of these phosphorylation events remain to be established, hypotheses concerning the most intriguing potential targets of AMPK that have been identified by this search are discussed herein. Our data suggests that signaling by AMPK in brain is likely to be involved in the regulation of pathways that have not yet been linked to this kinase.

AMP-activated protein kinase regulates the vacuolar H+-ATPase via direct phosphorylation of the A subunit (ATP6V1A) in the kidney

“…We also showed that the V-ATPase A subunit in the cytoplasmic V 1 sector is phosphorylated in vitro and in HEK cells by both PKA and AMPK. Our results thus confirmed prior findings that the V-ATPase A subunit was a potential direct AMPK phosphorylation target in an unbiased substrate screen (52). The present study uncovers a molecular mechanism of V-ATPase regulation by AMPK in kidney intercalated cells via direct phosphorylation of the V-ATPase A subunit by this metabolic sensing kinase.…”

“…We have demonstrated that in kidney intercalated cells and in epididymal clear cells, which share a common developmental origin in the Wolffian duct, the V-ATPase redistributes from the apical membrane to the cytosol with stimulation of the metabolic sensor AMPK (16,18). In our previous studies we first identified the V-ATPase A subunit as an AMPK substrate using the unbiased "MudSeeK" proteomic screening method and subsequently showed that the V-ATPase A subunit is phosphorylated directly by AMPK in kidney cells (18,52). AMPK is an important cell energy-sensing kinase that has been shown to downregulate several kidney membrane transport proteins (17,39).…”

AMPK in Health and Disease

“…Another possibility is that AMPK regulation of mTOR may be critical seeing that the inhibition of mTOR using leucine, leptin, or rapamycin reduces food intake (109). While a recent proteomics screen has identified 12 in vitro substrates of AMPK in the brain, many of which were related to glycolysis (504), future studies are required to determine whether AMPK directly affects neuropeptide gene expression or whether this is via indirect mechanisms possibly involving ACC and mTOR.…”

PKA Regulates Vacuolar H+-ATPase Localization and Activity via Direct Phosphorylation of the A Subunit in Kidney Cells

“…Increased [HCO 3 Ϫ ] may in turn activate the sAC/cAMP/PKA pathway and the V-ATPase at the apical membrane of A-intercalated cells resulting in enhanced proton extrusion and subsequent basolateral recovery of HCO 3 Ϫ into the extracellular space (19). The role of direct phosphorylation of V-ATPase subunits in H ϩ secretion in mammalian epithelial cells has not been well characterized (19,34,35). Although PKA agonists have been shown to regulate the V-ATPase in a variety of systems, it was not until recently that the direct phosphorylation of V-ATPase subunits by this kinase was linked to its regulation.…”