AMP-activated protein kinase phosphorylates R5/PTG, the glycogen targeting subunit of the R5/PTG-PP1 holoenzyme and accelerates its downregulation by the laforin-malin complex

Abstract

R5/PTG is one of the glycogen targeting subunits of type 1 protein phosphatase, a master regulator of glycogen synthesis. R5/PTG recruits the phosphatase to the places where glycogen synthesis occurs, allowing the activation of glycogen synthase and the inactivation of glycogen phosphorylase, thus increasing glycogen synthesis and decreasing its degradation. In this report, we show that the activity of R5/PTG is regulated by AMP-activated protein kinase (AMPK). We demonstrate that AMPK interacts physically with R5/PTG and modifies its basal phosphorylation status. We have also mapped the major phosphorylation sites of R5/PTG by mass spectrometry analysis, observing that phosphorylation of Ser-8 and Ser-268 increased upon activation of AMPK. We have recently described that the activity of R5/PTG is down-regulated by the laforin-malin complex, composed of a dual specificity phosphatase (laforin) and an E3-ubiquitin ligase (malin). We now demonstrate that phosphorylation of R5/PTG at Ser-8 by AMPK accelerates its laforin/malin-dependent ubiquitination and subsequent proteasomal degradation, which results in a decrease of its glycogenic activity. Thus, our results define a novel role of AMPK in glycogen homeostasis.

Previous SectionNext SectionGlycogen homeostasis depends mainly on the activity of the enzymes involved in its synthesis (glycogen synthase (GS)2) and its degradation (glycogen phosphorylase). These activities are regulated by a complex mechanism involving both allosteric regulation and phosphorylation (1, 2). Interestingly, although there are several kinases (AMPK, PKA, CKI, and glycogen synthase kinase 3) that inhibit glycogen synthesis through the phosphorylation of GS, there is only one known phosphatase (type 1 protein phosphatase (PP1)) that dephosphorylates both GS (leading to its activation) and glycogen phosphorylase (leading to its inactivation), which results in glycogen accumulation (1, 2). PP1 is recruited to glycogen by a family of glycogen targeting proteins, including: GM, GL, R5/PTG, R6, and R3E (3–7). The regulation of the activity of the holoenzyme formed by the PP1 catalytic subunit and one of these glycogen targeting subunits is different in each case. The glycogenic activity of GM is down-regulated by its PKA-dependent phosphorylation; PKA phosphorylates GM at a site in its PP1 binding motif, which leads to its dissociation from PP1. The glycogenic activity of the GL-PP1c holoenzyme is down-regulated by an allosteric mechanism involving phosphorylase-a (8–10). Little is known about the regulation of the holoenzymes involving R6 or R3E, but in the case of R5/PTG, we and others have recently described that its glycogenic activity is down-regulated by the laforin-malin complex, composed of a dual specificity phosphatase (laforin) and an E3-ubiquitin ligase (malin), which recognizes and ubiquitinates R5/PTG and targets it for proteasomal-dependent degradation (11–13).

Laforin and malin are two key proteins related to Lafora disease (LD, OMIM 254780), an autosomal recessive neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions named Lafora bodies. Although the role of these two proteins in cellular physiology is still poorly understood, several reports indicate that both the enzymatic activity of each protein and the physical interaction between them are critical parameters for the pathogenesis of LD. The fact that one of the histological determinants that characterize LD is the accumulation of glycogen-like intracellular inclusions suggests a direct involvement of the laforin-malin complex in the regulation of glycogen biosynthesis (14).

Contrary to mammalian cells, yeast glycogen synthesis is stimulated by nutrient limitation. However, the activity of yeast glycogen synthase (Gsy2) is similarly regulated by phosphorylation. The dephosphorylation and activation of Gsy2 is carried out mainly by the PP1 holoenzyme composed by Gac1 (glycogen-targeting subunit) and the phosphatase catalytic subunit Glc7 (15). It has been described that the Snf1 kinase (yeast orthologue of mammalian AMP-activated protein kinase (AMPK), see below) activates the Gac1/Glc7 protein phosphatase complex, thus facilitating the activation of Gsy2 under conditions of Snf1 activation (15, 16).

AMPK is a serine/threonine protein kinase that acts as a sensor of cellular energy status. Once activated, it switches on catabolic pathways and switches off many ATP-consuming processes (anabolic pathways) (see Refs. 17 and 18 for reviews). AMPK is a heterotrimer of three different subunits, α, β, and γ, each of which has different isoforms (α1, α2, β1, β2, γ1, γ2, and γ3). AMPKα is the catalytic subunit of the AMPK complex, AMPKγ is involved in AMP binding, and AMPKβ functions as a scaffold to assemble α and γ subunits and also determines the subcellular localization and substrate specificity of the complex (17, 18).

Because the yeast Snf1 orthologue of AMPK is involved in the regulation of the yeast PP1 holoenzyme involved in glycogen synthesis (Gac1/Glc7; see above), in this work we studied whether AMPK could regulate similar PP1 holoenzymes in mammalian cells. We found that AMPK interacted physically with the R5/PTG glycogen-targeting subunit and made it a better substrate for its laforin/malin-dependent degradation, resulting in a decrease of its glycogenic activity