AMP-activated Protein Kinase Phosphorylates R5/PTG, the Glycogen Targeting Subunit of the R5/PTG-Protein Phosphatase 1 Holoenzyme, and Accelerates Its Down-regulation 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.Glycogen 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: G M , G L , 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 G M is down-regulated by its PKA-dependent phosphorylation; PKA phosphorylates G M at a site in its PP1 binding motif, which leads to its dissociation from PP1. The glycogenic activity of the G L -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 c...

Protein Degradation and Quality Control in Cells from Laforin and Malin Knockout Mice

“…However, laforin may have other functions independent of malin and may not always act as a complex with malin, as has been suggested (17)(18)(19). Based primarily on experiments using cell culture systems, several substrates of malin have been proposed, mainly relevant to glycogen metabolism; laforin (18), glycogen synthase (17), PTG (18), AGL (19), AMP-activated protein kinase (40), and neuronatin (41). In 3-month-old Epm2b Ϫ/Ϫ mice, we found no evidence for alterations in the levels of PTG, AGL, or glycogen synthase activity (13), arguing against these proteins being malin substrates.…”

Insulin-induced hypoglycemia increases hepatic sensitivity to glucagon in dogs

“…To our knowledge, there is no evidence that AMPK can directly phosphorylate GP. However, a recent in vitro study suggested that AMPK may indirectly reduce PP1-mediated dephosphorylation of GS and GP, the net effect of which would favor glycogenolysis (23). In addition, AMPK can directly phosphorylate GS at Ser7 (24).…”

The Laforin-Like Dual-Specificity Phosphatase SEX4 from Arabidopsis Hydrolyzes Both C6- and C3-Phosphate Esters Introduced by Starch-Related Dikinases and Thereby Affects Phase Transition of α-Glucans

“…Furthermore, functional laforin has been reported to partially complement a SEX4-deficient Arabidopsis mutant (Gentry et al, 2007), suggesting a close functional similarity between this type of plant and mammalian DSPs. However, laforin is known to exert phosphatase activity on various phosphoproteins and to undergo additional protein-protein interactions such as homodimerization (Vilchez et al, 2007;Worby et al, 2008;Puri et al, 2009;Vernia et al, 2009). Currently, it is totally unknown whether or not the mammalian laforin and the plant SEX4 protein share functional similarities even in some of these (phospho) protein-related actions.…”