Structure-Function Analysis of PPP1R3D, a Protein Phosphatase 1 Targeting Subunit, Reveals a Binding Motif for 14-3-3 Proteins which Regulates its Glycogenic Properties

Abstract

Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. It plays a key role in regulating glycogen synthesis, by dephosphorylating crucial enzymes involved in glycogen homeostasis such as glycogen synthase (GS) and glycogen phosphorylase (GP). To play this role, PP1 binds to specific glycogen targeting subunits that, on one hand recognize the substrates to be dephosphorylated and on the other hand recruit PP1 to glycogen particles. In this work we have analyzed the functionality of the different protein binding domains of one of these glycogen targeting subunits, namely PPP1R3D (R6) and studied how binding properties of different domains affect its glycogenic properties. We have found that the PP1 binding domain of R6 comprises a conserved RVXF motif (R102VRF) located at the N-terminus of the protein. We have also identified a region located at the C-terminus of R6 (W267DNND) that is involved in binding to the PP1 glycogenic substrates. Our results indicate that although binding to PP1 and glycogenic substrates are independent processes, impairment of any of them results in lack of glycogenic activity of R6. In addition, we have characterized a novel site of regulation in R6 that is involved in binding to 14-3-3 proteins (RARS74LP). We present evidence indicating that when binding of R6 to 14-3-3 proteins is prevented, R6 displays hyper-glycogenic activity although is rapidly degraded by the lysosomal pathway. These results define binding to 14-3-3 proteins as an additional pathway in the control of the glycogenic properties of R6.

The interaction between AMPKβ2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content

“…4D). Since the R6-substrate-binding motif partially overlaps with the CBM domain (23), it is conceivable that glycogen competes with R6 binding to AMPK. Thus, our data suggest that low glycogen favors AMPKβ2/R6 interaction, whereas high glycogen content interferes with it.…”

“…R6 recruits PP1 phosphatase to its substrates (e.g., GS and GP) (23), thus playing a critical role in the regulation of glycogen metabolism. Most of the PP1-glycogen-targeting subunits exert their actions through binding to PP1 via a conserved Nterminal PP1-binding motif (RVXF), and interact with PP1-substrates via a conserved C-terminal substrate-binding (WXNXGNYX(L/I)) motif (23). We have recently shown that R6 utilizes this conserved region to bind to its glycogenic substrates GS and GP (23).…”

“…Most of the PP1-glycogen-targeting subunits exert their actions through binding to PP1 via a conserved Nterminal PP1-binding motif (RVXF), and interact with PP1-substrates via a conserved C-terminal substrate-binding (WXNXGNYX(L/I)) motif (23). We have recently shown that R6 utilizes this conserved region to bind to its glycogenic substrates GS and GP (23). In line with these results, our work indicates that AMPKβ/R6 interaction occurs also via the R6 substrate-binding motif (Fig.…”

“…2B). R6-RARA, a mutant known to have lost its capacity to bind to endogenous PP1c but not to PP1-substrates (23), presented a similar AMPKβ2-binding profile compared to wild-type, non-mutated R6 (R6-WT; Fig. 2B; right panel).…”

“…Recently, a number of functionally distinct protein domains have been identified in the R6 glycogentargeting subunit of PP1 (23). R6 is composed of domains that mediate binding to carbohydrates (via its CBM), binding to the PP1 catalytic subunit (PP1c; RVXF motif), and to the glycogen metabolism-related substrates of PP1 (via the highly conserved WDNND motif).…”