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.
Highlights
The control of glycogen homeostasis occurs via an exquisite coordination of events
Protein phosphatase 1 (PP1) plays a crucial role in regulating glycogen synthesis. It dephosphorylates key enzymes involved in glycogen homeostasis, such as glycogen synthase (GS) and glycogen phosphorylase (GP), leading to the activation of the former and the inactivation of the latter, resulting in glycogen accumulation
Consistent with its binding properties, this domain is well exposed to the solvent in both the model we present for R6 (Fig 1B) and in the case of GL
Summary
The control of glycogen homeostasis occurs via an exquisite coordination of events. These events comprises from the regulation of glucose intake to the control of glycogen synthesis and breakdown, amongst others. By the protein phosphatase 1 (PP1) results in the stimulation of glycogen synthesis by activating GS, and the prevention of glycogen breakdown by inactivating GP, which leads to the net accumulation of the polysaccharide [1] These PP1 glycogenic substrates establish only weak interactions with the phosphatase catalytic subunit (PP1c), the process requires the mediation of PP1 regulatory subunits to allow an efficient dephosphorylation ([2], [3]). In this context, it has been described until now seven glycogen targeting subunits [PPP1R3A (GM), PPP1R3B (GL), PPP1R3C (R5/PTG), PPP1R3D (R6), PPP1R3E (R3E), PPP1R3F (R3F) and PPP1R3G (R3G); [1], [3]] that serve as scaffold proteins. To accomplish their function, the glycogen targeting subunits need to bind to the PP1c catalytic subunit, to the PP1 glycogenic substrates and to the glycogen particle ([1], [2], [3])
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