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

RESEARCH ARTICLE 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 Carla Rubio-Villena, Pascual Sanz*☯, Maria Adelaida Garcia-Gimeno☯ Instituto de Biomedicina de Valencia, CSIC, and Centro de Investigación en Red de Enfermedades Raras (CIBERER), Jaime Roig 11, Valencia, Spain a11111 ☯ These authors contributed equally to this work. * Abstract OPEN ACCESS Citation: Rubio-Villena C, Sanz P, Garcia-Gimeno MA (2015) 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. PLoS ONE 10(6): e0131476. doi:10.1371/journal.pone.0131476 Editor: Houhui Xia, Louisiana State University Health Sciences Center, UNITED STATES Received: March 27, 2015 Accepted: June 1, 2015 Published: June 26, 2015 Copyright: © 2015 Rubio-Villena et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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. Data Availability Statement: All relevant data are within the paper. Funding: This work has been supported by grants from the Spanish Ministry of Education and Science SAF2011-27442 and a grant from Generalitat Valenciana (PrometeoII/2014/029). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Introduction 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. The key enzymes involved in glycogen metabolism are the glycogen synthase (GS) and glycogen phosphorylase (GP). The dephosphorylation of these enzymes PLOS ONE | DOI:10.1371/journal.pone.0131476 June 26, 2015 1 / 17 Structure-Function Analysis of PPP1R3D (R6) 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]. However, these PP1 glycogenic substrates establish only weak interactions with the phosphatase catalytic subunit (PP1c), thus 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. These glycogen targeting subunits not only provide additional docking sites for PP1 glycogenic substrates but also recruit the phosphatase to the glycogen particle, where the concentration of the substrates is higher. Therefore, to accomplish their function, the glycogen targeting subunits need to bind to the PP1c catalytic subunit, to the PP1 glycogenic substrates and also to the glycogen particle ([1], [2], [3]). PP1c is one of the major protein phosphatase involved in many different processes in eukaryotic cells. The specificity for the substrates that is able to dephosphorylate is given by its binding to a particular regulatory subunit. At present, more than one hundred different PP1 regulatory subunits have been defined [4], and although they do not show any overall degree of homology, most of them share a common docking motif for PP1 binding, named the RVXF motif ([2], [3]). This motif is present in the glycogen targeting subunits described above [5], although its functionality has only been proven in GM (R63VSF) ([6], [7]), GL (R62VSF) ([6], [7]), R5/PTG (R84VVF) [8] and R3F (R36VLF) [9]. These glycogenic subunits also bind to the PP1 substrates (i.e., GS and GP) to allow their efficient dephosphorylation by the PP1 phosphatase. It was postulated that binding of glycogen targeting subunits to these substrates was mediated by a conserved sequence WXNXGNYX(L/I) [5]. However, at present, the functionality of this domain has only been demonstrated in the case of GM (W219SNNN, [10]) and R5/PTG (W222DSNR, [11]). Finally, these glycogenic subunits contain a carbohydrate binding module of the CBM21 type ([12], [13]) that allows their binding to the glycogen particle [5]. This property is crucial for the localization of the PP1 phosphatase to this specific subcellular compartment where the glycogenic substrates are present. In this work, we have characterized the different binding domains of the glycogen targeting subunit PPP1R3D (R6) and have evaluated their functionality in regulating glycogen production. R6 is a glycogenic subunit of 33 kDa widely distributed in a variety of tissues, including liver, skeletal muscle, pancreas and brain ([14], [15]). In muscle cells R6 has a clear glycogenic activity, which is higher than GM but lower that R5/PTG [16]. We have recently described that the glycogenic activity of R6 is regulated by ubiquitination: R6 interacts with laforin, a dual specificity phosphatase involved in Lafora disease (a type of progressive myoclonus epilepsy), which targets R6 to malin, an E3-ubiquitin (...truncated)