The interaction between AMPK beta 2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content

11 páginas, 7 figuras.AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKβ at threonine-148 (Thr-148), which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKβ1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. Here, we further investigated the interaction of R6 with AMPKβ and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid analyses and co-immunoprecipitation of the overexpressed proteins in HEK293T cells revealed that both AMPKβ1 and β2 wild-type (WT) isoforms bind to R6. The AMPKβ/R6 interaction was stronger with the muscle-specific β2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKβ2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKβ2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced AMPKβ2/R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low glucose medium. These data are in accordance with R6 binding directly to AMPKβ2 when both proteins detach from the diminishing glycogen particle, which is simultaneous to increased AMPKβ2 Thr-148 autophosphorylation. Such model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle.DN is recipient of a VIDI-Innovational Research Grant from the Netherlands Organization of Scientific Research (NWO-ALW Grant no. 864.10.007). This work has further been supported by grants from the Spanish Ministry of Education and Science SAF2014-54604-C3-1-R and a grant from Generalitat Valenciana (PrometeoII/2014/029) to PS.Peer reviewe

TRIM32 and Malin in Neurological and Neuromuscular Rare Diseases

Tripartite motif (TRIM) proteins are RING E3 ubiquitin ligases defined by a shared domain structure. Several of them are implicated in rare genetic diseases, and mutations in TRIM32 and TRIM-like malin are associated with Limb-Girdle Muscular Dystrophy R8 and Lafora disease, respectively. These two proteins are evolutionary related, share a common ancestor, and both display NHL repeats at their C-terminus. Here, we revmniew the function of these two related E3 ubiquitin ligases discussing their intrinsic and possible common pathophysiological pathways

Primers used for site directed mutagenesis.

<p>Primers used for site directed mutagenesis.</p

Subcellular localization of R6 S25A and R6 S74A mutated forms.

<p>N2a cells were transfected with pYFP empty plasmid, pYFP-R6 wild type, pYFP-R6 S25A or pYFP-R6 S74A plasmids. The subcellular localization of R6 forms and glycogen granules was carried out as described in Materials and Methods. Images were obtained by using confocal microscopy (bars indicate 10 μm). Images corresponding to visible, YFP (in yellow) and glycogen (in red) fluorescences are shown.</p

Schematic representation of the different binding regions in R6.

<p>R6 possesses three separated interaction domains: PP1c binding motif (R₁₀₂VRF), PP1 substrate binding region (W₂₆₇DNND) and 14-3-3 binding motif (RARS₇₄LP). GS, glycogen synthase; GP, glycogen phosphorylase.</p

Analysis of the interacting properties of different domains of R6 by yeast two-hybrid analyses.

<p>Upper panels: yeast THY-AP4 strain was transformed with plasmids pBTM-R6 wt (LexA-R6), pBTM-R6 RARA and pBTM-R6 RAHA (A), pBTM-R6 WDNAD, and pBTM-WANNA (B) or pBTM-R6 S25A and pBTM-R6 S74A (C) and with pACT2 (GAD), pACT2-PP1α (GAD-PP1α), pACT2-laforin (GAD-laforin) or pACT2-14-3-3ε (GAD-14-3-3ε). Protein interaction was estimated by measuring the β-galactosidase activity. Values correspond to means from at least 6 different transformants (bars indicate standard deviation). Lower panels: protein expression in yeast transformants was analyzed by Western blotting using anti-HA antibodies (for the GAD-fusions) and anti-LexA (for the LexA-fusions) in several transformants from each condition. A representative western blot of some of these transformants is shown.</p

Characterization and structural modelling of R6 domains.

<p>(A) Multiple sequence alignment of PPP1R3D (R6), PPP1R3C (R5) and PPP1R3B (GL) proteins sequences from <i>H</i>. <i>sapiens</i> (Uniprot entries: O95685, Q9UQK1 and Q86XI6, respectively) was performed using the Clustal Omega program (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131476#sec002" target="_blank">Materials and Methods</a>). Invariance and conservation are highlighted by black and gray shadowing respectively. Amino acid sequences for the binding to 14-3-3 proteins, PP1 catalytic subunit and PP1 glycogenic substrates are enclosed in yellow, orange and cyan boxes respectively. Brackets indicate the modeled sequence of R6 shown in panel B. The CBM21 domain of R6 (according to Uniprot) is underlined in orange. Red triangles point at the mutated residues obtained in this study. (B) Homology model of the CBM21 domain of R6 was based on the template of GL (pdb entry: 2EEF). The amino acids corresponding to the PP1 glycogenic substrate binding domain (W₂₆₇DNND) and to the putative RVXF (R₂₅₂VHF) are shown in sticks and colored in cyan or orange, respectively (left panel). The N- and C-terminus of the model is also indicated. A representation of the surface of the R6 homology model to show the exposed amino acids to the solvent is presented in the right panel. Images were generated using PyMol (DeLano Scientific LLC, USA).</p

Binding of 14-3-3 proteins to R6 prevents its lysosomal degradation.

<p>A) R6-S74A mutant possesses a shorter half-life than wild type protein. Hek293 cells were transfected with pFLAG-R6 wt or pFLAG-R6-S74A plasmids. 24 hours after transfection, cells were treated with cycloheximide (300 μM) to block protein synthesis. At the indicated times, cell extracts (30 μg) were analyzed by Western blotting using anti-FLAG and anti-tubulin (as loading control). A representative western blot is shown on the left panel. On the right panel, the intensity of the bands related to the levels of tubulin is plotted and normalized respect to the values at time 0 (bars indicate the standard deviation of at least three independent experiments; **p < 0.01). B) R6-S74A protein is degraded by the lysosomal pathway. Hek293 cells were transfected with pFLAG-R6-S74A plasmid. Eighteen hours after transfection, cells were treated with ammonium chloride (20 mM)/ leupeptin (100 μM) or MG132 (5 μM) for six hours. Then, cells were lysed and extracts (30 μg) were analyzed by immunoblotting using anti-FLAG antibody and anti-tubulin as loading control. The intensity of the bands related to the levels of tubulin is plotted (bars indicate standard deviation of at least three independent experiments; *p < 0.05).</p

Glycogenic activity of different mutated forms of R6.

<p>(A) Measurement of glycogenic activity of different R6 mutated forms. N2a cells were transfected using 1 μg of pFLAG plasmid (negative control), pFLAG-R6 plasmid or its corresponding mutants. Forty-eight hours after transfection, the amount of glycogen was determined as described in Materials and Methods and represented as μg of glucose/mg of protein/relative amount of R6 respect to actin (wild type value considered as 1). Bars indicate standard deviation of three independent experiments (**p<0.01 or ***p<0.001, compared with control cells transfected with an empty plasmid; ##p<0.01, compared with cells expressing R6-WT). An inset with the mean values +/- standard deviation is included. (B) Protein levels of FLAG-R6 forms. A representative western blot analysis is shown. Cell extracts (30 μg) were analyzed using the corresponding anti-FLAG and anti-actin antibodies.</p

Analysis of the interacting properties of different domains of R6 by immunoprecipitation (GFP-Trap) in mammalian cells.

<p>Hek293 cells were transiently transfected with expression vectors coding for YFP, YFP-R6 wild type, and the corresponding mutants YFP-R6 RARA and YFP-R6 RAHA (A), YFP-R6 WDNAD and YFP-R6 WANNA (B), or YFP-R6 S25A and YFP-R6 S74A plasmids (C). Immunoprecipitation analyses were performed using GFP-Trap system (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131476#sec002" target="_blank">Materials and Methods</a> section). 40 μL of eluted beads and thirty micrograms of total protein from the soluble fraction of cell lysates (input) were analyzed by SDS-PAGE and Western blotting using appropriated antibodies.</p