-WANNA mutant. Alternatively, we decided to replace the second asparagine residue (Asn270) to alanine, resulting in R6-WDNAD mutant. We expressed these mutated forms in yeast and analyzed their interaction with PP1c, laforin and 14-3-3 proteins by yeast twohybrid. We observed that the R6-WDNAD mutant presented a comparable interaction pattern as wild variety with each of the studied proteins: PP1c, laforin and 14-3-3 proteins (Fig 2B). On the other hand, the R6-WANNA mutant did not interact with any from the studied proteins, despite being expressed in yeast (Fig 2B). As a way to study the interaction profile of these mutants within a mammalian system, we constructed the corresponding YFP-fusion proteins (YFP-R6-WDNAD and YFP-R6-WANNA) and expressed them in Hek293 cells. As shown in Fig 3B, the R6-WDNAD mutant was in a position to interact with endogenous PP1c, GS, GP and 14-3-3 proteins, suggesting that the mutation had not affected the binding properties of R6. Around the contrary, the R6-WANNA mutant, although conserved the capability to interact with endogenous PP1c and 14-3-3 proteins, the binding to the glycogenic substrates GP and GS was severely impaired (Fig 3B). These results confirmed the functionality on the W267DNND motif of R6 in substrate binding. Taking all these results collectively, we suggest that binding of R6 to PP1c occurs through the R102VRF motif and binding of R6 to PP1 substrates happens inside a region comprising the R252VHF as well as the W267DNND motifs, becoming the binding to PP1c and PP1 substrates independent from each other. On the other hand, binding of R6 to 14-3-3 proteins is independent from these defined regions of R6.
Analysis with the interacting properties of distinct domains of R6 by immunoprecipitation (GFP-Trap) in mammalian cells. Hek293 cells have been transiently transfected with expression vectors coding for YFP, YFP-R6 wild type, and also 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 have been performed utilizing GFP-Trap technique (see Materials and Approaches section). 40 L of eluted beads and thirty micrograms of total 864863-72-9 protein from the soluble fraction of cell lysates (input) were analyzed by SDS-PAGE and Western blotting utilizing appropriated antibodies.
It truly is recognized that 14-3-3 proteins bind to Ser/Thr phosphorylated residues [19]. So, so that you can discover the putative 14-3-3 binding domain in R6, we searched inside the databases for reports around the phosphorylation of R6 and located that it could be potentially phosphorylated in different residues: Ser23, Ser25, Ser28, Ser46, Ser74, Ser77, Ser78 and Ser133 ([32], [33], [34], [35]). Nonetheless, only two of those web pages, Ser25 and Ser74, could form part of the main putative 14-3-3 protein binding consensus motif-RSXpSXP- [19] 
(Fig 1A, yellow boxes). So as to study the functionality 21593435 of those web sites on 14-3-3 protein binding, we made non-phosphorylatable mutants in which Ser25 or Ser74 were changed to alanine (S25A, S74A). Then, we assessed the binding properties of your mutated types by yeast two-hybrid analysis. As shown in Fig 2C, binding of each R6-S25A and R6-S74A towards the PP1c catalytic subunit and to laforin was equivalent to wild variety. Nonetheless, even though mutation at Ser25 did not influence the interaction with 14-3-3 proteins, mutation at Ser74 totally eliminated this interaction (Fig 2C). To confirm these results inside a mammalian program, we constructed the corresponding YFP-fusion proteins (YFP-R6-S25A and YFP-R6-S7