-WANNA mutant. Alternatively, we decided to replace the second asparagine residue (Asn270) to alanine, resulting in R6-WDNAD mutant. We expressed these 934369-14-9 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 related interaction pattern as wild sort with each of the studied proteins: PP1c, laforin and 14-3-3 proteins (Fig 2B). Nevertheless, the R6-WANNA mutant didn’t interact with any of the studied proteins, in spite of being expressed in yeast (Fig 2B). So that you can study the interaction profile of these mutants in 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 able 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, while conserved the ability 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 outcomes confirmed the functionality in the W267DNND motif of R6 in substrate binding. Taking all these benefits collectively, we suggest that binding of R6 to PP1c occurs through the R102VRF motif and binding of R6 to PP1 substrates occurs inside a area comprising the R252VHF as well as the W267DNND motifs, being 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.
Evaluation of the interacting properties of unique 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 applying GFP-Trap system (see Materials and Methods section). 40 L of eluted beads and thirty micrograms of total protein in the soluble fraction of cell lysates (input) had been analyzed by SDS-PAGE and Western blotting applying appropriated antibodies.
It truly is recognized that 14-3-3 proteins bind to Ser/Thr phosphorylated residues [19]. So, in order to obtain the putative 14-3-3 binding domain in R6, we searched in the databases for reports on the phosphorylation of R6 and located that it might be potentially phosphorylated in distinct residues: Ser23, Ser25, Ser28, Ser46, Ser74, Ser77, Ser78 and Ser133 ([32], [33], [34], [35]). However, only two of these web pages, Ser25 and Ser74, could kind part of the key putative 14-3-3 protein binding consensus motif-RSXpSXP- [19] (Fig 1A, yellow boxes). So as to study the functionality 21593435 of these web-sites on 14-3-3 protein binding, we created non-phosphorylatable mutants in which Ser25 or Ser74 have been changed to alanine (S25A, S74A). Then, we assessed the binding properties of the mutated forms by yeast two-hybrid analysis. As shown in Fig 2C, binding of both R6-S25A and R6-S74A for the PP1c catalytic subunit and to laforin was comparable to wild kind. On the other hand, even though mutation at Ser25 didn’t have an effect on the interaction with 14-3-3 proteins, mutation at Ser74 fully eliminated this interaction (Fig 2C). To confirm these benefits in a mammalian system, we constructed the corresponding YFP-fusion proteins (YFP-R6-S25A and YFP-R6-S7