Publication details
Phosphomimicking mutations ≠ phosphorylation - a case study of 14-3-3 protein
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| Year of publication | 2022 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | Protein phosphorylation is one of the most common posttranslational modifications that affects protein structure, interactions, or localization. To study the effect of phosphorylation on protein properties, a fully and specifically phosphorylated sample is usually required, although not always achievable. Therefore, phosphorylation is often replaced by phosphomimicking mutation, i. e. mutation of phosphorylatable Ser/Thr/Tyr by negatively charged Asp or Glu [1]. However, how reliable is this approximation of phosphorylation? In this study, we have focused on dimeric 14-3-3 proteins, regulatory hubs interacting with hundreds of phosphorylated partners [2]. Phosphorylation of 14-3-3 protein at Ser58 has been proposed to induce 14-3-3 monomerization and changes in protein function [3,4]. However, difficulties with preparation of the phosphorylated sample often led to the usage of phosphomimicking and monomeric mutants [5,6]. Here, we have prepared the 14-3-3? protein fully and specifically phosphorylated at Ser58 and we have compared its properties with the phosphomimicking mutants (S58D, S58E), frequently used in the literature. We have revealed significant differences in protein oligomeric state, thermal stability, and hydrophobicity [4,7]. For instance, we have observed disparity in the dimerization dissociation constants of four orders of magnitude. For this reason, we encourage proper verification of protein properties before employment of phosphomimicking mutants. 1. G. Pérez-Mejías, A. Velázquez-Cruz, A. Guerra-Castellano, B. Banos-Jaime, A. Díaz-Quintana, K. González-Arzola, M. Ángel De la Rosa, I. Díaz-Moreno, Comput. Struct. Biotechnol. J., 18, (2020), 1852–1863. 2. N.N. Sluchanko, D.M. Bustos, Prog. Mol. Biol. Transl. Sci., 166, (2019), 19–61. 3. J.M. Woodcock, J. Murphy, F.C. Stomski, M.C. Berndt, A.F. Lopez, J. Biol. Chem., 278, (2003), 36323–36327. 4. Z. Trošanová, P. Louša, A. Kozeleková, T. Brom, N. Gašparik, J. Tungli, V. Weisová, E. Župa, G. Žoldák, J. Hritz, J. Mol. Biol., 434, (2022), 167479. 5. N.N. Sluchanko, I.S. Chernik, A.S. Seit-nebi, A. V Pivovarova, D.I. Levitsky, N.B. Gusev, Arch. Biochem. Biophys., 477, (2008), 305–312. 6. N.N. Sluchanko, M. V Sudnitsyna, A.S. Seit-nebi, A.A. Antson, N.B. Gusev, Biochem., 50, (2011), 9797–9808. 7. A. Kozeleková, A. Náplavová, T. Brom, N. Gašparik, J. Šimek, J. Houser, J. Hritz, Front. Chem., 10, (2022), in press. This study was financed by the Czech Science Foundation (no. GF20-05789L). AK acknowledges the Grant Agency of Masaryk University (MU) for the support of an excellent diploma thesis within the rector’s program (no. MUNI/C/1562/2019). We acknowledge CEITEC (Central European Institute of Technology) Proteomics Core Facility and Biomolecular Interactions and Crystallization Core Facility of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2018127) and European Regional Development Fund-Project ‘UP CIISB’ (CZ.02.1.01/0.0/0.0/18_046/0015974). We acknowledge the CEITEC Core Facility Cellular Imaging supported by MEYS CR (LM2018129 Czech-BioImaging). |
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