Phosphomimicking Mutations ≠ Phosphorylation – a
case study of 14‑3‑3 protein
A. Kozeleková1,2, A. Náplavová1, T.
Brom2, Z. Trošanová1,2, P. Louša1,2, J. Hritz1,3
Institute of Technology, Masaryk University, Kamenice 5, Brno, 625 00, Czechia
2National Centre for
Biomolecular Research, Masaryk University, Kamenice 5,
Brno, 625 00, Czechia
Chemistry, Faculty of Science, Masaryk University, Kamenice
5, Brno, 625 00, Czechia
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 . 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 .
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
14‑3‑3ζ 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.
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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).