The kinetics of phosporylation of tyrosine hydroxylase and its interaction with 14‑3‑3 zeta protein studied by NMR

Petr Louša, Hana Nedozrálová, Jiří Nováček, Erik Župa, Jozef Hritz

CEITEC MU, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic


Human tyrosine hydroxylase 1 (hTH1) activity is regulated by phosphorylation of its N-terminus and by an interaction with regulatory 14‑3‑3 protein. In order to monitor structural changes within the regulatory domain of hTH1 (RD‑hTH1, region of first 169 residues) caused by phosphorylation of S19 and S40 we have assigned NMR spectra by two different approaches. The non-uniform sampling approach based on sparse multidimensional Fourier transform allowed efficient acquisition of high dimensional NMR spectra. Increased dimensionality (5D) provided significant speed up of backbone and side-chain assignment of the unstructured RD-hTH1 region (~70 residues). The rest (structured parts) of RD-hTH1 was assigned by conventional set of 3D NMR experiments.

We also measured kinetic rates of phosphorylation of RD-hTH1 by PKA and PRAK kinase to gain better understanding of phosphorylation process. Kinetic series were derived from intensity changes of peaks of selected residues close to phosphorylation site. To reveal interactions between RD-hTH1 and protein 14-3-3 zeta we performed titration of doubly phosphorylated RD­hTH1 with highly concentrated 14-3-3. Changes in the peak intensity indicated the regions mostly involved in the interaction.

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The project is financed from the SoMoPro II programme. The research leading to this invention has acquired a financial grant from the People Programme (Marie Curie action) of the Seventh Framework Programme of EU according to the REA Grant Agreement No. 291782. The research is further co-financed by the South-Moravian Region. The article/paper reflects only the author´s views and the Union is not liable for any use that may be made of the information contained therein. In addition, this work was also supported by Czech Science Foundation (15-34684L). The computational simulations were realized in the National Supercomputing Center IT4Innovations, which is supported by The Ministry of Education, Youth and Sports from the Large Infrastructures for Research, Experimental Development and Innovations project „IT4Innovations National Supercomputing Center – LM2015070“. Further computational resources were provided by the MetaCentrum under the program LM2015042 and the CERIT Scientific Cloud under the program LM2015085, provided under the programme "Projects of Large Infrastructure for Research, Development, and Innovations". This research was carried out under the project CEITEC 2020 (LQ1601) with financial support from the Ministry of Education, Youth and Sports of the Czech Republic under the National Sustainability Programme II.