Basic Study
Copyright ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Biol Chem. May 27, 2021; 12(3): 38-51
Published online May 27, 2021. doi: 10.4331/wjbc.v12.i3.38
Polyglutamylase activity of tubulin tyrosine ligase-like 4 is negatively regulated by the never in mitosis gene A family kinase never in mitosis gene A -related kinase 5
Talita Diniz Melo-Hanchuk, Jörg Kobarg
Talita Diniz Melo-Hanchuk, Faculty of Pharmaceutical Sciences, Unicamp, Campinas 13083-862, Brazil
Jörg Kobarg, Faculty of Pharmaceutical Sciences, University of Campinas, Campinas 13083-862, Brazil
Author contributions: Melo-Hanchuk TD performed the experiments and interpreted them together with Kobarg J; both authors wrote the manuscript; Kobarg J supervised the project.
Supported by Fundação de Amparo à Pesquisa do Estado São Paulo (FAPESP; São Paulo, Brazil) through Grant Temático, No. 2017/03489-1.
Institutional review board statement: The study was reviewed and approved by the Institutional review board of UNICAMP.
Conflict-of-interest statement: Both authors declare they have no conflict of interest.
Data sharing statement: Data will be made available upon reasonable request.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Jörg Kobarg, PhD, Full Professor, Faculty of Pharmaceutical Sciences, University of Campinas, 200 Cândido Portinari, Campinas 13083-862, Brazil.
Received: October 25, 2020
Peer-review started: October 25, 2020
First decision: December 24, 2020
Revised: January 6, 2021
Accepted: February 25, 2021
Article in press: February 25, 2021
Published online: May 27, 2021
Research background

Enzymes of the tubulin tyrosine ligase-like (TTLL) family are responsible for the polyglutamylation of tubulins and many other protein substrates. The never in mitosis gene A-related kinases (NEKs) are protein kinases involved in diverse aspects of regulation of the cell cycle, microtubules, primary cilia and the deoxyribonucleic acid damage response. Previous data from the literature and protein interaction data between TTLLs and NEKs suggested a possible crosstalk and regulatory connection between these two protein families.

Research motivation

In a yeast two-hybrid assay for protein interactors of human NEK5, TTLL4 was identified as a partner. Additionally, a previously report showed that purified extracts of NEK in Crithidia fasciculata was capable of glutamylating tubulins in vitro. Here, we set out to confirm the interaction between NEK5 and TTLL4 and to explore possible functional consequences of this interaction.

Research objectives

Confirm and map the interaction between TTLL4 and NEK5 proteins and explore a possible regulation mechanism of TTLL4 through phosphorylation.

Research methods

We used transient transfection of full-length TTLL4, deletions and point mutants in cells with stable expression of NEK5 as well as knock down of NEK5 expression by short hairpin ribonucleic acid. Site-directed mutagenesis was used to generate a series of point mutants of TTLL4. The polyglutamylation activity of TTLL4 variants was assessed by Western blot, using antibody GT335, which detects polyglutamylation of protein substrates.

Research results

We confirmed the interaction between TTLL4 and NEK5 through yeast two hybrid screening and imunoprecipitation. Furthermore, we showed that expression of NEK5 interferes negatively in the polyglutamylation activity of TTLL4 towards tubulins and other protein substrates, whereas NEK5 knock down or over-expression of a kinase dead variant of NEK5 result in the contrary: An increase in TTLL4 activity. Mass spectrometry showed phosphorylation of TTLL4 on specific Thr, Ser and Tyr residues. Modification of some of these residues affected TTLL4 activity.

Research conclusions

We describe, for the first time, the interaction between members of the NEK and TTLL families. A mechanism for regulation of TTLL4 activity through phosphorylation has emerged and NEK5 is a potential effector kinase, affecting polyglutamylation of many substrates.

Research perspectives

This is the first evidence of a functional and regulatory crosstalk between TTLL and NEK protein families. Members of both families have localization and important functions at microtubules, primary cilia and centrosomes. The functional interplay of the protein families in the context of the cell cycle and microtubule functions should be explored in further detail. This work opens a new perspective of study on the NEK family, mainly in areas related to polyglutamylation, such as cilia, neuronal, blood and muscle disorders.