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

Tubulins, building blocks of microtubules, are modified substrates of diverse post-translational modifications including phosphorylation, polyglycylation and polyglutamylation. Polyglutamylation of microtubules, catalyzed by enzymes from the tubulin tyrosine ligase-like (TTLL) family, can regulate interactions with molecular motors and other proteins. Due to the diversity and functional importance of microtubule modifications, strict control of the TTLL enzymes has been suggested.


To characterize the interaction between never in mitosis gene A-related kinase 5 (NEK5) and TTLL4 proteins and the effects of TTLL4 phosphorylation.


The interaction between NEK5 and TTLL4 was identified by yeast two-hybrid screening using the C-terminus of NEK5 (a.a. 260–708) as bait and confirmed by immunoprecipitation. The phosphorylation sites of TTLL4 were identified by mass spectrometry and point mutations were introduced.


Here, we show that NEK5 interacts with TTLL4 and regulates its polyglutamylation activity. We further show that NEK5 can also interact with TTLL5 and TTLL7. The silencing of NEK5 increases the levels of polyglutamylation of proteins by increasing the activity of TTLL4. The same effects were observed after the expression of the catalytically inactive form of NEK5. This regulation of TTLL4 activity involves its phosphorylation at Y815 and S1136 amino acid residues.


Our results demonstrate, for the first time, the regulation of TTLL activity through phosphorylation, pointing to NEK5 as a potential effector kinase. We also suggest a general control of tubulin polyglutamylation through NEK family members in human cells.

Keywords: Kinase, Polyglutamylation, Never in mitosis gene A-related kinase 5, Tubulin tyrosine ligase-like 4, Microtubules, Post translational regulation

Core Tip: Tubulins are modified extensively by post-translational processes such as polyglutamylation. Considering the diversity of microtubule polyglutamylation and the existence of many non-tubulin substrates, it is important to understand how the effector enzymes, the tubulin ligase-like (TTLL) proteins, are regulated. TTLL4 interacts with never in mitosis gene A (NIMA)-related kinase 5, a member of the mitotic NIMA-related kinases. We demonstrate that NIMA-related kinase 5 is a potential regulator of polyglutamylation through the control of TTLL4 activity. Here we show, for the time, the regulation of TTLL4 activity through phosphorylation, and demonstrate the potential control of polyglutamylation through NEK family members in human cells.