Published online May 27, 2021. doi: 10.4331/wjbc.v12.i3.38
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
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.
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.
Confirm and map the interaction between TTLL4 and NEK5 proteins and explore a possible regulation mechanism of TTLL4 through phosphorylation.
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.
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.
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.
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.