# Missense variant in TTBK2 kinase domain causes loss of function and impaired protein phosphorylation

## TL;DR

A missense variant in the TTBK2 kinase domain reduces its function and disrupts protein phosphorylation, potentially contributing to disease.

## Contribution

This study is the first to demonstrate the functional impact of a TTBK2 missense variant using a CRISPR/Cas9 knock-in model.

## Key findings

- TTBK2-L209F leads to reduced TTBK2 protein levels and impaired kinase activity toward TDP-43.
- Phosphoproteomic analysis reveals dysregulated pathways in gene regulation, protein degradation, and cytoskeletal organization.
- The variant alters levels of cytoskeleton-related proteins and affects TGF-β signaling.

## Abstract

Tau tubulin kinase 2 (TTBK2) is a ubiquitous serine-threonine protein kinase implicated in diverse cellular processes, including microtubule regulation, ciliogenesis, synaptic signaling, and the phosphorylation of key proteins like TDP-43. Despite its relevance, many aspects of TTBK2 function in both physiological and pathological conditions remain poorly understood. Truncating variants in TTBK2 gene cause spinocerebellar ataxia type 11 (SCA11), a rare form of autosomal dominant cerebellar ataxia. However, the functional consequences and pathogenic potential of missense variants have yet to be elucidated. In this study, we developed a CRISPR/Cas9 knock-in cell model harboring a missense variant in TTBK2 kinase domain (NM_173500.4:c.625 C > T; p.Leu209Phe) to evaluate its impact on TTBK2 expression, associated protein levels, and phosphoproteomic profiles. TTBK2 missense variant (TTBK2-L209F) was associated with reduced TTBK2 protein levels, altered levels of cytoskeleton-related proteins, and impaired kinase activity, namely toward TDP-43. Phosphoproteomic analyses identified dysregulation in pathways linked to gene regulation, protein degradation, cytoskeletal organization, and TGF-β signaling. These findings provide valuable insights into the biological roles of TTBK2 in cellular signaling. Moreover, this study underscores the importance of functional studies to better understand the consequences of TTBK2 missense variants, particularly those affecting the kinase domain, and their potential contribution to disease.

The online version contains supplementary material available at 10.1038/s41598-025-32288-0.