NEK1 autophosphorylation is disrupted by amyotrophic lateral sclerosis-associated missense variants: activity biomarkers and structural insights
- 1University of Dundee
Abstract
Rare variants in NEK1, encoding a serine/threonine kinase, are amongst the most consistently implicated genetic contributors to amyotrophic lateral sclerosis (ALS), reported in approximately 2-3% of cases. Yet, whilst recent studies have characterised the cell biological consequences of NEK1 loss-of-function, the biochemical effects of ALS-associated missense variants on kinase activity have not been directly investigated. This distinction is mechanistically important, because missense alleles encode mutant proteins rather than simply reducing protein dosage. Here, we provide the most comprehensive cell-based phosphoproteomic map of NEK1 phosphorylation to date, identifying ten recurrent phosphorylation sites across independent expression and acquisition conditions. We experimentally assign pSer14, pThr156 and pSer418 as NEK1 autophosphorylation sites using kinase-dead controls, targeted extracted ion chromatogram analysis, phosphosite mutagenesis and phosphospecific antibodies. Leveraging activation-loop pThr156 as a readout of NEK1 activity, we assessed nine ALS-associated missense variants spanning the major functional regions of the protein. Amongst catalytic-domain variants, R261C produced the most robust reduction in pThr156 autophosphorylation, R232C produced a smaller reduction, and R232H increased pThr156; the basic-region variant, A313T, also showed a smaller reduction. Structural modelling provides a mechanistic framework for understanding these variant-specific effects. Our study establishes the first activity-based framework for functional classification of NEK1 missense variants, and provides direct evidence that ALS-associated missense variants can alter NEK1 autophosphorylation through a mechanism distinct from simple haploinsufficiency. The phosphospecific antibodies, isogenic cell lines and curated phosphoproteomic datasets generated here provide a community resource for future studies of NEK1 regulation, variant interpretation and therapeutic target validation.
Related
- Receptor-like kinase RLK15 binds a fungal RNase effector to enhance autophosphorylation and activate host defense
- Familial, neuropathological and cellular analysis identify ARPP21 as a major amyotrophic lateral sclerosis associated gene in French cohorts
- IRAK4 autophosphorylation functions as a molecular switch to regulate death domain assembly and Myddosome maturation
- OphthoEvidence report: Baseline structural optical coherence tomography biomarkers as predictors of visual outcomes following full-thickness macular hole surgery: A systematic review and meta-analysis protocol
- Multimodal Magnetic Resonance Imaging Biomarkers of Pediatric Traumatic Brain Injury Identified by Ensemble Learning Are Associated with Psychopathology
BibTeX
@misc{agarwal2026nek1,
title = {{NEK1 autophosphorylation is disrupted by amyotrophic lateral sclerosis-associated missense variants: activity biomarkers and structural insights}},
author = {S. Agarwal and S. A. Abdul Rehman and I. M. Munoz and A. Knebel and P. J. Hop and R. Gourlay and F. Brown and T. Macartney and I. Squires and J. H. Veldink and K. P. Kenna and J. Rouse and A. R. Mehta},
howpublished = {bioRxiv},
year = {2026},
month = jun,
doi = {10.64898/2026.06.17.733013},
url = {https://www.biorxiv.org/content/10.64898/2026.06.17.733013v2},
note = {CC BY},
}
RIS
TY - UNPB
TI - NEK1 autophosphorylation is disrupted by amyotrophic lateral sclerosis-associated missense variants: activity biomarkers and structural insights
AU - S. Agarwal
AU - S. A. Abdul Rehman
AU - I. M. Munoz
AU - A. Knebel
AU - P. J. Hop
AU - R. Gourlay
AU - F. Brown
AU - T. Macartney
AU - I. Squires
AU - J. H. Veldink
AU - K. P. Kenna
AU - J. Rouse
AU - A. R. Mehta
PY - 2026
DA - 2026/06/17
PB - bioRxiv
DO - 10.64898/2026.06.17.733013
UR - https://www.biorxiv.org/content/10.64898/2026.06.17.733013v2
AB - Rare variants in NEK1, encoding a serine/threonine kinase, are amongst the most consistently implicated genetic contributors to amyotrophic lateral sclerosis (ALS), reported in approximately 2-3% of cases. Yet, whilst recent studies have characterised the cell biological consequences of NEK1 loss-of-function, the biochemical effects of ALS-associated missense variants on kinase activity have not been directly investigated. This distinction is mechanistically important, because missense alleles encode mutant proteins rather than simply reducing protein dosage. Here, we provide the most comprehensive cell-based phosphoproteomic map of NEK1 phosphorylation to date, identifying ten recurrent phosphorylation sites across independent expression and acquisition conditions. We experimentally assign pSer14, pThr156 and pSer418 as NEK1 autophosphorylation sites using kinase-dead controls, targeted extracted ion chromatogram analysis, phosphosite mutagenesis and phosphospecific antibodies. Leveraging activation-loop pThr156 as a readout of NEK1 activity, we assessed nine ALS-associated missense variants spanning the major functional regions of the protein. Amongst catalytic-domain variants, R261C produced the most robust reduction in pThr156 autophosphorylation, R232C produced a smaller reduction, and R232H increased pThr156; the basic-region variant, A313T, also showed a smaller reduction. Structural modelling provides a mechanistic framework for understanding these variant-specific effects. Our study establishes the first activity-based framework for functional classification of NEK1 missense variants, and provides direct evidence that ALS-associated missense variants can alter NEK1 autophosphorylation through a mechanism distinct from simple haploinsufficiency. The phosphospecific antibodies, isogenic cell lines and curated phosphoproteomic datasets generated here provide a community resource for future studies of NEK1 regulation, variant interpretation and therapeutic target validation.
ER -