Updated project metadata. Rare variants in NEK1, encoding a serine/threonine kinase, are established contributors to amyotrophic lateral sclerosis (ALS), but the biochemical effects of ALS-associated NEK1 missense variants on kinase activity have not been directly investigated. This project maps the phosphorylation landscape of human NEK1 and identifies recurrent phosphorylation sites using cell-based and recombinant phosphoproteomic approaches. GFP-tagged wild-type and kinase-dead NEK1 were analysed in a NEK1 knockout U-2 OS background, and recombinant NEK1/C21ORF2 complexes were also examined. LC-MS/MS phosphosite mapping, targeted extracted ion chromatogram analysis, phosphosite mutagenesis and phosphospecific antibody validation were used to assign pSer14, pThr156 and pSer418 as NEK1 autophosphorylation sites. Activation-loop pThr156 was then used as a readout of NEK1 kinase activity to assess ALS-associated NEK1 missense variants. The dataset from Mehta Lab provides a curated phosphoproteomic resource for NEK1 regulation, kinase activity measurement and functional interpretation of ALS-associated NEK1 variants.