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. 2023 Dec 12;14:8248. doi: 10.1038/s41467-023-43865-0

Fig. 6. Phosphorylation of ECSIT may regulate the binding affinity for ACAD9.

Fig. 6

A ECSIT treated with MKK6-activated p38α MAP kinase reveals a different mobility shift compared to the non-treated protein on a native gel. B Radioblotting assays of active P38α MAP kinase and ECSIT p38α-treated sample corroborate ECSIT phosphorylation. Experiments repeated twice with similar results. C Diagram of the phosphorylation sites identified by mass spectrometry. ECSITCTER (res. 320–334) observed by cryo-EM is highlighted in purple and the Thr320 phosphosite in green. The interaction of ECSITCTER Thr320 with Gln331 suggests that a Thr320 phosphate group would generate a steric clash resulting incompatible for ACAD9 binding. D Mass photometry reveals that the ECSITT320E phosphomimetic mutant affects complex formation with ACAD9, resulting in large particles with the major species being an ACAD9 homodimer with unbound ECSIT. Experiments were repeated thrice with similar results. E Assays with H4 human neuroglioma cells both wild-type (WT) and overexpressing human amyloid precursor protein carrying Alzheimer’s-related mutation KM670/671NL (APP). Top left, native gel showing immunopurified fully assembled CI (1 MDa) from both cell types and subjected to the activity assays. Top right, Aβ1-42 detection in mitochondria isolated from WT (green) and APP (red) cells by immunoassays, represented as mean values ± SD for n = 3 biological replicas. Statistical significance was calculated with unpaired t-test, ****p < 0.0001). Bottom, NADH-dehydrogenase activity assays of CI in WT (green) and APP (red) cells at different concentrations (0.5, 0.1, and 0.01 mg/ml, respectively). Data are presented as mean values ± SD of n = 3 biological replicas. Statistical significance was calculated with two-way ANOVA followed by Sidak’s multiple comparison. Significant p < 0.0001. n.s. non-significant. F Cell extracts from H4 cells, both WT and APP, incubated with ECSITCTER confirmed the phosphorylation of ECSITCTER ex cellulo by a kinase pool. G Quantitative mapping of the ECSITCTER phosphopeptides indicate a decreased phosphorylation level under amyloidogenic conditions, with Thr320 (in green) displaying the highest differential level. Data are presented as normalised values and median of n = 2 biological replicas. Source data are provided as a Source Data file and in Supplementary Data 2 and 3.