Figure 5

Figure 5 CDKL1 variants interfere with kinase function.

(A and B) In vitro kinase assays. HeLa (A) or HEK293T (B) cells were transiently transfected with CDKL1 and IME2 constructs or empty vectors (HA^control and EGFP^control). Fusion proteins were immunoprecipitated and subjected to kinase assays using peptide substrate RPRSPGARR and the ADP-Glo kinase assay (Promega). Luminescence intensities of individual measuring points were normalized to the entire luminescence signal of an experiment and to the amount of immunoprecipitated protein (n = 7, A; n = 4, B). Medians (50th percentiles, lines in boxes) as well as 0th, 25th, 75th, and 100th percentiles are given in the box plots. Small aliquots were removed from precipitates and subjected to immunoblotting using anti-HA (A) and anti-GFP (B) antibodies; representative blots are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, 1-way ANOVA with Dunnett’s post hoc multiple-comparison test. (C) CDKL1 serine/threonine phosphorylation profiling. EGFP-tagged CDKL1 variants were expressed in HEK293T cells, purified using GFP-Trap (ChromoTek), and applied to STK-PamChip arrays (PamGene). Seventy-one peptides passed quality control. The heatmap displays average log₂-transformed signal intensities for indicated CDKL1 variants. The signals were sorted from high (red) to low (blue) intensity that corresponds to phosphorylation levels. To visualize overall sample variance and group differences, peptide phosphorylation and overall kinase activity are shown in box plot representation. (D) Peptide substrates with significantly changed phosphorylation. The heatmap shows significantly differentially phosphorylated peptides between samples treated with disease-associated CDKL1 variants versus CDKL1^WT. The effects of CDKL1 variants are log ratios [log₂(disease-associated CDKL1 variant)/log₂(CDKL1^WT)]. Blue and red indicate reduced and increased phosphorylation, respectively. Peptides that did not pass the significance threshold (P < 0.05, disease-associated CDKL1 variant vs. CDKL1^WT) are black. Peptide numbers correspond to those in Supplemental Table 2, where details on the substrates are also described.