Fig. 6: PI3Kα signalling induces CEP170 phosphorylation and PKCι activation.

a, b Phosphoproteomic analysis of hTERT-RPE1 cells serum-starved for 24 h and stimulated with 1938 or insulin ± BYL719 for 15 min (p-value calculated using the group comparison function within MSstats and adjusted using the Benjamini-Hochberg procedure, n = 5 independent experiments). a Volcano plot of phosphosites differentially regulated by 1938 or insulin ± BYL719 relative to DMSO-treated cells. Numbers in the top corners indicate numbers of phosphosites significantly up- or down-regulated relative to DMSO b Log2(FC) of pCEP170(S466), bars: mean ± SD, *p < 0.05 (1938 vs DMSO p = 0.01438, insulin vs DMSO p = 0.04240). c Sequence alignment of human and mouse CEP170(S466/S463). HEK293 cells were transfected with HA-CEP170 or HA-CEP170(S466D) plasmids, serum-starved for 48 h, fixed and stained with HA and (d) pericentrin or (e) ARL13B antibodies and DAPI, bar: 10 μm, arrows indicate centrosomes, arrowheads: cilia axonemes. d Representative of n = 3 independent experiments. e The % of ciliated transfected cells was scored, bars: mean ± SD, ≥21 transfected cells scored/condition for n = 3 independent experiments, *p < 0.05 (two-sided Student’s t-test, p = 0.0221). f, g KSEA (OmniPath) of kinases for which the substrate groups are differentially regulated by 15 min or 4 h 1938 or Insulin stimulation ± BYL719 in 24 h serum-starved hTERT-RPE1 cells relative to DMSO. As few phosphosites were altered by 1938 or insulin treatment, kinases for which the raw p-values relative to DMSO (using the Kolmogorov–Smirnov test) relative to DMSO control were p < 0.05 were considered significantly regulated. f Heatmap. g Venn diagram showing overlap of kinases differentially regulated in KSEA (OmniPath vs Edges vs PhosphoSitePlus database). h In vitro kinase assay for purified recombinant PKCι kinase domain (phosphorylated at activation loop and turn motif priming sites) with β-PSS positive control (pseudosubstrate sequence of PKCβ, with alanine mutated to phosphoacceptor serine, containing the PKCι recognition motif; Phenylalanine at -5 and Arginine at -3 with respect to the phospho-acceptor) and CEP170(S466) peptides, bars represent mean ± SD, n = 3 technical replicates, data representative of 2 independent experiments. Source data are provided as a Source Data file.