Fig. 3

PI3KC2α regulates tubulin autoregulation by sequestering TTC5. A Schematic images of TTC5-mediated tubulin autoregulation. B HeLa cells stably expressing GFP-α-tubulin were transfected with PI3KC2α siRNA for 24, 48, or 72 h. Total lysates were analyzed by immunoblotting analysis. C Physical interactions of α-tubulin with TTC5 were measured using co-IP experiments after transfecting the cells with PI3KC2α siRNA. D and E Interaction between PI3KC2α and TTC5 recognized by a PLA probe. After ligation and amplification, PLA signals (green dots) were counted. The nuclei were stained with DAPI (blue). Data are shown as mean ± standard deviation (n = 15). F and G CARM1 WT or KO MEFs were transfected with the myc-PI3KC2α vector. The cell lysates were immunoprecipitated with an anti-TTC5 antibody. The input and immunoprecipitates were immunoblotted to detect the indicated proteins (F). Mature Tuba1a and Pik3c2a mRNA levels in each group were measured (G). H to J Confocal images (H and I) and immunoblot analysis (J) showing α-tubulin (red) levels in CARM1 KO MEF cells transfected with WT, R175K-, or R175A-mutant of myc-PI3KC2α (green). K Schematic representation of CARM1–PI3KC2α axis in tubulin autoregulation pathway. Unmethylated PI3KC2α stabilizes free tubulin and disrupts tubulin autoregulation by sequestering TTC5. When PI3KC2α becomes unstable and degrades, TTC5 is released and triggers tubulin autoregulation