Fig. 2.

Interactions between AURKA and INPP5E affect the activity of both proteins. (A) In vitro kinase assay. The upper panel indicates γ-[³²P]ATP signal corresponding to phosphorylated GST–INPP5E (∼110 kDa), auto-phosphorylated (ph)AURKA (∼50 kDa) and phosphorylated histone H3 (phHH3, ∼16 kDa). The lower panel shows input proteins western blotted as indicated. (B) Recombinant active AURKA activates the hydrolysis of PtdIns(3,4,5)P₃ by INPP5E (n = 3 for each sample). Data show the mean±s.e.m.; *P = 0.014 (Student's t-test). (C) Quantification of INPP5E 5-phosphatase activity against PtdIns(3,4,5)P₃ upon AURKA inhibition with the indicated concentrations of C1368 in HEK293 cells overexpressing HA–INPP5E or empty vector. Data show the mean±s.e.m.; *P<0.01 (Student's t-test). Results were normalized to those of the vector-expressing cells to reduce background caused by the presence of other phosphatases in the cells. Non-normalized data are shown in supplementary material Fig. S2D. (D) HEK293 cells were co-transfected with plasmids expressing the indicated proteins. Cell lysates were separated and probed with the antibodies indicated. Blots from three independent experiments were quantified and the relative phosphorylation of AURKA was calculated (lower panel). Data show the mean±s.e.m.; *P = 0.002 (Student's t-test). (E) HEK293 cells were co-transfected with the indicated plasmids. AURKA was immunoprecipitated (IP) and a fraction of the sample was used in an in vitro kinase assay to detect AURKA auto-phosphorylation and AURKA activity towards HH3. A second fraction was used to assess AURKA immunoprecipitation with HA–INPP5E. The kinase dead AURKA mutant K162D was used as a negative control for specificity of AURKA phosphorylation. (F) Graphs indicate the ratio of phosphorylated HH3 to total HH3 (upper panel) and of immunoprecipitated INPP5E to input in cell lysates. Data were quantified from three blots and show the mean±s.e.m.; *P<0.05 (Student's t-test). (G,H) Western blot analysis of lysates (G) and immunoprecipitates (H) from HEK293 cells co-transfected with RFP–AURKA and either wild-type (WT) INPP5E or the indicated INPP5E JBTS mutants. Plasmid V5-LacZ was used as a negative control for immunoprecipitation. (I) Western blot analysis of precipitates and lysates from HEK293 cells co-transfected with RFP–AURKA and the indicated phosphatases. Molecular masses are indicated in kDa.