Fig. 3. ALK directly phosphorylates CDK9 at Tyr19.
a, PARP inhibitor-resistant SKOV3 cells were infected with control short hairpin (sh)RNA (pLKO.1) or shRNAs targeting CDK9 (shCDK9 no. 1/ shCDK9 no. 2). CDK9 expression in stable clones was determined by WB. Data represent two repeats with similar results. b, Cell viability of CDK9-knockdown PARP inhibitor-resistant cells treated with the indicated concentration of talazoparib for 6 d. Error bar represents mean ± s.d. of n = 3 independent experiments, two-tailed, unpaired Student’s t-test. c, Chou–Talalay analysis of CDK9-knockdown (shCDK9 no. 1/shCDK9 no. 2) and control-knockdown (pLKO.1) PARP inhibitor-resistant cells treated with varying concentrations of talazoparib and CDK9 inhibitor for 6 d. The mean percentage of growth inhibition derived from n = 3 independent MTT experiments was used to calculate the CI value. Strong synergism showed as CI < 0.5 at an optimal effect level (Fa > 0.75, region highlighted in orange). The CI values at Fa > 0.75: pLKO.1 < 0.5 < shCDK9 no. 1 < shCDK9 no. 2. d, PARP inhibitor-resistant SKOV3 cells treated with or without 0.5 μM ALK inhibitor (lorlatinib) for 24 h. Detection of p-ALK and CDK9 binding (red dots) was performed by Duo-link assay. Insets: ×2 magnification. Scale bar, 20 µm. Bar diagram shows the percentage of cells with positive interaction calculated from n = 3 independent experiments. Error bar represents mean ± s.d. ***P = 0.00003, two-tailed, unpaired Student’s t-test. e, Left: schematics of CDK9 and different ALK-expressing plasmids. Right: WB of FLAG-tagged CDK9 in cells coexpressing WT, constitutively active (Phe1174Leu) or kinase-dead (Ile1250Thr) ALK with FLAG-tagged CDK9 after IP with the indicated antibodies. Data represent two repeats with similar results. f, WB of tyrosine phosphorylation (p-Tyr) signal and CDK9 in an in vitro kinase assay of purified ALK incubated with WT or Tyr/Phe-mutant (phenylalanine mutation of Tyr19/Tyr92/Tyr185/Tyr282) CDK9 protein. Data represent two repeats with similar results. g,h, Cells expressing exogenous WT, Tyr19Phe, Tyr92Phe, Tyr185Phe or Tyr282Phe CDK9 with or without constitutively active ALK. The p-Tyr signal and FLAG-tagged CDK9 were examined by WB after IP with FLAG antibody (g). The p-Tyr19-CDK9 expression of FLAG-tagged CDK9 was examined by WB (h). i, WB of indicated proteins in cells treated with or without 0.5 μM ALK inhibitor (lorlatinib) for 24 h. j, WB of in vitro kinase assay in which purified GST–CDK9 was incubated with constitutively active (Phe1174Leu) or kinase-dead ALK (Ile1250Thr) proteins. Data represent two repeats with similar results.