AKT phosphorylates Ser³⁴ and Thr¹²⁷ of CLK2. A, diagram of CLK2 with putative AKT phosphorylation sites. Amino acids 29–35, RRRSRSW, 31–37, RSRSWSS, and 122–128, RRRSRTF, that correspond to the AKT consensus phosphorylation motifs are labeled with arrows. B, the CLK2 phosphorylation sites targeted by active AKT. Wild-type (WT) CLK2 and S34A, S36A, and T127A mutants were transfected into HeLa cells. AKT kinase assays were performed with immunoprecipitated (IP) WT-CLK2 or mutants as the substrates. The WT-CLK2 and S36A mutant were phosphorylated by active AKT, but S34A and T127A mutants were not. C, the sites of CLK2 phosphorylation induced by insulin. Insulin-induced CLK2 phosphorylation in both WT-CLK2 and S36A, but not in S34A and T127A mutants. D, ionizing radiation phosphorylated Ser³⁴ and Thr¹²⁷ of CLK2. IB, immunoblot.

The phosphorylation of CLK2 is induced by ionizing radiation and insulin. A, the CCD-18Lu cells were irradiated with 0.05 or 2 Gy of γ-rays and incubated for the indicated time periods. The phosphorylations of CLK2 were detected using immunoprecipitation (IP) with anti-CLK2 antibody followed by Western blotting with anti-phospho-Ser/Thr antibody. β-Actin was used as a loading control. B, effects of PI3K/AKT inhibition on 0.05 Gy or insulin-induced CLK2 phosphorylation. CCD-18Lu cells were pretreated with LY294002 (50 μm) or wortmannin (200 nm) for 1 h and then exposed to 0.05 Gy or insulin treatment (10 μg/ml). CLK2 phosphorylation was detected using immunoprecipitation with anti-CLK2 antibody followed by Western blotting with anti-phospho-Ser/Thr antibody. C, effects of PI3K/AKT inhibition on 2 Gy or insulin-induced CLK2 phosphorylation. IB, immunoblot.

CLK2 phosphorylation in AKT knockdown CCD-18Lu cells. siNon-Target (Mock) or siRNA specific for AKT (siAKT) directed to the target sequence of human AKT1 cDNAs were transfected into CCD-18Lu cells. After 24 h, the cells were exposed to 2 Gy of radiation or insulin treatment (10 μg/ml) and lysed at 4 h after irradiation or 15 min after insulin treatment. CLK2 phosphorylation was detected using immunoprecipitation (IP) with anti-CLK2 antibody followed by Western blotting with anti-phospho-Ser/Thr antibody. Western blot was performed with indicated antibodies. IB, immunoblot.

AKT binds to and phosphorylates CLK2. A, CLK2 co-precipitates with GST-AKT, in vitro. ³⁵S-Labeled CLK2 (10 μl) produced from a CLK2 cDNA construct was used in an in vitro binding assay with GST-AKT. ³⁵S-Labeled p27 was used as a positive control for the in vitro binding assay. B, CLK2 binds to endogenous AKT. HeLa cells were transfected with control vector or myc-CLK2. AKT was immunoprecipitated followed by Western blotting with anti-Myc antibody. C, CLK2 was phosphorylated by recombinant active AKT protein. In vitro AKT kinase assays were performed with immunoprecipitated (IP) CLK2 with anti-CLK2 antibody from HeLa cells overexpressing Myc-CLK2. Western blotting was performed with anti-phospho-Ser/Thr or anti-Myc antibodies. GSK-3 fusion protein was used as a positive control for the in vitro AKT kinase assays. D, endogenous CLK2 binds to endogenous AKT. HeLa cells were transfected with siNon-Target (Mock) or siRNA specific for CLK2 (siCLK2). After 24 h of transfection, AKT was immunoprecipitated followed by Western blotting with anti-CLK2 antibody. E, active AKT phosphorylates CLK2. HeLa cells were transiently transfected with vector, WT-AKT1, or Myr-AKT1. After 24 h of transfection, the phosphorylation of CLK2 was detected using immunoprecipitation with anti-CLK2 antibody followed by Western blotting with anti-phospho-Ser/Thr antibody. IB, immunoblot.

The regulation of the sensitivity of CCD-18Lu cells to ionizing radiation is dependent on CLK2 phosphorylation. A, CCD-18Lu cells were transfected with vacant vector, WT-CLK2, or CLK2 mutants and the overexpressions of WT-CLK2 and CLK2 mutants were detected. B, the viabilities of vector, WT-CLK2, or mutant-CLK2 overexpressing cells were measured using MTT assays 48 h after 2 Gy of γ-ray. C, effect of CLK2 phosphorylation on 2 Gy-induced apoptosis. DNA fragmentation was detected with HT TiterTACS Assay Kit for the quantification of apoptosis. The absorbance (450 nm) of untreated vector control was 0.34. D, the viabilities of vacant vector, WT-CLK2, or mutant-CLK2 overexpressing cells after exposure to 0.05 Gy of γ-ray were measured by MTT assay. E, the cell proliferation after irradiation of 0.05 Gy in vacant vector, WT-CLK2, or mutant-CLK2 CCD-18Lu cells. Data represent mean ± S.D. (n = 3) and were analyzed by Dunnett's test for multiple comparison in analysis of variance to irradiated vector control (*, p < 0.05).

The effects of CLK2 on radiation sensitivity of CCD-18Lu cells. Cells were transfected with either vacant vector or myc-CLK2. At 24 h post-transfection, cells were exposed to 0.05–2 Gy of γ-rays and cell viability was assayed 48 h later. A, the overexpression of Myc-CLK2 at 72 h post-transfection. B, the cell viability of CLK2 overexpressing CCD-18Lu cells to ionizing radiation was determined by MTT assay. C, cell proliferation of CLK2 overexpressing cells after irradiation. D, effect of CLK2 overexpression on 2 Gy-induced apoptosis. DNA fragmentation was detected with HT TiterTACS Assay Kit for the quantification of apoptosis. B–D, data represent mean ± S.D. (n = 3) and were analyzed by the t test. Data showed a significant difference compared with vector control at the indicated dose (*, p < 0.01; **, p < 0.001). E, cells were transfected with siNon-Target, siCLK2, or siGAPDH and then Western blot was performed 72 h later. F, cells were exposed to 0.05–2 Gy of γ-rays at 24 h post-transfection. The radiation sensitivity in CLK2 knockdown cells was detected by MTT assay at 48 h after irradiation. G, the cell proliferation after irradiation in CLK2 knockdown cells. H, effect of CLK2 knockdown on 2 Gy-induced apoptosis. The apoptosis was assessed as described in panel D. F–H, data represent mean ± S.D. (n = 3) and were analyzed by Tukey's test for multiple comparison in analysis of variance at the indicated dose (*, p < 0.05). UN, untreated control.