Figure 6.

CDK12 phosphorylates PAK2 at threonine 134/169 and activates MAPK signaling pathway. A. Kinase assay of recombinant GST-tagged CDK12 with PAK2 in vitro. CDK12 (200 ng) and inactive PAK2 (400 ng) were mixed with ATP in kinase buffer at 30 ℃ for 30 min. Phospho-Threonine and GST were detected by western blots. B. Kinase assay of mutated PAK2 proteins (WT, T134A, T169A, 2A-T134A/T169A) with CDK12. Phospho-Threonine and GST were detected by western blots. C. Protein level of PAK2 was measured by western blotting after GFP-tagged lentiviral transfection of different mutation type (Vector, WT, 2A) of PAK2 in HGC27 cells. D. Cell viability of different mutation type (Vector, WT, 2A) PAK2 transfected cells was measured by MTT assay (72 h) and representative fluorescence of GFP are shown. E. Cell proliferation of different mutation type (Vector, WT, 2A) PAK2 transfected cells was determined by crystal violet foci formation assay and representative images are shown. F. Co-localization of CDK12 (orange), PAK2 (Vector, WT, 2A, red) and GFP (green) were measured by immunofluorescence after stable overexpression of PAK2 in HGC27. CDK12 and PAK2-WT are evenly distributed throughout the nucleus (DAPI: blue) and cytoplasm, while PAK2-V and PAK2-2A with CDK12 were mainly in nucleus. Representative images are shown. G. MAPK signaling pathway analysis by western blotting. Phospho-MEK and Phospho-ERK was measured by western blots in HEK293T cells with empty vector, PAK2-WT, or PAK2-2A transfected. Representative results are shown. H. Longitudinal tumor volume, tumor weight, final tumor size and percentage of mice with tumors less than 1000 mm³ at the endpoint were measured from empty vector, CDK12-WT, and CDK12-2A-overexpressed tumors of HGC27 in a xenograft model. Tumor size was measured every 5-7 days, the volume was calculated by V = (length) × (width) × (height). Tumors in each group are shown. Data represent means ±SD. P<0.05: *, p<0.01: **, p<0.001: ***. Significance determined by two-tailed Student's t test.