Fig. 2

Aurora-A phosphorylates PRMT5 at S103. A PRMT5 served as an in vitro substrate of Aurora-A. To perform an in vitro kinase reaction, immunoprecipitated PRMT5 or recombinant MBP was incubated with recombinant His-Aurora-A in kinase reaction buffer containing P³²-ATP. The resulting samples were applied to autoradiography or Western blots to examine the phosphorylation effect or the immunoprecipitation result. Pptase indicates alkaline phosphatase. Note, Aurora-A underwent autophosphorylation during the in vitro kinase reaction. B Aurora-A could not phosphorylate PRMT5 S103A. Immunoprecipitated EGFP-PRMT5 WT or S103A was incubated with recombinant His-Aurora-A for in vitro kinase reactions. Autoradiography or Western blots was conducted to examine the phosphorylation effect or the immunoprecipitation result. C Knockdown of Aurora-A decreased PRMT5 p103 in amount. HeLa cells harboring shLuc or shAurora-A were analyzed for the level of PRMT5 p103 and PRMT5 by Western blots. D Overexpression of Aurora-A increased the level of PRMT5 p103. 293 cells transfected with EGFP or EGFP-Aurora-A were surveyed for the level of PRMT5 p103 and PRMT5 by Western blots. E PRMT5 S103A could not methylate HURP. 293T cells were infected with shLuc or shPRMT5 first, and the shPRMT5 cells were further transfected with EGFP, EGFP-RPMT5 WT or S103A. These cells were then surveyed for the level of HURP m122 by Western blots. Three independent experiments were performed for all the results listed above