Fig. 3
From: The crescent-like Golgi ribbon is shaped by the Ajuba/PRMT5/Aurora-A complex-modified HURP
PRMT5-induced HURP methylation is required for Aurora-A-catalyzed HURP phosphorylation. A HURP p725 signal was barely detected on 122K mutant in cells. 293T cells transfected with EGFP-HURP WT or R122K were analyzed for the signal intensity of HURP p725 at the migration position on SDS-PAGE of EGFP-HURP. B HURP p725 signal was only detected on immunoprecipitated HURP m122. HURP m122 and HURP nm122, i.e. the HURP without methylation at R122, were immunoprecipitated in HeLa cells. Western blots were then followed to examine HURP p725 signal on the two precipitated HURPs. The HURP p725 signal was firstly normalized against HURP m122 or nm122; subsequently, the relative level of [nm122/m122]p725 was calculated and plotted. C No significant difference of HURP m122 signal on HURP WT, 725A and 725E. HeLa cells transfected with EGFP or various versions of EGFP-HURP were examined for the signal of HURP m122 at the EGFP-HURP position. The m122 signal was firstly normalized against EGFP-HURP; subsequently, the ratio of [m122/EGFP-HURP]725A or [m122/EGFP-HURP]725E to [m122/EGFP-HURP]WT was calculated and plotted. D Knockdown of Ajuba decreased the level of HURP m122, HURP p725, PRMT5 p103 and Aurora-A m304. HeLa cells harboring shLuc or shAjuba were surveyed for the level of HURP m122, HURP p725, HURP, PRMT5 p103, PRMT5, Aurora-A m304, Aurora-A. The intensity of each protein was normalized against that of tubulin, and [shAjuba/shLuc]Ajuba, [shAjuba/shLuc]HURP m122, [shAjuba/shLuc]HURP p725 and [shAjuba/shLuc]HURP, [shAjuba/shLuc]PRMT5 p103, [shAjuba/shLuc]PRMT5, [shAjuba/shLuc]Aurora-A m304, and [shAjuba/shLuc]Aurora-A were calculated and plotted. E The model for the component interaction in the Ajuba/PRMT5/Aurora-A/HURP complex. Ajuba keeps the two protein-modifying enzymes together, and PRMT5 and Aurora-A modify and activate each other. Subsequently, HURP is methylated by PRMT5 first, followed by Aurora-A (AurA)-mediated phosphorylation. Three independent experiments were performed for all the results listed above. * stands for statistical significance by Student’s t-test with p < 0.05