Fig 1: HO-1/CO triggers K142 polyubiquitination of PFKFB3.(a) Demethylation of PFKFB3 in U937 cells treated with haemin for 6 h. (b) Alterations in arginine methylation status of PFKFB3 in U937 cells stably transfected with WT and mutant (H25A) HO-1 that lacks in catalytic activity. Note that transfection with WT but not with H25A mutant HO-1 causes PFKFB3 demethylation. Lysates were analysed by western blotting with anti-dimethyl-PFKFB3, anti-PFKFB3 and anti-HO-1 antibodies. (c) Effects of CrMP, an HO inhibitor of methylation of PFKFB3, indicating that the catalytic activity of HO is necessary for PFKFB3 demethylation. (d) Temporal alterations in methylation of PFKFB3 in U937 cells transfected with siRNA (control or siCBS) exposed to 5 μM CO. Lysates were analysed by western blotting using anti-dimethyl-PFKFB3, anti-PFKFB3 and anti-CBS antibodies. In the right panels, note suppression of basal me-PFKFB3 signals and disappearance of the CO-induced signal inhibition in siCBS-transfected cells. (e) Effects of Cdh1-silencing in HeLa cells on R131/134 methylation of PFKFB3. Note that knockdown of Cdh1 increases methylated PFKFB3. (f) Role of HO-1 catalytic activities in the regulation of demethylation and polyubiquitination of PFKFB3. PFKFB3 endogenous polyubiquitination ((Ub)n-PFKFB3) was observed by WT, but not by H25A mutant HO-1 transfection. (g) Effects of PRMT1 knockdown on PFKFB3 polyubiquitination. U937 cells were transfected with siRNA against control (scramble) or against PRMT1. Lysates were immunoprecipitated with anti-polyubiquitin antibody. The eluates are analysed with anti-PFKFB3 antibody. Lysates for input were immunoblotted with anti-dimethyl-PFKFB3 (R131/134), anti-PFKFB3 and anti-PRMT1. The expression level of GAPDH is used as a loading control. (h) Roles of K142 in HO-1-triggered PFKFB3 polyubiquitination. (i) Roles of K142 in PRMT1-sensitive PFKFB3 polyubiquitination. Note that K142A mutant transfectants are insensitive to HO-1 induction or to PRMT1 overexpression. Lysates were immunoprecipitated with anti-FLAG antibody. The eluates are analysed with anti-ubiquitin antibody. The expression level of GAPDH is used as a loading control. (j) Effects of MG132, a proteasomal inhibitor, on arginine dimethylation of PFKFB3. MG132 (10 μM) was treated for 6 h. All data are representative of at least two separate experiments. Asterisks in (a,c,d,h) indicate non-specific bands resulting from the polyclonal antibody.