Phosphorylation-dependent sumoylation of the PDSM. (A) Cos-7 cells were transfected with WT HSF4b, S299A, or empty plasmid (M), labeled with 32P, and exposed to heat shock (HS; 1 h at 42°C) or left untreated (C). HSF4 was immunoprecipitated, and phosphorylation was detected by autoradiography. Quantification was made by phosphorimager (Fuji) from three independent experiments. Equal loading is shown by Western blotting with anti-HSF4 antibodies. (B) Phosphoamino acid analysis of the 32P-labeled HSF4b shows phosphorylation on serine residues in nonstressed (C) and heat-shocked (HS) cells. (C) Two-dimensional phosphopeptide maps of WT HSF4b and S299A from untreated (C) and heat-shocked (HS; 1 h at 42°C) samples. Arrows indicate phosphopeptides missing from the maps of HSF4b S299A. (D and E) Cos-7 cells were transfected with empty plasmid (mock), WT HSF4b, K294R, or S299A, together with GFP-SUMO-1 or His-SUMO-2 as indicated. Cells were exposed to heat shock (HS; 15 min at 42°C) or left untreated (C). HSF4b sumoylation was detected by Western blotting with anti-HSF4 antibodies. Hsc70 is shown for equal loading. (F) Phosphopeptide maps of 32P-labeled GATA-1 and MEF2A WT and S→ A mutants were made as in C. Arrows indicate phosphopeptides missing from the maps of the S→ A mutants. (G and H) Cos-7 cells were transfected with GATA-1-MycHis (WT, S142A, or S142D) or MEF2A-MycHis (WT, S408A, or S408D), together with GFP-SUMO-1 as indicated. Cells were lysed in Laemmli sample buffer. Sumoylated species were detected by Western blotting with anti-Myc antibodies. Hsc70 shows equal loading. (I) In vitro sumoylation of GATA-1. Recombinant SUMO-1, SAE1/2, Ubc9, and PIASy were incubated with 35S-labeled reticulocyte-lysate translated GATA-1 (WT or S142D) at 30°C for 2 h. After SDS/PAGE separation, sumoylation of GATA-1 was detected by fluorography.