Basal SUMO1 conjugation is required for inducible phosphorylation of p100. (A) HCT-116 cells were transfected with wild-type (WT) p100 and S4 and P2 mutants, with or without NIK. Phosphorylation of p100 was detected by immunoblotting (IB) with anti-phospho-p100-S866/870. Expression of HA-NIK was verified by immunoblotting with anti-HA. (B) In vitro translation and subsequent in vitro SUMOylation assay were performed as described in the Methods. HA, haemagglutinin; HCT-116 cells, human colon tumour 116 cells; NIK, NF-κB-inducing kinase; NS, non-specific; SUMO1, small ubiquitin-like modifier 1.

In vitro and in vivo SUMO1 modification of p100. (A) SUMO1-conjugated p100 was detected by immunoprecipitation (IP) with anti-p100 N (recognizing both p100 and p52) and subsequent immunoblotting (IB) with anti-SUMO1. (B) HCT-116 cells were transfected with siRNA UBC9 oligonucleotides and control oligonucleotides. At 72 h after transfection, the cells were collected for IP and IB. (C) In vitro SUMOylation assay was performed by using in vitro-translated c-Myc-p100 and p100 mutants with single mutated SUMO site. SUMO1-conjugated p100 and non-conjugated p100 were detected by IB with anti-c-Myc. (D) HCT-116 cells were transfected with p100 or p100 SUMO mutants along with UBC9 and SUMO1 expression plasmids. S3, p100-KKK90/298/689RRR; S3-E864Q, p100-KKK90/298/689RRR-E864Q; S4, p100-KKKK90/298/689/863RRRR. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HA, haemagglutinin; HCT-116, human colon tumour 116 cells; HEK 293, human embryonic kidney 293 cells; MEFs, murine embryonic fibroblast cells; NEM, N-ethylmaleimide; NIK, NF-κB-inducing kinase; NS, non-specific; siRNA, short interfering RNA; SUMO1, small ubiquitin-like modifier 1; UBC9, ubiquitin-conjugating enzyme 9; VC, vector control; WT, wild type.

SUMO1 modification is required for the induced p100 processing. (A) HCT-116 cells were transfected with wild-type (WT) p100 or p100 SUMO mutants along with empty vector or NIK. The K5 mutant stands for p100-KKKKK153/179/182/856/863RRRRR. (B) Left panel: the WT MEFs were transfected with WT p100 and S4 mutant, followed by treatment with recombinant LT-α1β2 proteins (1 μg/ml) for 16 h. Middle panel: TRAF-deficient MEFs were transfected with WT p100 and S4 mutant, with or without NIK. Right panel: HEK 293 cells were transfected with WT p100 and S4 mutant, followed by transfection with human CD40 expression vector. At 24 h after second transfection, the transfected cells were treated with anti-CD40 (10 μg/ml) for 5 h. Expression of CD40 was confirmed with immunoblotting with anti-CD40. (C) HEK 293 cells were transfected first with siRNA UBC9 and control oligonucleotides followed by transfection with p100 (WT and S4), and CD40 expression vector 24 h later. At 72 h after first transfection, the cells were stimulated with anti-CD40 (10 μg/ml) for a further 5 h. GAPDH, glyceraldehyd-3-phosphate dehydrogenase; HA, haemagglutinin; HCT-116 cells, human colon tumour 116 cells; HEK 293 cells, human embryonic kidney cells; IB, immunoblotting; MEF, murine embryonic fibroblast; NIK, NF-κB-inducing kinase; NS, non-specific; siRNA, short interfering RNA; SUMO1, small ubiquitin-like modifier 1; TRAF, tumour necrosis factor receptor-associated factor.

SUMO modification of p100 is required for activation of the alternative NF-κB pathway. (A) The HCT-116 cells were transfected with c-Myc-tagged wild-type (WT) p100 and S3 and S4 mutants along with HA-β-TrCP, with or without NIK. The interaction of p100 and β-TrCP was tested by immunoprecipitation (IP) with anti-p100 N and immunoblotting (IB) with anti-HA. (B) The HCT-116 cells were transfected with WT p100 and the S4 mutant along with NIK. Polyubiquitination of the p100 complex was examined by IP with anti-p100 N and IB with an antibody against ubiquitin (Ub). (C) Downregulation of UBC9 inhibits CD40-induced nuclear translocation of p52/RelB. HEK 293 cells were treated as described in Fig 2C. The CE and NE fractions were used for the IP with anti-c-Myc and subsequent IB with RelA, RelB and p100N. (D) The p100^−/− NIH 3T3 stable cell lines reconstituted with WT p100 or the S4 mutant were transfected with CD40 expression plasmid. At 48 h after transfection, the cells were subjected to anti-CD40 stimulation. The total messenger RNAs (mRNAs) and the whole-cell lysate were used for evaluating SKP2 mRNA and SKP2 protein level, respectively. To minimize the interference resulting from unequal mRNA input, the probes for β-actin and the probes for SKP2 were added to the same reverse transcription–PCR reaction tube. CE, cytoplasmic extract; HA, haemagglutinin; HC, heavy chain; HCT-116 cells, human colon tumour 116 cells; HEK 293 cells, human embryonic kidney cells; NE, nuclear extract; NF-κB, nuclear factor-κB; NIK, NF-κB-inducing kinase; NS, non-specific; siRNA, short interfering RNA; SKP2, S-phase kinase-associated protein 2; SUMO, small ubiquitin-like modifier; TrCP, transducin repeat containing protein; UBC9, ubiquitin-conjugating enzyme 9.