Figure 4: CSN6 enhances neddylation of Cullin-1 via its MPN domain.

(a) Neddylation status of Cullin in gel-filtration chromatography fractions from spleen extracts from Csn6^+/+ (wt) and Csn6^+/− mice. Extracts of spleen from CSN6^+/+ or Csn6^+/− mice (4 weeks old) were ground and subjected to lysis. Lysates were fractionated by gel-filtration chromatography. Fractions were resolved by SDS-PAGE, followed by immunoblotting with indicated antibodies. Molecular size of eluted fraction is indicated above. (b) Non-neddylated Cullin was increased in Csn6^+/− mice. Extracts of spleen from CSN6^+/+ (wt) or Csn6^+/− mice were fractionated by gel filtration chromatography. Representative fractions 36–54 from (a) were resolved by SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with anti-Cullin-1, anti-CSN6 and anti-CSN5 antibodies. (c) CSN6 competed with CSN5 for binding to Cullin-1 and affected Cullin-1 neddylation. 293T cells were transfected with His-Cullin-1 plus increasing amounts of GFP-CSN6 or Myc-CSN5. The neddylated Cullin-1 proteins were pulled down using Ni-NTA-agarose beads and detected with anti-Nedd8 antibody. The lysates were also immunoprecipitated with CSN6 antibody or CSN5 antibody and were subjected to immunoblotting with anti-Cullin-1. (d) MPN domain of CSN6 had an important role in competing with CSN5 for Cullin-1 binding and affected Cullin-1 neddylation. 293T cells were cotransfected with indicated plasmids. The lysates were immunoprecipitated with anti-Flag or anti-CSN5 antibody and immunoblotted with the indicated antibodies. (e) Mutation of conserved residues in the MPN domain of CSN6 compromised CSN6’s capacity for competing with CSN5. 293T cells were cotransfected with indicated CSN6 MPN mutant plasmids. The lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies. (f) CSN6 antagonized CSN5 in regulating Cullin-1 neddylation. U2OS cells were infected with the indicated lentivirus carrying CSN6 shRNA, CSN5 shRNA or luciferase control shRNA. Cell lysates were immunoblotted with the indicated antibodies. (g) The chimeric protein 6N5C expressing the CSN6 MPN domain functioned like wild-type CSN6 in increasing Cullin-1 neddylation. 293T cells were cotransfected with indicated wild-type- and chimeric protein-expressing plasmids. The lysates were pulled down with Ni-NTA-agarose beads and immunoblotted with anti-Nedd8. (h) Both chimeric protein 6N5C and wild-type CSN6 decreased the stability of Fbxw7α in a dose-dependent manner. 293T cells were cotransfected with indicated plasmids. The lysates were immunoblotted with indicated antibodies.