Fig 1.

Arkadia interacts noncovalently with SUMO through three SIMs. (A) Arkadia contains three SIMs in the region from amino acids 280 to 400. SIMs are localized at position 300 (SIM1, VVVI), position 326 (SIM2, VEIV), and position 382 (SIM3, VVDL) of the human protein. The RING domain is localized in the C-terminal region. Sequence alignment of the region from amino acids 280 to 400 of Arkadia orthologues from vertebrate species indicates that the three SIMs are highly conserved throughout evolution. Asterisks indicate conserved amino acids. The different Arkadia mutants generated are represented in the lower panel, where the mutated motifs are indicated in black. (B and C) Arkadia interacts with SUMO through its three SIMs. HEK293 cells were transfected with the indicated Flag-Ark constructs, and equal amounts of whole-cell lysates were subjected to GST pulldown experiments with immobilized GST, SUMO1-GST, or SUMO3-GST. The Arkadia protein was detected by Western blotting (WB) with anti-Flag antibody before (5%) and after pulldown of the lysate. Blots were stained with Ponceau S prior to immunostaining in order to control the amount of GST proteins in the experiment. (D) The three SIMs of Arkadia are required for interaction with SUMO2 modified proteins in vivo. HEK293 cells were transfected with empty vector, Flag-Ark-wt, Flag-Ark-SIM123* mutated in the SIM, or Flag-Ark-RING* mutated in the RING. Flag immunoprecipates from whole-cell extracts were analyzed by Western blotting using anti-Flag and anti-SUMO2/3 antibodies. The corresponding lysates (input) were analyzed using anti-SUMO2/3 antibody. IP, immunoprecipitation. (E) Arkadia and SUMO2 interact at an endogenous level. Whole-cell extracts of HeLa cells were immunoprecipitated with anti-rabbit IgG (control) or anti-SUMO2/3 and analyzed by Western blotting along with input (10% whole-cell lysate) using anti-SUMO2/3 and anti-Ark antibodies. Numbers to the left of the gels are molecular masses (in kDa).