PIASγ requires the ₂₀LXXLL₂₄ motif to interact, SUMOylate and repress Nurr1.

<p>(A) Schematic representation of full-length PIASγ indicating functional domains SAP: <i>Scaffold Attachment factor-A/B acinus and PIAS</i>; the PINIT domain; SP<i>-</i>(<i>Siz/PIAS</i>)<i>-</i>RING and SIM: <i>SUMO Interaction Motif</i>. PIASγ has two LXXLL motifs. Mutations changing leucines (L) by alanines (A) in each LXXLL domain are depicted for GST-PIASγmut1 and GST-PIASγmut2. (B) GST, GST-PIASγ, GST-PIASγmut1 and GST-PIASγmut2 retained in glutathione-agarose beads were incubated with extracts from COS-1 cells transfected with Myc-Nurr1_363–598. Retained proteins were fractionated in SDS-PAGE and western blot developed with anti-Myc monoclonal antibody, revealing that only GST-PIASγ and GST-PIASγmut2 are able to interact with Nurr1. Recombinant GST-PIASγ proteins were equally loaded in each GST-pull down assays as shown by western blot using anti-PIASγ antibody (bottom). (C) Luciferase reporter assay showing that full-length PIASγmut1 lose repressor capacity over Nurr1 transactivity. HEK293 cells were transfected with the NBRE-3X-tk-LUC along with HA-Nurr1, PIASγ or PIASγmut1. After 48 hours, cells extracts were assayed for luciferase activity. Data are expressed as percentage of Nurr1 transactivation and correspond to the mean ± S.E.M of 4 independent experiments each performed in triplicates. Statistical significance was estimated by the non-parametric Mann-Whitney U test. *p<0.05 (Nurr1+PIASγ v/s Nurr1+PIASγmut1). In the bottom, western blots showing the expression of recombinant proteins and actin used as loading control. (D) Total extracts of COS-7 cells transfected with HA-Nurr1, SUMO-2 and PIASγ or PIASγmut1 were fractionated in SDS-PAGE and western blots performed with anti-HA, anti-PIASγ and actin (load control) antibodies.