Fig. 3.

p57^Kip2 enhances DA cell differentiation in vitro and interacts directly with Nurr1. (A) MN9D cells were cotransfected with enhanced GFP (EGFP) expression vector together with expression vectors for Nurrl, p57^Kip2, or both. The number of differentiated EGFP-expressing cells was counted 3 days after transfection as described (22). Average values of quadruplicates are shown. Error bars represent standard deviation. (B) p57^Kip2 interacts with Nurr1. HEK 293 cells were transfected with the empty expression vector, expression vectors encoding Flag-Nurr1, HA-p57^Kip2, or both. Nuclear cell extracts from transfected cells were subjected to immunoblotting with anti-HA or anti-Flag antibodies (Top). Nuclear cell extracts were immunoprecipitated by using anti-Nurr1 (Middle) or anti-p57^Kip2 (Bottom) antibodies, and immune complexes were subjected to immunoblotting by using anti-HA or anti-Flag antibodies. (C) p57^Kip2 interacts with Nurr1 in extracts from the embryonal ventral midbrain. Total cell extracts were prepared from rat E15 ventral midbrain and immunoprecipitated by using anti-IgG control (left lane) or anti-Nurr1 antibodies (center lane). Immune complexes were subjected to immunoblotting by using anti-p57^Kip2 antibodies. Nuclear cell extract from HEK 293 cells transfected with expression vectors encoding p57^Kip2 was used as control (right lane). (D) p57^Kip2 and Nurr1 proteins interact in transfected cells. The interaction between p57^Kip2 and Nurr1 was confirmed by using a mammalian two-hybrid assay. HEK 293 cells were transfected with expression vectors VP16-p57^Kip2 and Gal4DBD-Nurr1 (1–262). These vectors were transfected either alone or as indicated in the figure together with a luciferase reporter gene driven by four UAS Gal4-binding sites. Relative light units (RLU) were computed after normalization to β-galactosidase activities. Gal4DBD-Nurr1 (1–262) activates the reporter gene due to the presence of a transactivation domain within the Nurr1 amino-terminal domain. Activation is strongly enhanced by cotransfection of VP16-p57^Kip2. (E) A gel-mobility shift assay demonstrated that in vitro transcribed and translated HA-p57^Kip2 formed complexes with in vitro translated Nurr1 bound to a ³²P-labeled NBRE DNA probe. The positions of Nurr1 (shift) and Nurr1/p57^Kip2 complexes (super shift) bound to NBREs are indicated. Coincubation with HA antibodies abolished the binding of p57^Kip2 to the Nurr1 bound to NBRE probe. (F) p57^Kip2 inhibits Nurr1 activation of a reporter gene containing Nurr1 DNA-binding sites (NBREs). MN9D cells were transfected with a luciferase NBRE reporter plasmid and expression vectors encoding either Nurr1 and/or p57^Kip2. (G) In a control experiment, MN9D cells were transfected with a luciferase reporter plasmid containing retinoic acid receptor DNA-binding sites (βREs) and expression vectors encoding p57^Kip2. Cells were treated with or without 1 μM all-trans-retinoic acid. RLUs were computed after normalization to β-galactosidase activities.