Fig 2: Schematic model of nuclear import regulation by the TCR-PKC-?-RanGAP1 axis.Upon TCR stimulation, PKC-? phosphorylates RanGAP1 to increase its association with Ubc9, thereby enhancing the sumoylation of RanGAP1, which is required for assembly of the RanBP2/RanGAP1-SUMO1/Ubc9 subcomplex. This complex then promotes the nuclear import of NF-ATc1, NF-?B, and AP-1.

Fig 3: N-terminal of importin ß1 interacts with RanBP2. (a) Scheme of full-length Imp-ß1 and four mutants with 6 × His tags. Numbers on the right refer to amino acids (left). An illustration of an interaction model between importin ß1 and RanBP2 (right). (b) In vitro expressed and purified 6 × His tagged Imp-ß1 fragments were mixed with mitotic HeLa lysates and pulled-down with Ni-NTA beads. Pulled-down lysates were analyzed by IB with anti-RanBP2 or anti-6 × His antibodies. Asterisks indicate the 6 × His fusion proteins. Numbers indicate molecular mass markers in kilodaltons. In lanes marked ‘Input,' 20 µl of the 500 µl of lysate used per pull-down were analyzed directly. (c) Quantification (relative percentage) of mitotic chromosomal defect phenotypes in FLAG-net-vector, FLAG-Imp-ß1-FL, FLAG-Imp-ß1-N1 and FLAG-Imp-ß1-N2 transfected cells. Values are based on three independent experiments, counting 100 mitotic cells in each experiment. Mean values±S.D. (error bars) are shown. All cells were treated with double thymidine block, stained with DAPI (4',6-diamidino-2-phenylindole) and visualized by confocal microscopy

Fig 4: Ran binding is important for the interaction between the N-terminal of RanBP2 and importin ß1. (a) Scheme of full-length RanBP2 and two C-terminal deletion mutants with GFP tags. Numbers on the right refer to amino acids. LRD, leucine-rich domain; Zinc, zinc finger motifs; E3, SUMO E3 ligase domain; CY, cyclophilin homology domain. (b) HeLa cells were transfected with a GFP, GFP-RanBP2-FL, RanBP2-N2 or RanBP2-N1 expression plasmid. After double thymidine block, lysates prepared from the cells were immunoprecipitated with anti-GFP antibody and then analyzed by IB with anti-Imp-ß1 or anti-GFP antibodies. Asterisks indicate the GFP fusion proteins. Numbers indicate molecular mass markers in kilodaltons. HC, IgG heavy chain; LC, IgG light chain. In lanes marked ‘Input,' 20 µl of the 500 µl of extract used per IP were analyzed directly. (c) Scheme of full-length RanBP2 and the N- and C-terminal deletion mutant (M1) with the GST tag. Numbers on the right refer to amino acids. (d) In vitro expressed and purified GST-RanBP2-M1, Imp-ß1-N2-His and His-Ran-GTP were mixed and pulled-down with glutathione-Sepharose beads. Pulled-down lysates were separated by SDS-PAGE and then either stained with Coomassie brilliant blue (CBB) or subjected to IB with anti-6 × His antibody. Numbers indicate molecular mass markers in kilodaltons. (e) Scheme of GFP-tagged full-length RanBP2 carrying the W1211R/K1212M mutation and FLAG-tagged Imp-ß1-FL carrying the I178D/Y255A mutation. (f and g) HeLa cells were transfected with a GFP-RanBP2-FL (wt), GFP-RanBP2-FL_W1211R/K1212M (mt), FLAG-Imp-ß1-FL (wt) or FLAG-Imp-ß1-FL_ I178D/Y255A (mt) expression plasmid. After double thymidine block, lysates prepared from the cells were immunoprecipitated with anti-GFP or anti-FLAG antibody and then analyzed by IB with anti-Imp-ß1, anti-GFP, anti-RanBP2 or anti-FLAG antibodies. In lanes marked ‘Input,' 20 µl of the 500 µl of extract used per IP were analyzed directly

Fig 5: Model for RanBP2 in proper mitotic progression and faithful chromosomal segregation. Absence of RanBP2 during mitosis caused abnormal mitotic spindles, supernumerary centrosomes, impaired chromosomal alignment, induced mitotic catastrophe and cell death