Journal: Cell

Article Title: Phosphorylation of Nup98 by multiple kinases is crucial for NPC disassembly during mitotic entry.

doi: 10.1016/j.cell.2011.01.012

Figure Lengend Snippet: Figure 5. Nup98 Is Modified at NPCs during Mitotic Entry (A) HeLa cells stably expressing 3EGFP-NLS were fixed, stained with DAPI, and analyzed by immu- nofluorescence using the indicated anti-Nup antibodies. Nup98(ab1) and (ab2) were raised against aa 581–880 and 598–616, respectively. Whereas Nup98(ab2) recognizes Nup98 at the nuclear rim at the end of prophase, the epitope recognized by Nup98(ab1) is masked. This change in the accessibility of Nup98 correlates with nuclear efflux of 3EGFP-NLS. Bar, 10 mm. (B) HeLa cells were arrested prior to S phase or in mitosis by treatment with thymidine or nocoda- zole, respectively. Cell lysates were analyzed by western blotting using the two different Nup98 antibodies. Loading control: anti-a-tubulin. See also Figure S5 and Figure S7.

Article Snippet: Mouse a-Nup98 (against aa 581–880, ab1) was from Santa Cruz, a-a-tubulin was from Sigma, and secondary antibodies were from Molecular Probes.

Techniques: Stable Transfection, Expressing, Staining, Western Blot, Control

Journal: Cell

Article Title: Phosphorylation of Nup98 by multiple kinases is crucial for NPC disassembly during mitotic entry.

doi: 10.1016/j.cell.2011.01.012

Figure Lengend Snippet: Figure 6. Nup98 Is Phosphorylated on Multiple Sites during Mitosis (A) HeLa cells were arrested prior to S phase or in mitosis by treatment with thymidine or nocodazole, respectively. Cells were lysed and either mock-treated or incubated with 400 U l protein phosphatase. Samples were analyzed by western blotting using a-Nup98, a-Rae1, and a-a-tubulin antibodies. Nup98 from mitotic cells shows a retarded migration that is reverted by phosphatase treatment. (B) Schematic depiction of the domain organization of Nup98 and phosphorylation sites (lines) that were identified in Nup98 by mass spectrometry from nocodazole-arrested HeLa cells. Note that all phosphosites are found within or close to the C-terminal domain of Nup98. (C) Phosphomimetic (PM) mutants of Nup98 are inefficiently targeted to the NPC. HeLa cells were transiently transfected with GFP-tagged Nup98 wild-type, GFP-Nup98 4PM (Nek; S494E, S591E, S822E, and S861E), or GFP-Nup98 13PM and analyzed by confocal microscopy. (D) Quantification of NE over cytoplasmic localization of Nup98 variants from the experiment presented in (C). Each value represents the mean ratio of three independent experiments; error bars indicate the SEM. (E) In vitro kinase assay comparing phosphorylation of Nup98(506–863) wild-type and a 11A mutant lacking all identified phosphorylation sites in the NPC-targeting domain of Nup98. Two micrograms of purified His-tagged Nup98(506–863) WT or 11A (Coomassie stain on the left) were incubated with PKCbII (12 ng), CDK1/cyclin B1 (24 ng), Nek2 (100 ng), Nek6 (100 ng), Nek7 (100 ng), or Plk1 (200 ng) in the presence of g-[32P]ATP. Note that the strong autophos- phorylation of Nek6 (asterisk) was observed with various Nek6 preparations from different origin (bacteria, insect cells) and is also observed using the Nek6 model substrate casein (not shown). The relative phosphorylation of Nup98 11A and Nup98 WT was quantified by phosphoimaging and Image J. See also Figure S1, Figure S3, Figure S4, Table S1, and Table S2.

Article Snippet: Mouse a-Nup98 (against aa 581–880, ab1) was from Santa Cruz, a-a-tubulin was from Sigma, and secondary antibodies were from Molecular Probes.

Techniques: Incubation, Western Blot, Migration, Phospho-proteomics, Mass Spectrometry, Transfection, Confocal Microscopy, In Vitro, Kinase Assay, Mutagenesis, Staining, Bacteria

Journal: Cell

Article Title: Phosphorylation of Nup98 by multiple kinases is crucial for NPC disassembly during mitotic entry.

doi: 10.1016/j.cell.2011.01.012

Figure Lengend Snippet: Figure 7. Nup98 Phosphorylation Is Required for Efficient NPC Disassembly (A) Nup98 13A persists longer on chromatin during mitotic entry. HeLa cells stably expressing GFP-Nup98 WT or GFP-Nup98 13A were transfected with H2B-mCherry, synchronized by a double-thymidine block, and released. Cells were imaged during mitotic entry by confocal microscopy (LSM710, 403 objective). (B) Nup98 association with chromatin was scored for different stages of mitotic entry (late G2 [interphase], early prophase, late prophase, and prometaphase) by assigning cells into one of three categories, each linked to a numerical score reflecting the strength and continuity of the Nup98 signal at the chromatin periphery (2–strong, continuous rim staining, 1–weak, interrupted, 0–no visible rim). The numerical average was plotted for the different stages (5 experiments, total n > 50 cells; error bars indicate the SEM). (C) Nup98 phosphorylation is rate limiting for NPC disassembly in vitro. Mitotic extract was added to semipermeabilized HeLa cells stably expressing wild-type GFP-Nup98 or the GFP-Nup98 13A mutant. NE permeabilization was monitored as in Figure 1. (D) Quantification was performed as in Figure 1 (11 positions, n > 66 GFP-Nup98-positive cells). The error bars indicate the SEM. (E) Quantification of t50 as in Figure 2C. Error bars indicate the standard deviation. See also Figure S6.

Article Snippet: Mouse a-Nup98 (against aa 581–880, ab1) was from Santa Cruz, a-a-tubulin was from Sigma, and secondary antibodies were from Molecular Probes.

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Transfection, Blocking Assay, Confocal Microscopy, Staining, In Vitro, Mutagenesis, Standard Deviation