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First published online 29 May 2007
doi: 10.1242/jcs.03458

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Characterization of NIP2/centrobin, a novel substrate of Nek2, and its potential role in microtubule stabilization

Department of Biological Sciences and Research Center for Functional Cellulomics, Seoul National University, Seoul 151-742, Korea

View larger version (40K): [in this window] [in a new window]   Fig. 1. Immunoblot analysis of NIP2. (A) Lysates of 293T cells transfected with Myc-NIP2 were used for immunoblot analysis with antibodies specific to NIP2 or a Myc tag. The arrow indicates endogenous NIP2. (B) Lysates of HeLa cells were immunoprecipitated (IP) with pre-immune serum or the NIP2 anti-serum, followed by immunoblotting with the NIP2 antibody. The arrows indicate specific bands for NIP2 and the IgG heavy chain. (C) Selected cell lines from human (293T, HeLa, U2OS), rhesus monkey (COS7), mouse (NIH3T3), and Chinese hamster (CHO) origins were subjected to immunoblot analysis with the NIP2 antibody. The same blot was re-used for detection of β-tubulin. (D) The cell cycle of HeLa cells was arrested at G1/S phase with either 2.5 mM thymidine (TH) or 4 mM hydroxyurea (HU), or at G2/M phase with either 100 µg/ml nocodazole (NZ) or 1 µM taxol (TX). An exponentially growing cell population without any treatment (Exp) was included as a control. The cell lysates were subjected to immunoblot analysis with antibodies against NIP2, Nek2, C-NAP1 and phospho-histone H3 (pHH3).

View larger version (60K): [in this window] [in a new window]   Fig. 2. The C-terminal end of NIP2 is critical for centriolar localization. (A) U2OS cells were placed on ice for 30 minutes to disrupt microtubules or treated with 1 µg/ml taxol for 2 hours to stabilize them, and were immunostained with antibodies against NIP2 and β-tubulin. Arrows indicate the endogenous NIP2 staining at the centrosomes. Bar, 5 µm. (B) Several NIP2-truncated mutants with the GFP tags were expressed in HeLa cells. The β-tubulin was detected as a control. (C) HeLa cells were transfected with the NIP2-truncated mutant genes and immunostained with antibodies against GFP (green) and -tubulin (red). Arrows indicate the centrosomes. Bar, 10 µm. (D) Myc-tagged NIP2 truncated mutant proteins (Myc-NIP2, Myc-NIP2445-903, and Myc-NIP21-523) were co-expressed with GFP-centrin 2 (green) in HeLa cells and detected with the Myc antibody (red). The insets are magnified views of the centrosomes.

View larger version (34K): [in this window] [in a new window]   Fig. 3. NIP2 proteins outside the centrosome. (A) KE37 cell lysates were fractionated by sucrose gradient centrifugation for enrichment of the centrosome. Immunoblot analysis was carried out using antibodies against NIP2, Nek2 and -tubulin. Whole cell lysates (WCL) were used as a control. (B) Immunoblot analysis was carried out using soluble and insoluble fractions of whole cell lysates (WCL) and the centrosome fraction, and using antibodies against C-Nap1, NIP2, Plk1 and Nek2. (C) U2OS cells at mitosis were immunostained with NIP2 antibody (green). Antibodies against tyrosinated and acetylated tubulins (red) were used for staining dynamic and stable microtubules, respectively. (D) U2OS cells at interphase were immunostained with antibodies against NIP2 (red) and acetylated tubulin (green). Arrows indicate NIP2 signals in the cytoplasm. Note that centrosomal signals are out of focus in this figure. Bar, 10 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 4. NIP2 was phosphorylated by Nek2 in vitro and in vivo. (A) In vitro kinase assay. The wild-type (Nek2RHA1) and kinase-defective (Nek2KHA5) Nek2 proteins were prepared from transiently transfected 293T cells. As substrates, GST-NIP21-523 was purified from bacterial lysates and Myc-NIP21-523 was prepared by immunoprecipitation from transfected 293T cells. Casein was a control substrate for Nek2. Specific bands on the autoradiogram are marked. The bands marked with an asterisk are non-specific. (B) In vitro kinase assay carried out with wild-type (WT) or kinase-defective (KD) Nek2. The substrates were prepared from bacterially expressed GST fusion proteins linked to NIP21-193, NIP2194-560 or NIP2561-903. The separated gel was stained with Coomassie Blue and autoradiographed. The arrowheads indicate the substrate proteins and the asterisk indicates autophosphorylation of Nek2. (C) In vivo labeling of NIP2. HeLa cells transfected with pNek2RHA1 or pNek2KHA5 were incubated in the presence of 32P-orthophosphate (250 µCi/ml) for 3 hours. Endogenous NIP2 was immunoprecipitated (IP) and subjected to immunoblot analysis as well as autoradiogram. Exogenous Nek2 was also immunoprecipitated with the HA antibody, followed by immunoblot analysis and autoradiogram. A faint band of HA immunoprecipitate from untransfected cells corresponds to the IgG heavy chain.

View larger version (68K): [in this window] [in a new window]   Fig. 5. Effects of Nek2 on ectopic NIP2 proteins. (A) 293T cells were co-transfected with GFP-NIP2 and kinase-defective Nek2KHA5 (KD) or varying amounts of wild-type Nek2RHA1 (WT). Twenty-four hours later, cell lysates were prepared using a lysis buffer containing 1% NP40 and separated into the insoluble pellet (P) and soluble supernatant (S) by centrifugation. Equal amounts of pellet and supernatant proteins were subjected to immunoblot analysis using antibodies against GFP and the HA-tag for detection of ectopic NIP2 and Nek2 proteins, respectively. (B) Myc-NIP2 was co-transfected into HeLa cells with either Nek2RHA1 or Nek2KHA5. Co-immunostaining was carried out using NIP2 (green) and HA (red) antibodies. (C) HeLa cells that had been co-transfected with GFP-NIP2 and Nek2RHA1 were co-immunostained with antibodies against GFP (green) and Nek2 (red) or β-tubulin (red). DNA was stained with DAPI. Bars, 10 µm. (D) HeLa cells were transfected with Myc-NIP2445-903. Twenty-four hours after transfection, nocodazole (100 µg/ml) was added for 2 hours and cells were immunostained with antibodies against NIP2 (green) and acetylated -tubulin (Ac-tubulin, red). (E) HeLa cells with GFP or GFP-NIP2445-903 proteins were lysed in the presence of nocodazole or taxol and then fractionated into supernatant (S) and pellet (P). β-tubulin was used as a marker for fractionation.

View larger version (37K): [in this window] [in a new window]   Fig. 6. Suppression of NIP2 expression caused abnormality in spindle formation. (A) Two different amounts of siRNAs specific to NIP2 (siNIP2), Nek2 (siNek2) or non-specific control siRNA (siCTL) were transfected into HeLa cells. Forty-eight hours after transfection, immunoblot analysis was carried out to determine the cellular levels of NIP2 and Nek2 proteins. Acetylated tubulin and β-tubulin were detected as a control. (B) Twenty-four hours after transfection of siCTL or siNIP2, HeLa cells were co-immunostained with antibodies against NIP2 and -tubulin. (C) NIP2-suppressed HeLa cells were co-immunostained with antibodies against NIP2 and β-tubulin. The sizes of the cells were measured using Image-pro software and analyzed statistically. For statistical analysis of this and the following experiments, over 300 cells were counted for each group and the experiments were repeated three times. The results were presented as mean±s.e.m. (D) Microtubule re-growth assay. HeLa cells transfected with siCTL or siNIP2 were placed on ice for 30 minutes to disrupt microtubules and transferred immediately to 37°C for microtubule re-growth. Five minutes after the temperature shift, cells were fixed and co-immunostained with antibodies against NIP2 and β-tubulin. (E) Forty-eight hours after transfection with siCTL or siNIP2, U2OS cells were co-immunostained with antibodies against NIP2 (red) and β-tubulin (green). The number of mitotic cells with abnormal spindles was counted. (F) The nuclei of NIP2-suppressed U2OS cells were stained with DAPI and the number of cells with abnormal nuclear morphology was counted. (G) HeLa cells transfected with siCTL or siNIP2 were cultured for 24, 48 or 72 hours and subjected to FACS analyses to determine the DNA content of each cell population. (H) Ninety-six hours after transfection of siCTL or siNIP2, the MTT assay was carried out to determine the number of live cells. The proportion of the live cells was determined by counting the MTT-stained cells in the culture dishes.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Nek2 controls centrosomal NIP2 levels. (A) HeLa cells were transfected with siCTL, siNIP2 or siNek2. Twenty-four hours after transfection, cells were co-immunostained with antibodies against NIP2 and Nek2. Insets are magnified views of the centrosomes. (B) HeLa cells were transfected with siCTL or siNek2, and their cell cycles were arrested at the G1/S boundary with a double-thymidine block. The cells were then immunostained with NIP2 antibody to determine centrosomal NIP2 levels. More than 300 cells were analyzed per group in three independent experiments. FACS histograms show synchronization of HeLa cells at the G1/S boundary. (C) The amount of cellular Nek2 increased due to overexpression of HA-tagged wild-type Nek2 (Nek2RHA1) or decreased due to transfection of siNek2. Twenty-four hours after transfection, cells were immunostained with antibodies against NIP2 and HA. The amount of the centrosomal NIP2 was determined by fluorescent intensity. More than 300 cells were counted in three independent experiments.

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