QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 7 April 2009
doi: 10.1242/jcs.044321

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jang, C.-Y. Articles by Fang, G. Search for Related Content PubMed PubMed Citation Articles by Jang, C.-Y. Articles by Fang, G. Social Bookmarking                         What's this?

Plk1 and Aurora A regulate the depolymerase activity and the cellular localization of Kif2a

¹ Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
² Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA
³ Genentech, South San Francisco, CA 94080, USA

View larger version (38K): [in this window] [in a new window]   Fig. 1. Kif2a interacts with Plk1 during mitosis. (A) HeLa S3 cells stably expressing GFP-S-Plk1 were synchronized by a double-thymidine arrest and harvested at 8.5 hours post-release to enrich G2 and M cells. The Plk1 complex was tandem-affinity-purified and the associated proteins were identified by mass spectrometry (Seki et al., 2008a; Seki et al., 2008b). Peptides of Kif2a identified in the Plk1 complex are listed together with their XCorr and DeltaCN scores. (B) Shown are maximum projections from deconvolved z-stacks of representative HeLa cells stained for Kif2a (green), Plk1 (red), and DNA (blue). Bar, 5 µm. (C) Myc-Plk1 was co-transfected with GFP-Kif2a or GFP into 293T cells. Twenty hours after transfection, cells were treated with 100 ng/ml nocodazole for 18 hours and harvested. Cell lysates and the anti-GFP immunoprecipitates (IP) were analyzed by western blotting. (D,E) HeLa S3 cells were synchronized at the G1-S boundary by a double-thymidine treatment (TT), released into fresh medium and collected at the indicated times (D). Alternatively, HeLa S3 cells were synchronized at prometaphase by a thymidine-nocodazole block (TN), released into fresh medium, and harvested at indicated times (E). The cell cycle profile was analyzed by fluorescence-activated cell sorting (FACS) with the mitotic index (MI) determined by an antibody (MPM2) specific to mitotic phospho-proteins. Cell lysates and the anti-Plk1 immunoprecipitates (IP) were analyzed by western blotting. p38MAPK served as a loading control. Kif2a was co-precipitated with the anti-Plk1 antibody, but not with a non-specific IgG (data not shown). The kinase activity of Plk1 was measured on immunopurified Plk1 using casein as a substrate (Seki et al., 2008b). AS, asynchronous cells. (F) The Plk1-Kif2a complex was immunoprecipitated with an anti-Plk1 antibody from HeLa S3 cells harvested after a thymidine-nocodazole treatment (TN0) or collected at 9 hours after the release from a double-thymidine arrest (TT9). The immunoprecipitates were incubated with or without -phosphatase (-PPase), washed to remove dissociated Kif2a, and then analyzed by western blotting. (G) HeLa S3 cells were synchronized at prometaphase by a thymidine-nocodazole arrest and then released into fresh medium containing the proteasome inhibitor MG132 (20 µM) and one of the following kinase inhibitors: 1 µM BI 2536 [a Plk1 inhibitor (Lenart et al., 2007)], 25 µM Purvalanol A [a Cdk1 inhibitor (Skoufias et al., 2007)] and 5 µM VX680 [an Aurora A inhibitor (Harrington et al., 2004)]. Cells were harvested 2 hours later. Cell lysates and the anti-Plk1 immunoprecipitates were analyzed by western blotting.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Plk1 directly phosphorylates Kif2a and activates its depolymerase activity. (A) Recombinant His-Kif2a was incubated with wild-type (WT) or kinase-dead (KD) Plx1 in the presence of radioactive ATP. Casein, a common substrate of Plx1, was included as a control. The amounts of His-Plx1 and His-Kif2a used in the reaction were visualized by silver staining (for Plx1) and by Coomassie blue staining (for Kif2a). (B) Recombinant His-Kif2a (10 nM) was phosphorylated with wild-type or kinase-dead Plx1 and the reaction mix was incubated with Taxol-stabilized MTs for 3 minutes. MTs and Kif2a were then analyzed in a co-pelleting assay, followed by detection of MTs with silver staining and detection of Kif2a and Plx1 with western blotting. sup., supernatant. (C) His-Kif2a (10 nM) was phosphorylated by Plx1 and the reaction mix was then incubated with Taxol-stabilized MTs, fixed, and sedimented onto coverslips. Shown are representative immunofluorescence images of β-tubulin. Scale bar: 20 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Plk1 promotes the recruitment of Kif2a to spindle MTs and to spindle poles. (A-E) HeLa cells were treated with 100 nM BI 2536 for 2 hours. (A) Maximum projections from deconvolved z-stacks of representative DMSO- or BI 2436-treated HeLa cells stained for Kif2a (green), β-tubulin (red), and DNA (blue). (B) The levels of endogenous proteins in treated cells were analyzed by western blotting. (C-E) Total immunofluorescence intensities for β-tubulin (MT) and for spindle pole signals from Plk1, Kif2a (C), -tubulin (D) and NuMA (E) in treated metaphase cells were quantified and plotted (n=10 cells for each quantification). In C, *P<1.2x10-3; #P<8.9x10-4; ##P<1.4x10-2; ###P<1.7x10-7. In D, *P<8.3x10-2; **P<2.0x10-4 (two-tailed t-test). A.U., arbitrary units. Scale bar: 5 µm.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Plk1 is targeted to centrosomes independent of Kif2a. (A-F) HeLa cells were either control-transfected or transfected with a siRNA specific for Kif2a (siKif2a) and analyzed at 62 hours post-transfection (A,C-F); for B, 100 ng/ml nocodazole was added at 40 hours post-transfection and mitotic cells were harvested at 62 hours post-transfection. (A) Maximum projections from deconvolved z-stacks of representative cells stained for Aurora A (green), Plk1 (red), and DNA (blue). (B) Control or Kif2a-knockdown cells treated with nocodazole were analyzed by western blotting for endogenous levels of indicated proteins. (C-F) Asynchronous cells were stained for various spindle and spindle pole antigens and immunofluorescence images of metaphase cells were acquired under a constant exposure for each antigen. Total immunofluorescence intensities for β-tubulin (MT) and for spindle pole signals from Kif2a, Plk1 (C), -tubulin (D), NuMA (E), and Aurora A (F) in metaphase cells were quantified and plotted (n=10 cells for each quantification). In C, *P<4.3x10-7; **P<4.8x10-2 (two-tailed t-test). A.U., arbitrary unit. Scale bar: 5 µm.

View larger version (43K): [in this window] [in a new window]   Fig. 5. Aurora A colocalizes and interacts with Kif2a and inhibits its recruitment and its depolymerase activity. (A) Maximum projections from deconvolved z-stacks of representative HeLa cells stained for Kif2a (green), Aurora A (red) and DNA (blue). (B) Myc-Aurora A was co-transfected with GFP-Kif2a or GFP into 293T cells. Twelve hours after transfection, cells were treated with 100 ng/ml of nocodazole for 18 hours. Cell lysates and anti-GFP immunoprecipitates (IP) were analyzed by western blotting. (C-E) HeLa cells were treated with 500 nM VX680 for 15 minutes, fixed and stained for Kif2a (green), β-tubulin (red) and DNA (blue). (C) Maximum projections from deconvolved z-stacks of representative control or VX680-treated HeLa cells. (D) Immunofluorescence intensities for β-tubulin and Kif2a on metaphase spindle were quantified and plotted (n=10 cells for each quantification). (E) Levels of endogenous proteins in VX680-treated cells were analyzed by western blotting with p38MAPK as a loading control. In D, *P<6.6x10-4; **P<8.5x10-5; ***P<1.1x10-4 (two-tailed t-test). A.U., arbitrary unit. (F) Recombinant His-Kif2a was incubated with recombinant His-Aurora A with or without VX680 in the presence of radioactive ATP and phospho-Kif2a was analyzed by SDS-PAGE. The amount of His-Kif2a used in the reaction was visualized by Coomassie blue staining. (G) Recombinant His-Kif2a (25 nM) was phosphorylated by His-Aurora A with or without VX680 and the reaction mix was incubated with Taxol-stabilized MTs for 10 minutes either in the presence or absence of VX680. MTs were then analyzed in a co-pelleting assay, followed by detection of MTs with silver staining and detection of Kif2a and Aurora A with western blotting. As controls, incubation of MTs with Aurora A alone or with VX680 alone did not alter the amounts of MTs pelleted (data not shown). sup., supernatant; -/-, no VX680 added; -/+, VX680 added during the incubation with MTs, but not during the phosphorylation reaction with Aurora A; +/+, VX680 present both during the phosphorylation reaction and during the incubation with MTs. (H) His-Kif2a (25 nM) was phosphorylated by Aurora A and the reaction mix was then incubated with Taxol-stabilized MTs for 10 minutes, fixed, and sedimented onto coverslips. Shown are representative immunofluorescence images of β-tubulin. For optimal analysis of the inhibition of Kif2a by Aurora A, a higher concentration of Kif2a and a longer incubation time were used for samples in G and H compared to those in Fig. 2B,C. Scale bars: 5 µm (A,C); 20 µm (H).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter