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First published online 11 November 2008
doi: 10.1242/jcs.035360

Journal of Cell Science 121, 3912-3921 (2008)
Published by The Company of Biologists 2008
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The NIMA-family kinase Nek6 phosphorylates the kinesin Eg5 at a novel site necessary for mitotic spindle formation

Joseph Rapley1, Marta Nicolàs2, Aaron Groen3, Laura Regué2, M. Teresa Bertran2, Carme Caelles2,4, Joseph Avruch1,* and Joan Roig2,*,{ddagger}

1 Department of Molecular Biology and Medical Services, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, MA 02114, USA
2 Cell Signaling Group, Molecular Medicine Program, Institute for Research in Biomedicine (IRB Barcelona), 08028 Barcelona, Spain
3 Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA
4 Department of Biochemistry and Molecular Biology (Pharmacy), Universitat de Barcelona, 08028 Barcelona, Spain


Figure 1
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  		Fig. 1. Eg5 interacts with Nek6 and Nercc1. (A) Immunoprecipitates (IP) were prepared from exponentially growing (Exp) or nocodazole-arrested mitotic (M) U2OS cell extracts using either normal rabbit IgG (NIgG), anti-Nek6 or anti-Nercc1 antibodies and analyzed by immunoblot (W) with the indicated antibodies. Eg5 in the corresponding extracts is shown in the lower panel. (B) Immunoprecipitates were prepared from exponentially growing or nocodazole-arrested mitotic U2OS cells using NIgG or anti-Nercc1 antibody, and immunoblotted for Nek6 and Nercc1.

 

Figure 2
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  		Fig. 2. Nek6 phosphorylates Eg5 in vitro at a conserved site that is phosphorylated during mitosis. (A) Recombinant GST-HsEg5 or His6-XlEg5 were phosphorylated by FLAG-Nek6. The insets show 32P incorporation with time (upper gel, left to right with no kinase in rightmost lane) and a Coomassie-blue stain of the substrate (lower gel). (B) Myelin basic protein (MBP, positive control), maltose-binding protein (MalBP), or MalBP fusion proteins containing the Eg5 segments indicated were incubated with [{gamma}-32P]ATP/Mg2+ and either FLAG-Nek6 (odd-numbered lanes) or pre-activated FLAG-Nercc1 (even-numbered lanes) for 30 minutes followed by SDS-PAGE. The upper panels show Coomassie-blue stains of the gels and the lower panels the corresponding autoradiographs. White arrowheads indicate Nercc1 and black arrowheads indicate Nek6. Asterisks indicate the location of MBP (lanes 3 and 4) or MalBP fusion proteins (7-12). The left gel is 12% acrylamide, the right gel 7.5%; Nek6 is run off the latter. (C) HsEg5, human Eg5; MmEg5, mouse Eg5; XlEg5, X. laevis Eg5; KLP61F, D. melanogaster KLP61F; bmk-1, C. elegans BMK-1; BimC, A. nidulans BimC; KIP1/Cin8p, S. cerevisiae KIP1/Cin8p; Cut7, S. pombe Cut7. Identical and conserved residues are shaded; regions conserved around the C-terminal CDK1 site (BimC box) and Nek6 site (LXXS*) are boxed. (D) Using normal IgG (NIgG) and anti-HsEg5 antibodies, immunoprecipitates were prepared from extracts of 32P-labelled U2OS cells growing exponentially (Exp) or incubated overnight in 0.25 mM nocodazole (M), and subjected to SDS-PAGE; a PhosphoImage of the gel is shown. (E) Left, two-dimensional tryptic phosphopeptide map of endogenous 32P-Eg5 immunoprecipitated from 32P-labelled mitotic U2OS cells, visualized by PhosphorImager. Right, phosphopeptide map of purified recombinant 32P-GST-Eg5 phosphorylated in vitro by immunoprecipitated mitotic CDK1. The gray arrows mark phosphopeptides attributed to CDK1; the black arrow indicates a 32P-peptide evident in digests of mitotic Eg5 that is not present in digests of CDK1-phosphorylated 32P-Eg5. (F) Top left, two-dimensional phosphopeptide map of endogenous Eg5 immunoprecipitated from 32P-labelled mitotic U2OS cells; right (top and bottom), two-dimensional maps of recombinant GST-Eg5 wild type (wt; top right) and GST-Eg5[Ser1033Ala] (bottom right) phosphorylated in vitro by FLAG-Nek6; note in the lower right map the absence of the most anodally migrating 32P-peptide seen in the upper right map, which encompasses Ser1033-P. Bottom left, a mixture of comparable amounts of the digests of mitotic 32P-Eg5 and FLAG-Nek6-phosphorylated 32P-Eg5. The arrow marks the phosphopeptide common to the two digests, which contains Ser1033. In the digests of mitotic 32P-Eg5 (E, left; F, upper left), this spot contains ~3% of total 32P, estimated by PhosphorImager.

 

Figure 3
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  		Fig. 3. Eg5[Ser1033] phosphorylation in vivo. (A) U2OS cells were co-transfected with either myc-Eg5 wild type (lanes 1-3) or myc-Eg5[Ser1033Ala] (lane 4) and empty plasmid (lane 1), FLAG-Nek6 wild type (lanes 2 and 4) or FLAG-Nek6 [Lys74Met Lys75Met] (lane 3). 24 hours later, anti-Myc immunoprecipitates were subjected to immunoblot with anti-Myc (lower panel) and anti-Eg5[Ser1033-P] (top panel) antibodies. (B) U2OS cells growing exponentially were either untreated (first lane, Exp) or arrested with nocodazole (0.25 mM overnight; lane 2); arrested cells were allowed to exit mitosis in nocodazole-free medium and extracted at the times indicated (lanes 3-7). Eg5 immunoprecipitates (top two rows) and cell extracts (bottom seven rows) were analyzed by immunoblotting with the indicated antibodies. a-P-Eg5(S1033), anti-Eg5[Ser1033-P]; a-P-CDK1, anti-CDK1[Tyr15-P]; a-P-Nercc1, anti-Nercc1[Thr210-P]; a-P-Nek6, anti-Nek6[Ser206-P]. (C) Extracts from XL177 X. laevis cells were resolved by SDS-PAGE and immunoblotted with either anti-XlEg5 (left) or anti-XlEg5[Ser1046-P] (right). (D) XL177 cells growing exponentially were fixed and stained with antibodies to XlEg5 (lower two rows), XlEg5[Ser1046-P] (upper two rows) and either β- or {gamma}-tubulin as indicated. DNA is stained with DAPI. Representative cells in metaphase are shown. Scale bar: 10 µm.

 

Figure 4
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  		Fig. 4. Phosphoserine 1046 immunolocalization in XL177 cells in interphase and different phases of mitosis. As in Fig. 3D. Scale bar: 10 µm.

 

Figure 5
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  		Fig. 5. Eg5[Ser1033] phosphorylation is required for normal Eg5 function in mitotic spindle assembly. (A) U2OS cells transiently expressing Myc-Eg5 wild type, Myc-Eg5[Thr926Ala] or Myc-Eg5[Ser1033Ala] were stained for Myc, β-tubulin and DNA. A representative transfected cell in mitosis is shown in each case, except for Myc-Eg5[Ser1033Ala], for which a mitotic cell plus two transfected interphase cells is shown. (B) HeLa cells were co-transfected with either control oligonucleotides (lanes 1-6) or siRNA oligonucleotides directed against human Eg5 (lanes 7-12), together with empty plasmid (lanes 1 and 7) or plasmids encoding Myc-Eg5 wild type (wt) that was unmodified (lanes 2 and 8) or Myc-Eg5 variants that were rendered RNAi resistant (rrEg5 wt, Eg5 wild type; rrEg5 TA, Eg5[Thr926Ala]; rrEg5 SA, Eg5[Ser1033Ala]; rrEg5 SD, Eg5[Ser1033Asp]). Cell lysates were immunoblotted with anti-Myc (upper panel) or anti-Eg5 (middle panel) to determine recombinant and total Eg5 expression, respectively, and with anti-{alpha}-tubulin antibodies to evaluate loading (bottom panel). (C) HeLa cells were co-transfected with siRNA oligonucleotides directed against human Eg5 and with plasmids encoding Myc-GFP (control) or several RNAi-resistant Myc-Eg5 (rrEg5) variants. At 48 hours after transfection, cells were fixed and stained with anti-Myc and anti-β-tubulin antibodies, and with DAPI. Myc-positive cells were scored as being in interphase (gray columns), in normal mitosis (white columns) or in an abnormal mitosis (mostly monopolar spindles; black columns). The figure represents the mean of three independent experiments, wherein >100 cells were scored for each point in each of the experiments; error bars indicate s.e.m. The fraction of abnormal mitoses in cells expressing rrEg5 wild type was compared with each of the other conditions by Student's t-test; *P<0.05. The comparison of Ser1033Ala with Ser1033Asp (S1033D) gave a P value of 0.068.

 

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© The Company of Biologists Ltd 2008