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First published online 30 October 2007
doi: 10.1242/jcs.014795

Journal of Cell Science 120, 4060-4070 (2007)
Published by The Company of Biologists 2007
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Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

Feng Yang1, David G. Camp, II1, Marina A. Gritsenko1, Quanzhou Luo1, Ryan T. Kelly1, Therese R. W. Clauss1, William R. Brinkley2, Richard D. Smith1 and David L. Stenoien3,*

1 Environmental Molecular Sciences Laboratory and Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA
2 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA
3 Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA


Figure 1
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  		Fig. 1. P190 immunostaining colocalizes with the CPC in human cells. (A) Immunofluorescence staining of mitotic HeLa cells shows P190 immunoreactivity (green) on kinetochores from prophase to metaphase, in the midzone during anaphase to telophase and in the midbody at the completion of mitosis. With the exception of residual midbody staining, no P190 immunoreactivity is observed during interphase. (B) Nocodazole treatment results in a large number of cells in prophase that progress through mitosis after nocodazole has been removed for 40 or 80 minutes. (C) Dual immunofluorescence shows that P190 (green) colocalizes with Aurora-B (red) throughout mitosis. (D,E) P190 immunoreactivity (green) colocalizes with INCENP (red) in HMECs (D) but not in mouse RAW macrophages (E). DNA is labeled with DAPI (blue). Bars, 10 µm.

 

Figure 2
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  		Fig. 2. Recognition of multiple mitotic phosphoproteins by P190. (A) On western blots, P190 recognizes several phosphoproteins in nocodazole-arrested cells (lane 2) but not in unsynchronized (lane 1) or thymidine-treated (lane 3) cells. Molecular weight markers are shown in kD. (B) P190 recognizes a subset of mitotic phosphoproteins that are distinct from those recognized by MPM-2 and CC3 and that show differential sensitivity to CDK inhibition and phosphatase treatments. Shown are western blots on lysates from cells treated with nocodazole (N), roscovitine (R) or alkaline phosphatase (AP), as indicated, and probed with P190, MPM-2 or CC3 antibodies. (C) Strategy for identifying protein complexes and phosphorylation sites containing the P190 phosphoepitope. (D) Immunoprecipitation with P190 (lanes 1 and 3) or control antibody (lanes 2 and 4) eluted with 8 M urea (lanes 1 and 2) or boiling in SDS gel loading buffer (lanes 3 and 4).

 

Figure 3
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  		Fig. 3. Validation of proteomic results. (A) SMAD2 and phospho-SMAD2 are specifically immunoprecipitated from HeLa mitotic cell lysates using the P190 antibody. (B) SMAD2 phosphorylation on the P190 motif is increased in nocodazole (N)-arrested versus thymidine (T)-arrested HeLa cells. (C) FZR1 is immunoprecipitated by P190, but CDC27, which contains a related motif, is not. (D) RIC8A (red) faintly colocalizes with P190 (green) during prophase and metaphase, and the amount of colocalization increases at later stages of cell division. (E) DLG1 (green) localizes to the cleavage furrow. DNA is labeled with DAPI (blue). Bars, 10 µm.

 

Figure 4
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  		Fig. 4. Identification of the phosphorylation motif in PLK3. (A) Phosphorylated PLK3 (PLK3pS284) but not non-phosphorylated PLK3 (PLK3npS284) or PLK1 is present in P190 IPs. (B) Phosphorylated PLK3 localizes to centrosomes, spindle poles and midbodies during mitosis. By contrast, non-phosphorylated PLK3 is more diffusely distributed than this during mitosis. The staining pattern of phosphorylated PLK3 is unaffected by blocking with the nonphosphorylated peptide (NP-Peptide) but disappears in the presence of the phosphorylated peptide (P-Peptide). (C) Phosphorylated PLK3 (green) localization partially overlaps with PLK1 (red) at centrosomes, spindle poles and midbodies, and with the CPC at midbodies.

 

Figure 5
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  		Fig. 5. Confirmation of INCENP as a P190-reactive phosphoprotein. (A) INCENP and Aurora-B are specifically immunoprecipitated from HeLa mitotic cell lysates using the P190 antibody. (B) P190 recognizes bands corresponding to the molecular weight of INCENP (arrow) in both INCENP and Aurora-B IPs. (C) Site-directed mutagenesis of serine 197 to alanine (S197A) or aspartate (S197D) prevents P190 binding. The arrow shows the location of GFP-INCENP. (D) Mutation of serine 197 to alanine, but not to aspartate, prevents INCENP from properly localizing to the midbody. The arrow denotes the midbody in the merged panel and the region containing the midbody was enlarged to clearly demonstrate that S197A (green) is not present in the midbody containing Aurora-B (red). Bars, 10 µm.

 




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