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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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Research Article

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

* Author for correspondence (e-mail: david.stenoien{at}pnl.gov)

Accepted 11 September 2007

The chromosomal passenger complex (CPC) is a crucial regulator of chromosome, cytoskeleton and membrane dynamics during mitosis. Here, using liquid chromatography coupled to mass spectrometry (LC-MS), we identified phosphopeptides and phosphoprotein complexes recognized by a phosphorylation-specific antibody that labels the CPC. A mitotic phosphorylation motif {PX[G/T/S][L/M]S(P) P or WGLS(P) P} was identified by MS in 11 proteins, including FZR1 (Cdh1) and RIC8A–two proteins with potential links to the CPC. Phosphoprotein complexes contained the known CPC components INCENP, Aurora-B (Aurkb) and TD-60 (Rcc2, RCC1-like), as well as SMAD2, 14-3-3 proteins, PP2A and Cdk1 (Cdc2a), a probable kinase for this motif. Protein sequence analysis identified phosphorylation motifs in additional proteins, including SMAD2, PLK3 and INCENP. Mitotic SMAD2 and PLK3 phosphorylation was confirmed using phosphorylation-specific antibodies, and, in the case of Plk3, phosphorylation correlated with its localization to the mitotic apparatus and the midbody. A mutagenesis approach was used to show that INCENP phosphorylation is required for its localization to the midbody. These results provide evidence for a shared phosphorylation event that regulates localization of crucial proteins during mitosis.

Key words: Phosphorylation, Mitosis, Proteomics, Chromosomal passenger complex






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