Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

Author:

Yang Feng1,Camp David G.1,Gritsenko Marina A.1,Luo Quanzhou1,Kelly Ryan T.1,Clauss Therese R. W.1,Brinkley William R.2,Smith Richard D.1,Stenoien David L.3

Affiliation:

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

Abstract

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.

Publisher

The Company of Biologists

Subject

Cell Biology

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