Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus.
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.
Yang
Feng
F
Environmental Molecular Sciences Laboratory and Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Camp
David G
DG
Gritsenko
Marina A
MA
Luo
Quanzhou
Q
Kelly
Ryan T
RT
Clauss
Therese R W
TR
Brinkley
William R
WR
Smith
Richard D
RD
Stenoien
David L
DL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008938:Mitosis; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D040901:Proteomics; D015203:Reproducibility of Results; D008941:Spindle Apparatus / Q000378:metabolism*; D025521:Tumor Suppressor Proteins; D000097868:Polo-like Kinases
D000911:Antibodies, Monoclonal; D002868:Chromosomal Proteins, Non-Histone; D010455:Peptides; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D025521:Tumor Suppressor Proteins; C083767:INCENP protein, human; C486256:PLK3 protein, human; D000097868:Polo-like Kinases