Specific primary sequence requirements for Aurora B kinase-mediated phosphorylation and subcellular localization of TMAP during mitosis.
During mitosis, regulation of protein structures and functions by phosphorylation plays critical roles in orchestrating a series of complex events essential for the cell division process. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a novel player in spindle assembly and chromosome segregation. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis. However, the mechanisms and functional importance of phosphorylation at most of the sites identified are currently unknown. Here, we report that TMAP is a novel substrate of the Aurora B kinase. Ser627 of TMAP was specifically phosphorylated by Aurora B both in vitro and in vivo. Ser627 and neighboring conserved residues were strictly required for efficient phosphorylation of TMAP by Aurora B, as even minor amino acid substitutions of the phosphorylation motif significantly diminished the efficiency of the substrate phosphorylation. Nearly all mutations at the phosphorylation motif had dramatic effects on the subcellular localization of TMAP. Instead of being localized to the chromosome region during late mitosis, the mutants remained associated with microtubules and centrosomes throughout mitosis. However, the changes in the subcellular localization of these mutants could not be completely explained by the phosphorylation status on Ser627. Our findings suggest that the motif surrounding Ser627 ((625) RRSRRL (630)) is a critical part of a functionally important sequence motif which not only governs the kinase-substrate recognition, but also regulates the subcellular localization of TMAP during mitosis.
Kim
Hyun-Jun
HJ
Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Jangangu, Suwon, Republic of Korea.
Kwon
Hye-Rim
HR
Bae
Chang-Dae
CD
Park
Joobae
J
Hong
Kyung U
KU
D064107:Aurora Kinase B; D064127:Aurora Kinases; D015153:Blotting, Western; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D024001:HMGN Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering
C114988:CKAP2 protein, human; D003598:Cytoskeletal Proteins; D024001:HMGN Proteins; D034741:RNA, Small Interfering; C576921:AURKB protein, human; D064107:Aurora Kinase B; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases