Analysis of mitotic phosphorylation of borealin.
BACKGROUND
The main role of the chromosomal passenger complex is to ensure that Aurora B kinase is properly localized and activated before and during mitosis. Borealin, a member of the chromosomal passenger complex, shows increased expression during G2/M phases and is involved in targeting the complex to the centromere and the spindle midzone, where it ensures proper chromosome segregation and cytokinesis. Borealin has a consensus CDK1 phosphorylation site, threonine 106 and can be phosphorylated by Aurora B Kinase at serine 165 in vitro.


RESULTS
Here, we show that Borealin is phosphorylated during mitosis in human cells. Dephosphorylation of Borealin occurs as cells exit mitosis. The phosphorylated form of Borealin is found in an INCENP-containing complex in mitosis. INCENP-containing complexes from cells in S phase are enriched in the phosphorylated form suggesting that phosphorylation may encourage entry of Borealin into the chromosomal passenger complex. Although Aurora B Kinase is found in complexes that contain Borealin, it is not required for the mitotic phosphorylation of Borealin. Mutation of T106 or S165 of Borealin to alanine does not alter the electrophoretic mobility shift of Borealin. Experiments with cyclohexamide and the phosphatase inhibitor sodium fluoride suggest that Borealin is phosphorylated by a protein kinase that can be active in interphase and mitosis and that the phosphorylation may be regulated by a short-lived phosphatase that is active in interphase but not mitosis.


CONCLUSIONS
Borealin is phosphorylated during mitosis. Neither residue S165, T106 nor phosphorylation of Borealin by Aurora B Kinase is required to generate the mitotic, shifted form of Borealin. Suppression of phosphorylation during interphase is ensured by a labile protein, possibly a cell cycle regulated phosphatase.
Kaur
Harpreet
H
Department of Biological Sciences, University of Toledo, Toledo, OH 43606, USA. hkaur@utnet.utoledo.edu <hkaur@utnet.utoledo.edu>
Stiff
Andrew C
AC
Date
Dipali A
DA
Taylor
William R
WR
D019943:Amino Acid Substitution; D064107:Aurora Kinase B; D064127:Aurora Kinases; D001549:Benzamides / Q000494:pharmacology; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002503:Centromere / Q000378:metabolism / Q000648:ultrastructure; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D020090:Chromosome Segregation / Q000502:physiology*; D016384:Consensus Sequence; D003513:Cycloheximide / Q000494:pharmacology; D016195:G2 Phase; D006367:HeLa Cells; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D007399:Interphase; D008677:Metaphase; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008938:Mitosis; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012969:Sodium Fluoride / Q000494:pharmacology; D008941:Spindle Apparatus / Q000378:metabolism / Q000648:ultrastructure; D013379:Substrate Specificity; D000077022:Survivin; D014162:Transfection; D003521:Cyclopropanes; D011720:Pyrazoles; D011743:Pyrimidines
D064107:Aurora Kinase B; D064127:Aurora Kinases; D001549:Benzamides; D016203:CDC2 Protein Kinase; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D003513:Cycloheximide; D006576:Heterocyclic Compounds, 4 or More Rings; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D009363:Neoplasm Proteins; D015739:Nocodazole; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D010768:Phosphoserine; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011500:Protein Synthesis Inhibitors; D011799:Quinazolines; D011993:Recombinant Fusion Proteins; D012969:Sodium Fluoride; D000077022:Survivin; C474722:4-(4-(N-benzoylamino)anilino)-6-methoxy-7-(3-(1-morpholino)propoxy)quinazoline; C107489:BIRC5 protein, human; C490157:CDCA8 protein, human; C083767:INCENP protein, human; C472645:blebbistatin; C484810:tozasertib; D003521:Cyclopropanes; D011720:Pyrazoles; D011743:Pyrimidines; C576921:AURKB protein, human