Effects of phosphatase and proteasome inhibitors on Borealin phosphorylation and degradation

J Biochem. 2012 Apr;151(4):361-9. doi: 10.1093/jb/mvs015. Epub 2012 Feb 29.

Abstract

The chromosomal passenger complex (CPC) senses tension defects at the kinetochore to activate the spindle assembly checkpoint, and helps to position the cleavage furrow. The CPC, consisting of INCENP, Survivin, Borealin and Aurora B localizes to the inner centromere at metaphase and re-localizes to the spindle midzone at anaphase; several CPC functions are regulated by post-translational modification. Borealin is phosphorylated at multiple sites and phosphorylation at S219 causes Borealin to migrate more slowly upon electrophoresis. Here we find that Cdk1 can induce a mobility shift of Borealin, suggesting that S219 phosphorylation is under Cdk1 control. However, Cdk1 is inefficient at phosphorylating purified Borealin in vitro. A yeast orthologue of Borealin, Npl1, is dephosphorylated by the phosphatase Cdc14. We find no difference in the mobility shift of Borealin in human cells lacking either Cdc14A or Cdc14B. In contrast, the phosphatase inhibitor okadaic acid does delay the dephosphorylation of Borealin as cells exit mitosis. The proteasome inhibitor MG132 reduces Borealin phosphorylation in mitosis and increases the steady-state level of Borealin, especially in mutants lacking the C-terminus. However, a second, structurally unrelated proteasome inhibitor, lactacystin did not up-regulate Borealin. These results suggest that the effect of MG132 on Borealin is due to the inhibition of an intracellular protease other than the proteasome.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetylcysteine / analogs & derivatives
  • Acetylcysteine / pharmacology
  • Amino Acid Substitution
  • CDC2 Protein Kinase / chemistry
  • CDC2 Protein Kinase / genetics
  • CDC2 Protein Kinase / metabolism
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Dual-Specificity Phosphatases / genetics
  • Dual-Specificity Phosphatases / metabolism
  • Gene Expression
  • HeLa Cells
  • Humans
  • Leupeptins / pharmacology*
  • Okadaic Acid / pharmacology*
  • Peptide Fragments / metabolism
  • Phosphoric Monoester Hydrolases / antagonists & inhibitors*
  • Phosphorylation
  • Proteasome Inhibitors*
  • Protein Processing, Post-Translational*
  • Protein Transport
  • Proteolysis / drug effects*
  • Ubiquitin / metabolism

Substances

  • CDCA8 protein, human
  • Cell Cycle Proteins
  • Leupeptins
  • Peptide Fragments
  • Proteasome Inhibitors
  • Ubiquitin
  • lactacystin
  • Okadaic Acid
  • CDC2 Protein Kinase
  • Phosphoric Monoester Hydrolases
  • CDC14B protein, human
  • Dual-Specificity Phosphatases
  • benzyloxycarbonylleucyl-leucyl-leucine aldehyde
  • Acetylcysteine