Requirements for protein phosphorylation and the kinase activity of polo-like kinase 1 (Plk1) for the kinetochore function of mitotic arrest deficiency protein 1 (Mad1)

J Biol Chem. 2008 Dec 19;283(51):35834-44. doi: 10.1074/jbc.M804967200. Epub 2008 Oct 15.

Abstract

Mitotic arrest deficiency protein 1 (Mad1) is associated with microtubule-unattached kinetochores in mitotic cells and is a component of the spindle assembly checkpoint (SAC). Here, we have studied the phosphorylation of Mad1 and mapped using liquid chromatography-tandem mass spectrometry several phosphorylated amino acids in this protein. One phosphorylated residue, Thr680, was characterized to be important for the kinetochore localization of Mad1 and its SAC function. We also found that in mitotic cells Mad1 co-immunoprecipitated with Plk1. Depletion of cellular Plk1 using small interfering RNAs and inhibition of the kinase activity of Plk1 using a kinase-dead mutant or a small molecule inhibitor attenuated Mad1 phosphorylation and its association with kinetochores. Collectively, these findings indicate mechanistic roles contributed by protein phosphorylation and Plk1 to the SAC activity of Mad1.

MeSH terms

  • Cell Cycle Proteins / antagonists & inhibitors
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Chromatography, Liquid
  • HeLa Cells
  • Humans
  • Kinetochores / metabolism*
  • Mass Screening
  • Mitosis / physiology*
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Phosphorylation / physiology
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proto-Oncogene Proteins / antagonists & inhibitors
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • RNA, Small Interfering / genetics

Substances

  • Cell Cycle Proteins
  • MAD1L1 protein, human
  • Nuclear Proteins
  • Proto-Oncogene Proteins
  • RNA, Small Interfering
  • Protein-Serine-Threonine Kinases
  • polo-like kinase 1