Polo-like kinase 1 creates the tension-sensing 3F3/2 phosphoepitope and modulates the association of spindle-checkpoint proteins at kinetochores

Curr Biol. 2005 Jun 21;15(12):1078-89. doi: 10.1016/j.cub.2005.05.026.

Abstract

Background: In mitosis, a mechanochemical system recognizes tension that is generated by bipolar microtubule attachment to sister kinetochores. This is translated into multiple outputs including the stabilization of microtubule attachments, changes in kinetochore protein dynamics, and the silencing of the spindle checkpoint. How kinetochores sense tension and translate this into various signals represent critical unanswered questions. The kinetochores of chromosomes not under tension are specifically phosphorylated at an epitope recognized by the 3F3/2 monoclonal antibody. Determining the kinase that generates the 3F3/2 phosphoepitope at kinetochores should reveal an important component of this system that regulates mitotic progression.

Results: We demonstrate that Polo-like kinase 1 (Plk1) creates the 3F3/2 phosphoepitope on mitotic kinetochores. In a permeabilized in vitro cell system, the depletion of Xenopus Plk1 from M phase extract leads to the loss of 3F3/2 kinase activity. Purified recombinant Plk1 is sufficient to generate the 3F3/2 phosphoepitope in this system. Using siRNA, we show that the reduction of Plk1 protein levels significantly diminishes 3F3/2 phosphoepitope expression at kinetochores. The consensus phosphorylation sites of Plk1 show strong similarity to the 3F3/2 phosphoepitope sequence determined by phosphopeptide mapping. The inhibition of Plk1 by siRNA alters the normal kinetochore association of Mad2, Cenp-E, Hec1/Ndc80, Spc24, and Cdc20 and induces a spindle-checkpoint-mediated mitotic arrest.

Conclusions: Plk1 generates the 3F3/2 phosphoepitope at kinetochores that are not under tension and contributes to the normal kinetochore association of several key proteins important in checkpoint signaling. Mechanical tension regulates Plk1 accumulation at kinetochores and possibly its kinase activity.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Animals
  • Antibodies, Monoclonal / metabolism
  • Cdc20 Proteins
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / immunology
  • Cell Cycle Proteins / metabolism*
  • Cells, Cultured
  • Chromosomal Proteins, Non-Histone / metabolism
  • Cytoskeletal Proteins
  • Epitopes / metabolism*
  • HeLa Cells
  • Humans
  • Kinetochores / metabolism*
  • Mad2 Proteins
  • Microtubule-Associated Proteins / metabolism
  • Mitosis
  • Nuclear Proteins / metabolism
  • Phosphorylation
  • Protein Kinases / genetics
  • Protein Kinases / immunology
  • Protein Kinases / metabolism*
  • Protein-Serine-Threonine Kinases
  • Proteins / metabolism*
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / immunology
  • Proto-Oncogene Proteins / metabolism*
  • RNA, Small Interfering / genetics
  • Spindle Apparatus / genetics
  • Spindle Apparatus / metabolism*
  • Xenopus

Substances

  • Antibodies, Monoclonal
  • Cdc20 Proteins
  • Cdc20 protein, mouse
  • Cell Cycle Proteins
  • Chromosomal Proteins, Non-Histone
  • Cytoskeletal Proteins
  • Epitopes
  • Mad2 Proteins
  • Mad2l1 protein, mouse
  • Mad2l2 protein, mouse
  • Microtubule-Associated Proteins
  • NDC80 protein, human
  • Nuclear Proteins
  • Proteins
  • Proto-Oncogene Proteins
  • RNA, Small Interfering
  • SPC24 protein, human
  • centromere protein E
  • Protein Kinases
  • Protein-Serine-Threonine Kinases
  • polo-like kinase 1