Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1

J Cell Biol. 2019 Apr 1;218(4):1235-1249. doi: 10.1083/jcb.201810058. Epub 2019 Feb 12.

Abstract

The ATR kinase controls cell cycle transitions and the DNA damage response. ATR activity is regulated through two ATR-activating proteins, ETAA1 and TOPBP1. To examine how each activator contributes to ATR signaling, we used quantitative mass spectrometry to identify changes in protein phosphorylation in ETAA1- or TOPBP1-deficient cells. We identified 724, 285, and 118 phosphosites to be regulated by TOPBP1, ETAA1, or both ATR activators, respectively. Gene ontology analysis of TOPBP1- and ETAA1-dependent phosphoproteins revealed TOPBP1 to be a primary ATR activator for replication stress, while ETAA1 regulates mitotic ATR signaling. Inactivation of ATR or ETAA1, but not TOPBP1, results in decreased Aurora B kinase activity during mitosis. Additionally, ATR activation by ETAA1 is required for proper chromosome alignment during metaphase and for a fully functional spindle assembly checkpoint response. Thus, we conclude that ETAA1 and TOPBP1 regulate distinct aspects of ATR signaling with ETAA1 having a dominant function in mitotic cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Video-Audio Media

MeSH terms

  • Antigens, Surface / genetics
  • Antigens, Surface / metabolism*
  • Antineoplastic Agents, Phytogenic / pharmacology
  • Ataxia Telangiectasia Mutated Proteins / genetics
  • Ataxia Telangiectasia Mutated Proteins / metabolism*
  • Aurora Kinase B / genetics
  • Aurora Kinase B / metabolism
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Chromosome Segregation* / drug effects
  • DNA Damage
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Enzyme Activation
  • Gene Expression Regulation, Neoplastic
  • Genomic Instability
  • HCT116 Cells
  • HEK293 Cells
  • Humans
  • M Phase Cell Cycle Checkpoints
  • Mitosis* / drug effects
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Paclitaxel / pharmacology
  • Phosphorylation
  • Protein Interaction Maps
  • Proteomics / methods
  • Signal Transduction

Substances

  • Antigens, Surface
  • Antineoplastic Agents, Phytogenic
  • Carrier Proteins
  • DNA-Binding Proteins
  • ETAA1 protein, human
  • Nuclear Proteins
  • TOPBP1 protein, human
  • ATR protein, human
  • AURKB protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Aurora Kinase B
  • Paclitaxel