SUMO regulates p21Cip1 intracellular distribution and with p21Cip1 facilitates multiprotein complex formation in the nucleolus upon DNA damage

PLoS One. 2017 Jun 5;12(6):e0178925. doi: 10.1371/journal.pone.0178925. eCollection 2017.

Abstract

We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus.

MeSH terms

  • Cell Nucleolus / genetics
  • Cell Nucleolus / metabolism*
  • Cyclin E / genetics
  • Cyclin E / metabolism
  • Cyclin-Dependent Kinase 2 / genetics
  • Cyclin-Dependent Kinase 2 / metabolism
  • Cyclin-Dependent Kinase Inhibitor p21 / deficiency
  • Cyclin-Dependent Kinase Inhibitor p21 / genetics*
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism
  • DNA Damage
  • Gene Expression Regulation*
  • HCT116 Cells
  • Humans
  • Karyopherins / genetics
  • Karyopherins / metabolism
  • Organelle Biogenesis
  • Organelles / genetics
  • Organelles / metabolism*
  • Proliferating Cell Nuclear Antigen / genetics
  • Proliferating Cell Nuclear Antigen / metabolism
  • Promyelocytic Leukemia Protein / genetics
  • Promyelocytic Leukemia Protein / metabolism
  • Protein Binding
  • Protein Multimerization
  • Protein Transport
  • Proto-Oncogene Proteins c-mdm2 / genetics
  • Proto-Oncogene Proteins c-mdm2 / metabolism
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism
  • SUMO-1 Protein / genetics
  • SUMO-1 Protein / metabolism*
  • Signal Transduction
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism
  • Ubiquitin-Conjugating Enzymes / deficiency
  • Ubiquitin-Conjugating Enzymes / genetics

Substances

  • CDKN1A protein, human
  • Cyclin E
  • Cyclin-Dependent Kinase Inhibitor p21
  • Karyopherins
  • Proliferating Cell Nuclear Antigen
  • Promyelocytic Leukemia Protein
  • Receptors, Cytoplasmic and Nuclear
  • SUMO-1 Protein
  • SUMO1 protein, human
  • Tumor Suppressor Protein p53
  • exportin 1 protein
  • PML protein, human
  • Ubiquitin-Conjugating Enzymes
  • MDM2 protein, human
  • Proto-Oncogene Proteins c-mdm2
  • CDK2 protein, human
  • Cyclin-Dependent Kinase 2
  • Cysteine Endopeptidases
  • SENP2 protein, human
  • ubiquitin-conjugating enzyme UBC9

Grant support

This work was supported by: Ministerio de Economia y Competitividad (www.mineco.gob.es) grants: SAF2013–42742-R and SAF2016-76239-R to NA, BFU2014-54754-P to ML and SAF2016-80626-R to RF; Red Temática de investigación cooperativa en cáncer grant RD 12/0036/0049 to NA (www.rticc.org). NAb was recipient of a pre-doctoral fellowship from the Ministerio de Educación y Ciencia (Spain). Funding for open access charge: Ministerio de Economia y Competitividad grant SAF2016-76239-R to NA and University of Barcelona contribution.