Casein kinase-2-mediated phosphorylation increases the SUMO-dependent activity of the cytomegalovirus transactivator IE2

J Biol Chem. 2019 Oct 4;294(40):14546-14561. doi: 10.1074/jbc.RA119.009601. Epub 2019 Aug 1.

Abstract

Many viral factors manipulate the host post-translational modification (PTM) machinery for efficient viral replication. In particular, phosphorylation and SUMOylation can distinctly regulate the activity of the human cytomegalovirus (HCMV) transactivator immediate early 2 (IE2). However, the molecular mechanism of this process is unknown. Using various structural, biochemical, and cell-based approaches, here we uncovered that IE2 exploits a cross-talk between phosphorylation and SUMOylation. A scan for small ubiquitin-like modifier (SUMO)-interacting motifs (SIMs) revealed two SIMs in IE2, and a real-time SUMOylation assay indicated that the N-terminal SIM (IE2-SIM1) enhances IE2 SUMOylation up to 4-fold. Kinetic analysis and structural studies disclosed that IE2 is a SUMO cis-E3 ligase. We also found that two putative casein kinase 2 (CK2) sites adjacent to IE2-SIM1 are phosphorylated in vitro and in cells. The phosphorylation drastically increased IE2-SUMO affinity, IE2 SUMOylation, and cis-E3 activity of IE2. Additional salt bridges between the phosphoserines and SUMO accounted for the increased IE2-SUMO affinity. Phosphorylation also enhanced the SUMO-dependent transactivation activity and auto-repression activity of IE2. Together, our findings highlight a novel mechanism whereby SUMOylation and phosphorylation of the viral cis-E3 ligase and transactivator protein IE2 work in tandem to enable transcriptional regulation of viral gene.

Keywords: SUMO-interacting motif (SIM); enzyme kinetics; host–pathogen interaction; nuclear magnetic resonance (NMR); phosphorylation; post-translational modification (PTM); sumoylation; transcription co-activator; viral protein; viral transcription.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Casein Kinase II / chemistry
  • Casein Kinase II / genetics*
  • Cytomegalovirus / enzymology
  • Cytomegalovirus / genetics
  • Gene Expression Regulation, Viral / genetics
  • Humans
  • Immediate-Early Proteins / chemistry
  • Immediate-Early Proteins / genetics*
  • Immediate-Early Proteins / metabolism
  • Kinetics
  • Phosphorylation / genetics*
  • Protein Interaction Domains and Motifs / genetics
  • Protein Processing, Post-Translational
  • SUMO-1 Protein / chemistry
  • SUMO-1 Protein / genetics*
  • SUMO-1 Protein / metabolism
  • Small Ubiquitin-Related Modifier Proteins / chemistry
  • Small Ubiquitin-Related Modifier Proteins / genetics
  • Sumoylation / genetics*
  • Trans-Activators / chemistry
  • Trans-Activators / genetics*
  • Trans-Activators / metabolism
  • Ubiquitin-Protein Ligases / chemistry
  • Ubiquitin-Protein Ligases / genetics
  • Virus Replication / genetics

Substances

  • IE2 protein, Cytomegalovirus
  • Immediate-Early Proteins
  • SUMO-1 Protein
  • Small Ubiquitin-Related Modifier Proteins
  • Trans-Activators
  • Ubiquitin-Protein Ligases
  • Casein Kinase II

Associated data

  • PDB/1Z5S
  • PDB/2VRR
  • PDB/4WJO
  • PDB/1WM3
  • PDB/6K5T
  • PDB/6K5R
  • PDB/2UYZ