Non-degradative Ubiquitination of Protein Kinases

PLoS Comput Biol. 2016 Jun 2;12(6):e1004898. doi: 10.1371/journal.pcbi.1004898. eCollection 2016 Jun.

Abstract

Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Binding Sites
  • Enzyme Activation
  • HEK293 Cells
  • Humans
  • Jurkat Cells
  • Models, Chemical
  • Molecular Docking Simulation / methods*
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Mapping / methods*
  • Protein Kinases / chemistry*
  • Protein Kinases / ultrastructure*
  • Substrate Specificity
  • Ubiquitinated Proteins / chemistry*
  • Ubiquitinated Proteins / ultrastructure*

Substances

  • Ubiquitinated Proteins
  • Protein Kinases