Two mutations impair the stability and function of ubiquitin-activating enzyme (E1)

J Cell Physiol. 2012 Apr;227(4):1561-8. doi: 10.1002/jcp.22870.


Protein ubiquitination plays critical roles in the regulation of multiple cellular processes including cell proliferation, signal transduction, oncogenesis, and hypoxic response. TS20 is a Balb3T3-derived cell line in which ubiquitination is inhibited by restrictive temperature. While TS20 has been used to elucidate the degradation of many important proteins including p53, p27, HIF-1α, and ornithine decarboxylase, the molecular basis of its temperature sensitivity has not been fully determined. We cloned full-length E1 cDNA from TS20. Sequencing analysis revealed two point mutations (nt736G to A and nt2313G to C) that lead to substitution of aa189A to T and aa714W to C, respectively. Transient transfection assays revealed that mutant E1 was less stable than its wild-type counterpart, and restrictive temperature (39°C) accelerated its degradation. Under permissive temperature, reverting aa714C to W significantly improved E1 stability and activity. Under restrictive temperature, reverting of both substitutions was required to fully restore E1 stability. Similar results were observed when the mutants were expressed in non-TS20 cells, indicating the mutations are sufficient for its temperature sensitive degradation observed in TS20 cells. Functionally, reverting aa714C to W was sufficient to facilitate the monoubiquitination of H2A and to support TS20 growth at 39°C. It also significantly improved the ubiquitination-dependent disposal of HIF-1α. Our data conclusively demonstrate that mutations introgenic to UVBE1 cause E1 instability, which leads to deficiency of E1 function. Our data establish the molecular basis for unambiguous interpretation of experimental data based on TS20 cells, and provide new insight into the structural determinants of E1 stability.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Apoptosis
  • BALB 3T3 Cells
  • Base Sequence
  • Cell Line
  • Cell Proliferation
  • DNA, Complementary / genetics
  • Enzyme Stability
  • HEK293 Cells
  • Humans
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics*
  • Mutant Proteins / metabolism*
  • Mutation, Missense*
  • Point Mutation*
  • Proteasome Endopeptidase Complex / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Temperature
  • Transfection
  • Ubiquitin-Activating Enzymes / chemistry
  • Ubiquitin-Activating Enzymes / genetics*
  • Ubiquitin-Activating Enzymes / metabolism*
  • Ubiquitination


  • DNA, Complementary
  • Mutant Proteins
  • Recombinant Proteins
  • Proteasome Endopeptidase Complex
  • Ubiquitin-Activating Enzymes