The acidic tail domain of human Cdc34 is required for p27Kip1 ubiquitination and complementation of a cdc34 temperature sensitive yeast strain

Karen Block, Srikanth Appikonda, Horng Ru Lin, Joanna Bloom, Michele Pagano, P. Renee Yew

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

Human Cdc34 is an ubiquitin conjugating enzyme or E2 that ubiquitinates substrates including p27Kip1, IκBα, Wee1, and MyoD. Cdc34 possesses a core catalytic domain encoding the active site cysteine and an acidic tail domain within the carboxyl terminal 36 amino acids. Studies suggest that Cdc34 is phosphorylated in mammalian cells at 5 potential residues within the tail domain. In order to study the biological significance of the Cdc34 acidic tail domain and the possible significance of phosphorylation within this region, we tested the ability of human Cdc34 mutants to complement the cdc34-2 temperature sensitive (ts) strain of Saccharomyces cerevisiae. Our studies indicated that complementation of the cdc34-2 ts strain was critically dependent upon the carboxyl-terminal 36 amino acids of human Cdc34, but did not require phosphorylation of human Cdc34 residues S203, S222, S231, T233, and S236. Further studies demonstrated that although a Cdc34 mutant bearing a deletion of the C-terminal 36 amino acids (Cdc34 1-200) was efficiently charged with ubiquitin by E1, it was severely reduced for the ability to ubiquitinate p27Kip1 in vitro compared to wildtype Cdc34. Both in vivo and in vitro binding studies indicated that Cdc34 1-200 bound to the E3-SCF components, Cul1 and Roc1, at levels comparable to the wildtype Cdc34. These studies suggest that the 36 amino acid acidic tail domain of human Cdc34 is critical for its ability to transfer ubiquitin to a substrate and is dispensable for the association of Cdc34 with Cul1 and Roc1. We postulate that the tail domain of Cdc34 may be important for its efficient dissociation from Cul1 and Roc1, an essential requirement for ubiquitination by the budding yeast Cdc34p, or it may be required more directly for ubiquitin transfer to the substrate.

Original languageEnglish (US)
Pages (from-to)1421-1427
Number of pages7
JournalCell Cycle
Volume4
Issue number10
DOIs
StatePublished - Oct 2005

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Keywords

  • Cdc34
  • Cul1
  • Roc1
  • Ubiquitin
  • p27Kip1

ASJC Scopus subject areas

  • Molecular Biology
  • Developmental Biology
  • Cell Biology

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