SCF E3-mediated autoubiquitination negatively regulates activity of Cdc34 E2 but plays a nonessential role in the catalytic cycle in vitro and in vivo

K. Matthew Scaglione, Parmil K. Bansal, Andrew E. Deffenbaugh, Alexi Kiss, Johnnie M. Moore, Sergey Korolev, Ross Cocklin, Mark Goebl, Katsumi Kitagawa, Dorota Skowyra

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

One of the several still unexplained aspects of the mechanism by which the Cdc34/SCF RING-type ubiquitin ligases work is the marked stimulation of Cdc34 autoubiquitination, a phenomenon of unknown mechanism and significance. In in vitro experiments with single-lysine-containing Cdc34 mutant proteins of Saccharomyces cerevisiae, we found that the SCF-mediated stimulation of autoubiquitination is limited to specific N-terminal lysines modified via an intermolecular mechanism. In a striking contrast, SCF quenches autoubiquitination of C-terminal lysines catalyzed in an intramolecular manner. Unlike autoubiquitination of the C-terminal lysines, which has no functional consequence, autoubiquitination of the N-terminal lysines inhibits Cdc34. This autoinhibitory mechanism plays a nonessential role in the catalytic cycle, as the lysineless K0Cdc34ΔC is indistinguishable from Cdc34ΔC in ubiquitination of the prototype SCFCdc4 substrate Sic1 in vitro, and replacement of the CDC34 gene with either the K0cdc34ΔC or the cdc34ΔC allele in yeast has no cell cycle phenotype. We discuss the implications of these findings for the mechanism of Cdc34 function with SCF.

Original languageEnglish (US)
Pages (from-to)5860-5870
Number of pages11
JournalMolecular and cellular biology
Volume27
Issue number16
DOIs
StatePublished - Aug 1 2007

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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    Scaglione, K. M., Bansal, P. K., Deffenbaugh, A. E., Kiss, A., Moore, J. M., Korolev, S., Cocklin, R., Goebl, M., Kitagawa, K., & Skowyra, D. (2007). SCF E3-mediated autoubiquitination negatively regulates activity of Cdc34 E2 but plays a nonessential role in the catalytic cycle in vitro and in vivo. Molecular and cellular biology, 27(16), 5860-5870. https://doi.org/10.1128/MCB.01555-06