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Published December 9, 2008 | Published + Supplemental Material
Journal Article Open

Regulation of peptide import through phosphorylation of Ubr1, the ubiquitin ligase of the N-end rule pathway


Substrates of the N-end rule pathway include proteins with destabilizing N-terminal residues. These residues are recognized by E3 ubiquitin ligases called N-recognins. Ubr1 is the N-recognin of the yeast Saccharomyces cerevisiae. Extracellular amino acids or short peptides up-regulate the peptide transporter gene PTR2, thereby increasing the capacity of a cell to import peptides. Cup9 is a transcriptional repressor that down-regulates PTR2. The induction of PTR2 by peptides or amino acids involves accelerated degradation of Cup9 by the N-end rule pathway. We report here that the Ubr1 N-recognin, which conditionally targets Cup9 for degradation, is phosphorylated in vivo at multiple sites, including Ser300 and Tyr277. We also show that the type-I casein kinases Yck1 and Yck2 phosphorylate Ubr1 on Ser300, and thereby make possible ("prime") the subsequent (presumably sequential) phosphorylations of Ubr1 on Ser296, Ser292, Thr288, and Tyr277 by Mck1, a kinase of the glycogen synthase kinase 3 (Gsk3) family. Phosphorylation of Ubr1 on Tyr277 by Mck1 is a previously undescribed example of a cascade-based tyrosine phosphorylation by a Gsk3-type kinase outside of autophosphorylation. We show that the Yck1/Yck2-mediated phosphorylation of Ubr1 on Ser300 plays a major role in the control of peptide import by the N-end rule pathway. In contrast to phosphorylation on Ser300, the subsequent (primed) phosphorylations, including the one on Tyr277, have at most minor effects on the known properties of Ubr1, including regulation of peptide import. Thus, a biological role of the rest of Ubr1 phosphorylation cascade remains to be identified.

Additional Information

© 2008 by the National Academy of Sciences. Contributed by Alexander Varshavsky, September 9, 2008 (received for review August 25, 2008). Published online before print November 25, 2008, doi: 10.1073/pnas.0808891105 We thank M. Longtine (Oklahoma State University, Stillwater, OK) for pFA6a-KanMX6 and pFA6a-SkHIS3MX6; L. C. Robinson (Louisiana State University, Shreveport, LA) for the LRB906 and LRB756 S. cerevisiae; K. W. Cunningham (Johns Hopkins University, Baltimore) for gsk3Δ S. cerevisiae; M. Snyder (Yale University, New Haven, CT) for a GST-kinase library; and G. Hathaway, J. Zhou, and S. Horvath (California Institute of Technology) for MS analyses and biotin/Tyr277-containing peptides. We thank T. Hunter for comments on the article. We are also grateful to the present and former members of the Varshavsky laboratory, particularly Z. Xia for advice and assistance, J. Sheng for permission to cite unpublished data, and to A. Shemorry for comments on the paper. This study was supported by grants from the National Institutes of Health (GM31530, DK39520, to A.V.), the Sandler Program in Asthma Research (A.V.), and the Ellison Medical Foundation (A.V.). Author contributions: C.-S.H. and A.V. designed research; C.-S.H. performed research; C.-S.H. and A.V. analyzed data; and C.-S.H. and A.V. wrote the paper. The authors declare no conflict of interest. This article contains supporting information online at www.pnas.org/cgi/content/full/0808891105/DCSupplemental.

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