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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 105; no. 49; pp. 19188 - 19193
• Main Authors: Hwang, Cheol-Sang, Varshavsky, Alexander
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 09.12.2008; National Acad Sciences
• Subjects: Amino acids; Antibodies; Binding Sites; Biological Sciences; Casein; Casein Kinase I - metabolism; Cell extracts; Cells; Dipeptides - metabolism; Escherichia coli - genetics; Eukaryotic cells; Gene expression regulation; Glucose - metabolism; Glycogen Synthase Kinase 3; Immunoblotting; Kinases; Mass Spectrometry; peptide transporter; Peptides; Phosphorylation; Protein-Tyrosine Kinases - metabolism; Proteins; Repressors; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Serine - metabolism; Substrate Specificity; Threonine - metabolism; Transcription; Tyrosine; Tyrosine - metabolism; Ubiquitin-protein ligase; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitins; Yeast; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0808891105

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.