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 Ser(300) and Tyr(277). We also show that the type-I casein kinases Yck1 and Yck2 phosphorylate Ubr1 on Ser(300), and thereby make possible ("prime") the subsequent (presumably sequential) phosphorylations of Ubr1 on Ser(296), Ser(292), Thr(288), and Tyr(277) by Mck1, a kinase of the glycogen synthase kinase 3 (Gsk3) family. Phosphorylation of Ubr1 on Tyr(277) 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 Ser(300) plays a major role in the control of peptide import by the N-end rule pathway. In contrast to phosphorylation on Ser(300), the subsequent (primed) phosphorylations, including the one on Tyr(277), 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.
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