Abstract
The c-Myc transcription factor is a key regulator of cell proliferation and cell fate decisions. c-Myc overexpression is observed in a variety of human tumors, revealing the importance of maintaining normal levels of c-Myc protein. c-Myc protein stability in mammalian cells is controlled by interdependent and sequential phosphorylation and dephosphorylation events on two highly conserved residues, serine 62 and threonine 58. Here we show that these sequential phosphorylation and dephosphorylation events and their effect on c-Myc stability also occurs in the model system Saccharomyces cerevisiae. These results suggest the presence of a conserved pathway in yeast that controls protein turnover in response to a specific phospho-degron sequence. These findings have implications regarding conserved pathways for regulated protein degradation, and they validate the use of genetically tractable yeast for the study of the turnover of proteins such as c-Myc that contain this motif.
Highlights
We and others have previously reported that the stability of c-Myc protein in mammalian cells is controlled by sequential phosphorylation and dephosphorylation events on two highly
A cis to trans isomerization at the bond proceeding serine 62 is catalyzed by the peptidylprolyl isomerase, Pin1, allowing the stabilizing serine 62 phosphate to be removed by the transspecific phosphatase, protein phosphatase 2A (PP2A)
28.1 Ϯ 1.0 min, a 3-fold increase when compared with mean c-Myc half-life in a wild type strain. These results demonstrate that in S. cerevisiae, Rim11p participates in the phosphorylation of c-Myc at Thr-58 and that this phosphorylation leads to c-Myc destabilization, similar to the activity of GSK3 on c-Myc in mammalian cells
Summary
Yeast Media and Reagents—All strains used were haploid and isogenic with BY4741 (Mata his3⌬1 leu2⌬0 met15⌬0 ura3⌬0) or W303 (Mata ade his ura leu112 trp can100). Cells were grown in selective media or 1% yeast extract, 2% peptone, adenine, 2% glucose at 23, 30, or 37 °C as indicated. Galactose was added to the media to a final concentration of 2% to induce expression of the c-myc gene from the GAL1 promoter for 1–3 h at the indicated temperatures. Primary antibodies were diluted in 1:1 Odyssey Blocking buffer:PBS with 0.05% Tween 20 or in 2.5% nonfat milk PBS, 0.05% Tween (P-Thr-58 antibody). Secondary antibodies were diluted 1:10,000 in 1:1 Odyssey Blocking buffer:PBS, 0.05% Tween or in 1.25% nonfat milk PBS, 0.05% Tween (P-Thr-58 antibody). C-Myc and Cdc protein levels were quantitated using LI-COR Odyssey Infrared Imager software Version 1.2. The TAP-Kss and TAP-Rim strains were grown overnight in 1% yeast extract, 2% peptone, adenine, 2% glucose to an optical density at 600 nm of 0.5.
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