Abstract
The target of rapamycin complex 1 (TORC1) is an evolutionarily conserved sensor of nutrient availability. Genetic and pharmacological studies in the yeast Saccharomyces cerevisiae have provided mechanistic insights on the regulation of TORC1 signaling in response to nutrients. Using a highly specific antibody that recognizes phosphorylation of the bona fide TORC1 target ribosomal protein S6 (Rps6) in yeast, we found that nutrients rapidly induce Rps6 phosphorylation in a TORC1-dependent manner. Moreover, we demonstrate that Ypk3, an AGC kinase which exhibits high homology to human S6 kinase (S6K), is required for the phosphorylation of Rps6 in vivo. Rps6 phosphorylation is completely abolished in cells lacking Ypk3 (ypk3Δ), whereas Sch9, previously reported to be the yeast ortholog of S6K, is dispensable for Rps6 phosphorylation. Phosphorylation-deficient mutations in regulatory motifs of Ypk3 abrogate Rps6 phosphorylation, and complementation of ypk3Δ cells with human S6 kinase restores Rps6 phosphorylation in a rapamycin-sensitive manner. Our findings demonstrate that Ypk3 is a critical component of the TORC1 pathway and that the use of a phospho-S6 specific antibody offers a valuable tool to identify new nutrient-dependent and rapamycin-sensitive targets in vivo.
Highlights
Target of rapamycin (TOR) is a highly conserved serine/threonine kinase that is found in two structurally and functionally distinct complexes termed TOR complex 1 (TORC1) and TORC2 of which TORC1 is uniquely sensitive to rapamycin [1]
Since TORC1 activity is controlled by nutrients, in particular by nitrogen source availability [14,17,18], we investigated if ribosomal protein S6 (Rps6) phosphorylation is regulated by the nitrogen source
Our findings further indicate that Ypk3 functions as S6 kinase (S6K) in S. cerevisiae downstream of TORC1
Summary
Target of rapamycin (TOR) is a highly conserved serine/threonine kinase that is found in two structurally and functionally distinct complexes termed TOR complex 1 (TORC1) and TORC2 of which TORC1 is uniquely sensitive to rapamycin [1]. While almost all eukaryotes have a single TOR gene, several yeasts such as S. cerevisiae and S. pombe have two TOR genes. In budding yeast, both TOR1 and TOR2 can be part of TORC1, but TORC2 contains only TOR2 [2,3]. TORC1 acts as a nutritional sensor that couples nutrient availability to protein synthesis and cell growth. This signaling pathway controls the activity of a wide variety of kinases that help to PLOS ONE | DOI:10.1371/journal.pone.0120250. This signaling pathway controls the activity of a wide variety of kinases that help to PLOS ONE | DOI:10.1371/journal.pone.0120250 March 13, 2015
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