Global Analysis of Phosphoproteome Regulation by the Ser/Thr Phosphatase Ppt1 in Saccharomyces cerevisiae

Abstract

Even though protein phosphatases are key regulators of signal transduction, their cellular mechanisms of action are poorly understood. Here, we undertook a large-scale proteomics survey to identify cellular protein targets of a serine/threonine phosphatase. We used SILAC-based quantitative MS to measure differences in protein expression and phosphorylation upon ablation of the serine/threonine phosphatase Ppt1 in Saccharomyces cerevisiae. Phosphopeptide fractionation by strong cation exchange chromatography combined with immobilized metal affinity chromatography (IMAC) enrichment enabled quantification of more than 8000 distinct phosphorylation sites in Ppt1 wild-type versus Ppt1-deficient yeast cells. We further quantified the relative expression of 1897 yeast proteins and detected no major protein changes accompanying Ppt1 deficiency. Notably, we found 33 phosphorylation sites to be significantly and reproducibly up-regulated while no phosphorylation events were repressed in cells lacking Ppt1. Ppt1 acted on its cellular target proteins in a sequence- and site-specific fashion. Several of the regulated phosphoproteins were involved in the response to heat stress in agreement with known Ppt1 functions. Additionally, biosynthetic enzymes were particularly prominent among Ppt1-regulated phosphoproteins, pointing to unappreciated roles of Ppt1 in the control of various metabolic functions. These results demonstrate the utility of large-scale and quantitative phosphoproteomics to identify cellular sites of serine/threonine phosphatase action in an unbiased manner.

2-Deoxyglucose Impairs Saccharomyces cerevisiae Growth by Stimulating Snf1-Regulated and α-Arrestin-Mediated Trafficking of Hexose Transporters 1 and 3

“…Hence, Ser447 is not an important site, or not a sufficient site, for the Snf1-dependent regulation of Rod1. Indeed, more-recent phosphoproteomic studies have identified 31 phosphorylation sites on Rod1 by mass spectroscopy (63)(64)(65)(66). Whether any of these sites are phosphorylated by Snf1 and regulate Rod1 function will be important to determine in subsequent studies.…”

Mechanistic aspects of the Hsp90 phosphoregulation

“…The finding that the phosphatase Ppt1 acts on two of the active sites further emphasizes its important role in the Hsp90 multichaperone complexes. 10 In summary, this study together with work from the Neckers lab 6,11 contributes to closing the gap in our understanding of the relationship between phosphorylation and Hsp90 chaperone activity. and the C-terminal domain of Hsp90 (s602 and s604) (left and middle part) (PdB 2CG9 of the full-length yeast Hsp90 crystal structure and PdB 2CGE of the Hsp90 M-C domain crystal structure).…”

Activation and inhibition of Snf1 kinase activity by phosphorylation within the activation loop

“…We purified the Snf1 heterotrimer containing the Gal83-N1 protein (Fig 5C) which lacks residues 12–141. In vitro kinase assays from our lab (13) as well as numerous mass spectrometry studies of the yeast phosphoproteome (21–26, 28–32) have localized the primary phosphorylation sites to this N-terminal region of Gal83 (Appendix A). When incubated with Sak1 kinase, we detect incorporation of labeled phosphate into the Snf1 subunit and the full-length Gal83 subunit (Fig.…”