Asc1p/RACK1 Connects Ribosomes to Eukaryotic Phosphosignaling

ABSTRACT

WD40 repeat proteins fold into characteristic β-propeller structures and control signaling circuits during cellular adaptation processes within eukaryotes. The RACK1 protein of Saccharomyces cerevisiae, Asc1p, consists exclusively of a single seven-bladed β-propeller that operates from the ribosomal base at the head region of the 40S subunit. Here we show that the R38D K40E ribosomal binding-compromised variant (Asc1DEp) is severely destabilized through mutation of phosphosite T143 to a dephosphorylation-mimicking alanine, probably through proteasomal degradation, leading to asc1⁻ phenotypes. Phosphosite Y250 contributes to resistance to translational inhibitors but does not influence Asc1DEp stability. Beyond its own phosphorylation at T143, Y250, and other sites, Asc1p heavily influences the phosphorylation of as many as 90 proteins at 120 sites. Many of these proteins are regulators of fundamental processes ranging from mRNA translation to protein transport and turnover, cytoskeleton organization, and cellular signaling. Our data expose Asc1p/RACK1 as a key factor in phosphosignaling and manifest it as a control point at the head of the ribosomal 40S subunit itself regulated through posttranslational modification.

KEY WORDS::

• Asc1/RACK1
• Saccharomyces cerevisiae
• protein phosphorylation
• quantitative proteomics
• ribosome
• signal transduction network

Supplemental material for this article may be found at https://doi.org/10.1128/MCB.00279-16.

ACKNOWLEDGMENTS

We thank Sabrina Sander for her experimental support and Mahdokht Kohansal Nodehi (Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany) for sharing her expertise in phosphopeptide enrichment with us. We thank Heike Krebber for providing plasmid pHK697 and the Rps3p antibody. We thank Andrew Link (Vanderbilt University Medical Center, Nashville, TN) and Hans-Ulrich Mösch (Philipps-Universität, Marburg, Germany) for providing Asc1p and Tec1p antibodies, respectively.

This work was supported by the Göttingen Graduate School for Neurosciences and Molecular Biosciences (DFG Grant GSC 226/2) and by DFG Grant VA352/2-1.