Convergence of Ubiquitylation and Phosphorylation Signaling in Rapamycin-Treated Yeast Cells

The target of rapamycin (TOR) kinase senses the availability of nutrients and coordinates cellular growth and proliferation with nutrient abundance. Inhibition of TOR mimics nutrient starvation and leads to the reorganization of many cellular processes, including autophagy, protein translation, and vesicle trafficking. TOR regulates cellular physiology by modulating phosphorylation and ubiquitylation signaling networks; however, the global scope of such regulation is not fully known. Here, we used a mass-spectrometry-based proteomics approach for the parallel quantification of ubiquitylation, phosphorylation, and proteome changes in rapamycin-treated yeast cells. Our data constitute a detailed proteomic analysis of rapamycin-treated yeast with 3590 proteins, 8961 phosphorylation sites, and 2299 di-Gly modified lysines (putative ubiquitylation sites) quantified. The phosphoproteome was extensively modulated by rapamycin treatment, with more than 900 up-regulated sites one hour after rapamycin treatment. Dynamically regulated phosphoproteins were involved in diverse cellular processes, prominently including transcription, membrane organization, vesicle-mediated transport, and autophagy. Several hundred ubiquitylation sites were increased after rapamycin treatment, and about half as many decreased in abundance. We found that proteome, phosphorylation, and ubiquitylation changes converged on the Rsp5-ubiquitin ligase, Rsp5 adaptor proteins, and Rsp5 targets. Putative Rsp5 targets were biased for increased ubiquitylation, suggesting activation of Rsp5 by rapamycin. Rsp5 adaptor proteins, which recruit target proteins for Rsp5-dependent ubiquitylation, were biased for increased phosphorylation. Furthermore, we found that permeases and transporters, which are often ubiquitylated by Rsp5, were biased for reduced ubiquitylation and reduced protein abundance. The convergence of multiple proteome-level changes on the Rsp5 system indicates a key role of this pathway in the response to rapamycin treatment. Collectively, these data reveal new insights into the global proteome dynamics in response to rapamycin treatment and provide a first detailed view of the co-regulation of phosphorylation- and ubiquitylation-dependent signaling networks by this compound

SPATA2 links CYLD to the TNF-α receptor signaling complex and modulates the receptor signaling outcomes

TNF‐α is a key regulator of innate immune and proinflammatory responses. However, the composition of the TNF‐α receptor‐associated signaling complexes (TNF‐RSC) and the architecture of the downstream signaling networks are incompletely understood. We employed quantitative mass spectrometry to demonstrate that TNF‐α stimulation induces widespread protein phosphorylation and that the scope of phosphorylation expands in a temporal manner. TNF‐α stimulation also induces rapid ubiquitylation of components of the TNF‐RSC. Temporal analysis of the TNF‐RSC composition identified SPATA2 as a novel component of the TNF‐RSC. The predicted PUB domain in the N‐terminus of SPATA2 interacts with the USP domain of CYLD, whereas the C‐terminus of SPATA2 interacts with HOIP. SPATA2 is required for recruitment of CYLD to the TNF‐RSC. Downregulation of SPATA2 augments transcriptional activation of NF‐κB and inhibits TNF‐α‐induced necroptosis, pointing to an important function of SPATA2 in modulating the outcomes of TNF‐α signaling. Taken together, our study draws a detailed map of TNF‐α signaling, identifies SPATA2 as a novel component of TNF‐α signaling, and provides a rich resource for further functional investigations

Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions

Acetylation is frequently detected on mitochondrial enzymes, and the sirtuin deacetylase SIRT3 is thought to regulate metabolism by deacetylating mitochondrial proteins. However, the stoichiometry of acetylation has not been studied and is important for under-standing whether SIRT3 regulates or suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoi-chiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By exam-ining acetylation changes in the liver, heart, brain, and brown adipose tissue of fasted mice, we found that SIRT3-targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3-dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetyla-tion sites, and greater sensitivity of SIRT3-targeted sites to chemi-cal acetylation in vitro and fasting-induced acetylation in vivo, suggest a nonenzymatic mechanism of acetylation. Our data indi-cate that most mitochondrial acetylation occurs as a low-level nonenzymatic protein lesion and that SIRT3 functions as a protein repair factor that removes acetylation lesions from lysine residues

A quantitative 14-3-3 interaction screen connects the nuclear exosome targeting complex to the DNA damage response

RNA metabolism is altered following DNA damage, but the underlying mechanisms are not well understood. Through a 14-3-3 interaction screen for DNA damage-induced protein interactions in human cells, we identified protein complexes connected to RNA biology. These include the nuclear exosome targeting (NEXT) complex that regulates turnover of noncoding RNAs termed promoter upstream transcripts (PROMPTs). We show that the NEXT subunit RBM7 is phosphorylated upon DNA damage by the MAPKAPK2 kinase and establish that this mediates 14-3-3 binding and decreases PROMPT binding. These findings and our observation that cells lacking RBM7 display DNA damage hypersensitivity link PROMPT turnover to the DNA damage response

The Spindle Assembly Checkpoint Is Not Essential for Viability of Human Cells with Genetically Lowered APC/C Activity

The anaphase-promoting complex/cyclosome (APC/C) and the spindle assembly checkpoint (SAC), which inhibits the APC/C, are essential determinants of mitotic timing and faithful division of genetic material. Activation of the APC/C is known to depend on two APC/C-interacting E2 ubiquitin-conjugating enzymes—UBE2C and UBE2S. We show that APC/C activity in human cells is tuned by the combinatorial use of three E2s, namely UBE2C, UBE2S, and UBE2D. Genetic deletion of UBE2C and UBE2S, individually or in combination, leads to discriminative reduction in APC/C function and sensitizes cells to UBE2D depletion. Reduction of APC/C activity results in loss of switch-like metaphase-to-anaphase transition and, strikingly, renders cells insensitive to chemical inhibition of MPS1 and genetic ablation of MAD2, both of which are essential for the SAC. These results provide insights into the regulation of APC/C activity and demonstrate that the essentiality of the SAC is imposed by the strength of the APC/C