Identification of ubiquitin Ser57 kinases regulating the oxidative stress response in yeast

Abstract

1Ubiquitination regulates many different cellular processes, including protein quality control, 2 membrane trafficking, and stress responses. The diversity of ubiquitin functions in the cell is 3 partly due to its ability to form chains with distinct linkages that can alter the fate of substrate 4 proteins in unique ways. The complexity of the ubiquitin code is further enhanced by post-5 translational modifications on ubiquitin itself, the biological functions of which are not well 6understood. Here, we present genetic and biochemical evidence that serine 57 (Ser57) 7 phosphorylation of ubiquitin functions in stress responses in Saccharomyces cerevisiae, 8including the oxidative stress response. We also identify and characterize the first known Ser57 9 ubiquitin kinases in yeast and human cells, and we report that two Ser57 ubiquitin kinases 10 regulate the oxidative stress response in yeast. These studies implicate ubiquitin 11 phosphorylation at the Ser57 position as an important modifier of ubiquitin function, particularly 12 in response to proteotoxic stress. 13

Regulation of ubiquitin and ubiquitin‐like modifiers by phosphorylation

“…We found that S57D-Ub was associated with several stressresistance phenotypes, conferring resistance to heat stress and toxic amino acid analogs such as canavanine and thialysine [35,36]. Interestingly, S57D-Ub also conferred hypersensitivity to hydroxyurea, while expression of S57A-Ub conferred hypersensitivity to heat and oxidative stress [36]. Together, these findings underscore the importance of the Ser57 position during conditions of stress.…”

“…More directed studies have uncovered phenotypes associated with both S57A (phosphorylation-resistant) and S57D (phosphomimetic) Ub variants. We found that S57D-Ub was associated with several stressresistance phenotypes, conferring resistance to heat stress and toxic amino acid analogs such as canavanine and thialysine [35,36]. Interestingly, S57D-Ub also conferred hypersensitivity to hydroxyurea, while expression of S57A-Ub conferred hypersensitivity to heat and oxidative stress [36].…”

“…The blue box highlights several important questions that remain to be answered in this new arena of regulatory biology. Gin4, Kcc4 [35,36] Thr12 Unknown Unknown (Fig. 2), a site for K63-linked Ub polymerization that modulates DNA repair [19], cell cycle control [20], transcriptional regulation [21], endocytic trafficking [22], and inflammatory signaling [23].…”

“…Ser57-phosphorylated ubiquitin (pS57-Ub) was the first phospho-Ub species discovered in a pioneering proteomic analysis of the ubiquitin-modified proteome of yeast [9]. Since then, other studies have reported pS57-Ub in yeast [34][35][36] as well as in different human cells and tissues-including breast cancer tissues [37], melanoma cells [38], and lung adenocarcinoma cells [39,40]. However, alanine scanning mutagenesis of ubiquitin had revealed that Ser57 is not required for the essential functions of ubiquitin in yeast [17].…”

“…This effort initially uncovered a single yeast kinase-the Snf1related kinase Vhs1-as a Ser57 ubiquitin kinase. However, subsequent rounds of screening uncovered additional yeast Snf1-related kinases capable of phosphorylating ubiquitin at the Ser57 positionincluding Sks1 (the Vhs1 paralog), Gin4, and Kcc4 [36]. Importantly, both Sks1 and Vhs1 were capable of generating pS57-Ub when expressed in yeast.…”