Ubiquitin is a post-translational modifier that regulates diverse cellular processes in eukaryotic cells. The broad utility of ubiquitin as a regulatory modification is due to the high degree of complexity associated with ubiquitin polymers, which are added to substrate proteins by the activity of ubiquitin conjugation machinery (E1-E2-E3 cascades) and removed from substrates by deubiquitylases (DUBs). Ubiquitin can be conjugated recursively at any of seven internal lysines or the N-terminus to generate polymers with distinct topological features (Herhaus and Dikic, 2015; Yau and Rape, 2016; Swatek and Komander, 2016). Complexity is further enhanced by the formation of mixed and branched polymers (Ohtake et al., 2018; Meyer and Rape, 2014; Swatek et al., 2019) and by post-translational modifications that can occur on ubiquitin itself (Herhaus and Dikic, 2015). For example, PINK1-mediated phosphorylation of ubiquitin at the Ser65 position plays an important role in mitophagy by regulating parkin-mediated ubiquitination of mitochondrial membrane proteins (Wauer et al., 2015; Ordureau et al., 2015; Koyano et al., 2014; Kazlauskaite et al., 2014; Kane et al., 2014; Ordureau et al., 2014). Phosphorylation of ubiquitin at the Ser57 has also been reported (Peng et al., 2003; Swaney et al., 2015; Lee et al., 2017), but the kinases that produce this modification and its regulatory significance remain unknown.

Many proteotoxic stresses activate ubiquitin networks to promote protein quality control and protect the cell from damage associated with systemic protein misfolding. Oxidative stress is highly damaging to the cell, triggering deployment and re-distribution of existing ubiquitin pools and induction of ubiquitin biosynthesis to promote survival by activating a repertoire of ubiquitin-mediated responses (Cheng et al., 1994). During oxidative stress, many proteins become damaged and misfolded, resulting in a global increase in K48-linked ubiquitin conjugation that targets substrates for clearance by proteasome-mediated degradation (Finley, 2009; Shang and Taylor, 2011). Oxidative stress also triggers translation arrest (Grant, 2011), resulting in K63-linked polyubiquitylation on ribosomes to stabilize the 80S complex and the formation of polysomes (Silva et al., 2015). Furthermore, oxidative damage of DNA activates signaling and repair processes that are tightly regulated by K63 ubiquitylation and deubiquitylation activities (Demple and Harrison, 1994; Bergink and Jentsch, 2009; Ng et al., 2016; Croteau and Bohr, 1997; Thorslund et al., 2015). Thus, ubiquitin networks regulate many cellular processes critical for survival during conditions of oxidative stress.

Although ubiquitin networks play a critical role in the eukaryotic cellular response to proteotoxic stress, precisely how these networks are tuned to enhance protein quality control and other protective functions remain unclear. Given that Ser65 phosphorylation of ubiquitin regulates the clearance of damaged mitochondria (Wauer et al., 2015; Ordureau et al., 2015; Koyano et al., 2014; Kazlauskaite et al., 2014; Kane et al., 2014; Ordureau et al., 2014), we hypothesized that phosphorylation at other positions may regulate ubiquitin function, particularly in conditions that promote protein damage and misfolding. Since it is the most abundant phosphorylated form (Swaney et al., 2015), we examined the biological functions of Ser57 phosphorylated ubiquitin in yeast, aiming to identify and characterize the molecular events and signaling processes that regulate its production.

To probe potential biological functions of Ser57 ubiquitin phosphorylation in yeast, we generated yeast strains expressing exclusively wildtype, Ser57Ala (phosphorylation resistant, or S57A) or Ser57Asp (phosphomimetic, or S57D) ubiquitin. It is important to emphasize that such complete ubiquitin replacement may exaggerate effects associated with physiological ubiquitin phosphorylation, which occurs at very low stoichiometry (Swaney et al., 2015; Lee et al., 2017) and probably in a highly localized manner (as exemplified by PINK1-mediated Ser65 phosphorylation of ubiquitin on damaged segments of mitochondrial membrane [Pickrell and Youle, 2015]). With these limitations in mind, we examined the growth of these yeast strains in the context of various stressors, including heat stress, DNA damage and replication stress (hydroxyurea and arsenate), and protein misfolding stress (canavanine and thialysine, which are toxic analogs of arginine and lysine, respectively). We found that expression of S57A or S57D ubiquitin did not affect growth at ambient temperature (26°C) (consistent with previous reports [Peng et al., 2003; Lee et al., 2017; Sloper-Mould et al., 2001]) or sensitivity to arsenate (Figure 1A). However, expression of S57D ubiquitin conferred sensitivity to hydroxyurea and resistance to canavanine (which was reported previously [Lee et al., 2017]) and thialysine (Figure 1A). We also noticed that expression of S57D ubiquitin enhanced both long-term and acute tolerance of thermal stress (Figure 1B–D) while yeast expressing S57A ubiquitin exhibited thermal sensitivity (Figure 1E and Figure 1—figure supplement 1). These findings indicate that Ser57 phosphorylation of ubiquitin promotes cellular tolerance of various proteotoxic stressors.

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Quantification and statistical analysis for viability stain measurements (Figure 1D) and cell growth measurements (Figure 1E and Figure 1—figure supplement 1).

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Next, we analyzed the role of Ser57 phosphorylation in the oxidative stress response. Wildtype yeast cells arrest growth in response to oxidative stress and activate responses that help cells cope with the proteotoxic and DNA damaging effects of oxidation (Silva et al., 2015; Shapira et al., 2004; Petti et al., 2011; Martindale and Holbrook, 2002). Interestingly, while yeast cells expressing wildtype or S57D ubiquitin arrested growth in response to moderate oxidative stress (>1 mM H₂O₂), cells expressing S57A ubiquitin were deficient in this response and only arrested growth in response to more severe oxidative stress (>2 mM H₂O₂) (Figure 2A). The failed growth arrest observed for cells expressing S57A ubiquitin correlated with decreased viability (Figure 2B). Since oxidative stress induces the production of both K48- and K63-linked ubiquitin conjugates (Shang and Taylor, 2011; Silva et al., 2015; Sun et al., 2009; Tsirigotis et al., 2001) we tested if the expression of S57A and S57D ubiquitin alters ubiquitin conjugation patterns in response to oxidative stress. We found that 30 min exposure to H₂O₂ (1.0 mM) resulted in an increased abundance of total ubiquitin conjugates (Figure 2C) as well as K48-linked polymers (Figure 2C–D), consistent with a previous report (Silva et al., 2015). Compared to cells expressing wildtype ubiquitin, we found that cells expressing S57D ubiquitin exhibited increased abundance of K48-linked polymers and decreased abundance of K63-linked polymers (Figure 2C–E). By contrast, cells expressing S57A ubiquitin did not exhibit a significant increase in the production of K48-linked polyubiquitin chains in response to oxidative stress (Figure 2C–D). Since substitutions at the Ser57 position of ubiquitin (S57A, S57D) might interfere with the recognition of ubiquitin polymers by linkage-specific antibodies, we used SILAC-MS to analyze how the expression of phosphomimetic (S57D) ubiquitin affects ubiquitin polymer linkage abundance compared to wildtype ubiquitin during conditions of oxidative stress. Consistent with the immunoblot results, this analysis revealed that S57D expression increases the production of K48-linked polymers by 39% during oxidative stress (Figure 2—figure supplement 1 and Supplementary file 1). (Notably, K63-linked polymers are a blind spot of this analysis, since the K63-linked ubiquitin remnant peptide also harbors the Ser57 position which is mutated to Asp in the light channel of this experiment.) It is worth noting that complete ubiquitin replacement in these cells exaggerates the impact physiological phosphorylation of ubiquitin is likely to have on global poly-ubiquitin linkage abundance. Indeed, we found that oxidative stress induced an approximately two-fold increase in phosphorylation of ubiquitin at the Ser57 position (Figure 2F and Figure 2—figure supplement 2). Based on previous measurements (Swaney et al., 2015; Lee et al., 2017), this stress-induced ubiquitin phosphorylation remains sub-stoichiometric and may have localized effects but is unlikely to alter global poly-ubiquitin linkage patterns. Ultimately, a deeper understanding of how Ser57 phosphorylated ubiquitin contributes to cellular stress responses will require the identification and characterization of the kinases that produce it.

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Quantification and statistical analysis for the growth (Figure 2A), cell viability (Figure 2B), and anti-K48/K63 western blot analysis (Figure 2D–E) of cells in the presence or absence of H₂O₂.

The source data for both replicates in Figure 2F are also included.

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To identify candidate ubiquitin kinases, we screened for Ser57 phosphorylation activity by co-expressing ubiquitin and yeast kinases in E. coli and immunoblotting lysates using an antibody specific for Ser57 phosphorylated ubiquitin. Initially, we focused on candidate kinases for which mutants exhibit phenotypes corresponding to those observed for cells expressing S57A or S57D ubiquitin. We found that co-expression of ubiquitin with the kinase Vhs1 resulted in immunodetection of Ser57 phosphorylated ubiquitin (Figure 3A and Figure 3—figure supplement 1). Vhs1 is part of the yeast family of Snf1-related kinases (Tumolo et al., 2020), and additional screening revealed three other kinases in this family that phosphorylated ubiquitin at the Ser57 position: Sks1 (which is 43% identical to Vhs1) (Figure 3B), Gin4 and Kcc4 (Figure 3—figure supplement 2). A previous study reported consensus phosphorylation motifs for Vhs1, Gin4, and Kcc4, and all bear similarity to the amino acid sequence surrounding Ser57 in ubiquitin (Supplementary file 2; Mok et al., 2010).

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Source measurements for the H:L ratio of ubiquitin peptides resolved in all replicate experiments of SUB280 cells overexpressing Sks1 (Figure 3E) or Vhs1 (Figure 3F).

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We further characterized the activity of Vhs1 and Sks1. Analysis of Vhs1 and Sks1 activity using Phos-tag acrylamide gels (Figure 3—figure supplement 3) confirmed the production of Ser57 phosphorylated ubiquitin. Using purified recombinant Vhs1 and Sks1, we reconstituted kinase activity toward Ser57 of ubiquitin and found that both kinases exhibited a preference for polymers (tri-ubiquitin > di ubiquitin > mono ubiquitin) although the activity of Sks1 was noticeably greater than that of Vhs1 (Figure 3C–D and Figure 3—figure supplement 4–5). Analysis of linkage specificity in the phosphorylation reaction revealed that Vhs1 is active toward linear (M1-linked), K29-, and K48-linked tetramers, while Sks1 is active toward linear, K48-, and K63-linked polymers (Figure 3—figure supplement 6). These differences in linkage-specific activities in vitro may underlie non-overlapping functions for these two kinases in vivo. To test if the observed in vitro activity correlated with in vivo activity, we used SILAC to quantify changes to ubiquitin modifications following over-expression of Vhs1 or Sks1. Importantly, we observed that overexpression of either Vhs1 or Sks1 increased ubiquitin phosphorylation at the Ser57 position (Figure 3E–F and Figure 3—figure supplement 7–8). Interestingly, this analysis also revealed a number of phosphopeptides derived from Vhs1 within its kinase domain (Ser86) and its Ser-rich C-terminus (Figure 3—figure supplement 9), suggesting that Vhs1 itself may be subject to complex phospho-regulation. Taken together our data indicate that Vhs1 and Sks1 are bona fide ubiquitin kinases that specifically phosphorylate the Ser57 position.

The family of Snf1-related kinases in yeast share homology with the human AMPK-related kinases (Tumolo et al., 2020). To test if human AMPK-related kinases exhibit activity toward ubiquitin, we performed in vitro kinase assays and found that a subset of this family phosphorylated ubiquitin at the Ser57 position specifically on tetramers (Figure 3G). This activity was apparent in the MARK kinases (MARK1-4) as well as related kinases SIK1 and SIK2. It is noteworthy that other candidate human ubiquitin kinases (initially identified by commercial screening services) did not exhibit Ser57 ubiquitin kinase activity in vitro (Figure 3—figure supplement 10). Mass spectrometry analysis confirmed production of Ser57 phosphorylated ubiquitin by MARK2 in vitro (Figure 3—figure supplement 11). Further analysis of MARK2 activity toward linkage-specific ubiquitin tetramers revealed a preference for linear, K11-, K29-, and K63-linked tetramers (Figure 3—figure supplement 12). Given that Ser57 ubiquitin phosphorylation activity was detected within yeast and human Snf1-related kinases (Figure 3—figure supplement 13), we propose that this is an evolutionarily conserved function for a subset of kinases within this family.

In an effort to understand the biological functions of Ser57-phosphorylated ubiquitin in yeast, we examined whether the deletion or overexpression of Ser57 ubiquitin kinases phenocopies expression of S57A or S57D ubiquitin, respectively (Figure 1A–B and Figure 2A). We did not observe heat tolerance phenotypes associated with deletion or overexpression of Ser57 ubiquitin kinases (Figure 4—figure supplement 1–2). However, yeast cells overexpressing VHS1 exhibited resistance to canavanine and thialysine (Figure 4A–B and Figure 4—figure supplement 3), reminiscent of canavanine and thialysine resistance conferred by S57D expression (Figure 1A; Lee et al., 2017). Overexpression of a catalytic dead variant (vhs1-K41R, which harbors a mutation in the conserved ATP binding pocket of the kinase domain) did not confer canavanine or thialysine resistance (Figure 4A–B and Figure 4—figure supplement 3). Importantly, the canavanine and thialysine resistance associated with VHS1 overexpression was suppressed in the presence of S57A ubiquitin (Figure 4C–D and Figure 4—figure supplement 4), indicating the phenotypes are driven by the production of Ser57-phosphorylated ubiquitin. We also observed that yeast cells overexpressing SKS1 exhibited hypersensitivity to hydroxyurea (Figure 4—figure supplement 5), consistent with the hydroxyurea sensitivity phenotype observed in yeast cells expressing S57D ubiquitin (Figure 1A). The hydroxyurea hypersensitivity associated with SKS1 overexpression was suppressed in the presence of S57A ubiquitin (Figure 4E), indicating the phenotype requires Ser57 phosphorylation of ubiquitin. Taken together, these phenotypic correlations suggest that Vhs1 and Sks1 exert stress phenotypes associated with the phosphorylation of ubiquitin at the Ser57 position.

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Quantification and statistical analysis for growth tolerance in canavanine (Figure 4B and D), thialysine (Figure 4—figure supplements 3 and 4), and hydrogen peroxide (Figure 4E and F).

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We also examined whether the deletion of Ser57 ubiquitin kinases phenocopied the oxidative stress response defect observed for yeast cells expressing S57A ubiquitin (Figure 2A). Notably, Δsks1Δvhs1 double mutant cells failed to arrest growth in response to oxidative stress, a phenotype that could only be complemented by the re-introduction of both VHS1 and SKS1 (Figure 4F). This phenotype of Δsks1Δvhs1 double mutant cells was also suppressed by the expression of S57D (but not WT or S57A) ubiquitin (Figure 4G) suggesting that the growth arrest defect in Δsks1Δvhs1 cells is linked to a deficiency in the production of ubiquitin phosphorylated at the Ser57 position. To explore this further, we used SILAC-MS to compare levels of Ser57 phosphorylated ubiquitin in untreated or H₂O₂-treated cells, however, this analysis did not reveal significant changes in Δsks1Δvhs1 cells (Figure 4H and Table 1). While these results indicate that Sks1 and Vhs1 are dispensable for production of Ser57 phosphorylated ubiquitin in the acute phase of the oxidative stress response, we cannot exclude roles for these kinases during prolonged exposure to oxidative stress or the possibility that they contribute to the phosphorylation of localized pools of ubiquitin.

The data presented here provide evidence that Ser57 phosphorylated ubiquitin and the kinases that produce it play an important role in several yeast stress responses, but the low stoichiometry of this modification suggests its effects are likely limited and localized in a physiological context. To the best of our knowledge, this study reports the first Ser57 ubiquitin kinases, and the only known ubiquitin kinases besides PINK1. Importantly, PINK1 activity is tightly regulated and highly localized to the outer membrane of damaged mitochondria. One potential explanation of the genetic and biochemical data presented here is that Sks1 and Vhs1 phosphorylate a localized pool (or pools) of ubiquitin in response to stress. However, the data also indicate that Sks1 and Vhs1 are not required for the production of Ser57 phosphorylated ubiquitin during normal growth or oxidative stress (Figure 4H and Table 1). This may be due to redundancy of kinase activities, possibly with Gin4, Kcc4, or other as-yet-unidentified ubiquitin kinases. However, such redundancy cannot explain the phenotypes observed during oxidative stress, since Δsks1Δvhs1 double mutant cells exhibit oxidative stress phenotypes that are suppressed by expression of phosphomimetic (S57D) ubiquitin (Figure 4G). In this case, we propose that a localized pool of ubiquitin phosphorylated by Sks1 and Vhs1 is critical for the oxidative stress response but is not resolved in our proteomic analysis, or only contributes a small fraction to a larger pool. This interpretation reconciles the genetic and biochemical data presented here, and it is consistent with the precedent established with PINK1, which is subject to tight regulation and contributes to localized production of Ser65 phosphorylated ubiquitin. However, further characterization of the Ser57 ubiquitin kinases reported here and analysis of localized ubiquitin pools will be required to validate this interpretation.

Genetic experiments presented here reveal that ubiquitin replacement with phosphomimetic ubiquitin phenocopies kinase gain of function (as is the case with canavanine, thialysine, and hydroxyurea sensitivities) while replacement with phosphorylation resistant ubiquitin phenocopies kinase loss of function (as is the case with sensitivity to H₂O₂). An important limitation of this genetic analysis is the built-in assumption that ubiquitin variants (phosphomimetic or phosphorylation resistant) behave as expected and do not alter other biochemical properties of ubiquitin in a cellular context. Additionally, such phenotypes associated with ubiquitin variants are likely to over-estimate the physiological effects of ubiquitin phosphorylation. In the case of phospho-mimetic (S57D) ubiquitin, this is because physiological ubiquitin phosphorylation is sub-stoichiometric. In the case of phosphorylation resistant (S57A) ubiquitin, this may be due to redundancy of kinases. Such redundancy seems likely since we identified multiple Ser57 ubiquitin kinases (Vhs1, Sks1, Gin4, and Kcc4) and the deletion of two kinases did not decrease the production of Ser57 phosphorylated ubiquitin (Figure 4H and Table 1). Furthermore, we cannot exclude the possibility that mutation of Ser57 alters the biochemical properties of ubiquitin in such a way as to phenocopy the effects associated with kinase deletion and/or overexpression. Going forward, these limitations present formidable challenges for dissecting the biological functions of ubiquitin phosphorylation, which is why the identification and characterization of ubiquitin kinases is critical. Ultimately, deeper characterization of Ser57 ubiquitin kinases – particularly to understand their localization and regulation in the context of proteotoxic stress – will likely be critical to understanding how phosphorylation regulates the biology of the ubiquitin code.

All data generated or analyzed during this study are included in the manuscript and supporting files.

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Author details

1. Nathaniel L Hepowit

   Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7614-2756

2. Kevin N Pereira

   Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States

   Contribution

   Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5377-397X

3. Jessica M Tumolo

   Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

4. Walter J Chazin

   Department of Biochemistry, Vanderbilt University, Nashville, United States

   Contribution

   Conceptualization, Resources

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2180-0790

5. Jason A MacGurn

   Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   jason.a.macgurn@vanderbilt.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5063-259X

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