Casein kinase 1 gamma integrates oxidative stress and innate immune responses


 Animals utilize associated pathways to elicit responses to oxidative stress and infection. The molecular mechanisms coordinating these pathways remain unclear. Here, using C. elegans we identified the highly conserved casein kinase 1 gamma CSNK-1 (also known as CK1g or CSNK1G), as a key regulator of these processes. csnk-1 interacted with the bli-3/tsp-15/doxa-1 dual oxidase genes by nonallelic noncomplementation to negatively regulate animal survival in excess iodide, an oxidative stressor. A conserved interaction was detected between DOXA-1 and CSNK-1 and between their human homologs DUOXA2 and CSNK1G2. csnk-1 deficiency resulted in upregulated expression of innate immunity genes and increased animal survival in the pathogenic Pseudomonas aeruginosa PA14. Phosphoproteomic analyses identified decreased phosphorylation of key innate immunity regulators NSY-1 MAPKKK and LIN-45 Raf in csnk-1(lf) mutants. Indeed, NSY-1 and LIN-45 pathways were required for the increased survival of csnk-1-deficient animals in PA14. Further analyses suggest that CSNK-1 and SKN-1 Nrf2 might act in parallel to regulate oxidative stress response. Together, we propose that CSNK-1 CSNK1G plays a novel pivotal role in integrating animal’s responses to oxidative stress and pathogens.


Introduction
previously isolated mutant (mac397) that can grow into adults in 5 mM NaI (Xu et al., 2018). 93 mac397 affected the conserved casein kinase 1 gamma gene csnk-1. We found that csnk-1 94 interacted with the bli-3/tsp-15/doxa-1 complex to negatively regulate animal survival in excess 95 iodide. Transcriptome analyses suggest that CSNK-1 was a negative regulator of innate 96 immune gene expression, which was validated by increased survival of csnk-1(lf) mutants in 97 the pathogenic bacteria Pseudomonas aeruginosa PA14. Phosphoproteomic analyses 98 identified decreased phosphorylation of NSY-1 and LIN-45 in csnk-1(lf) mutants, and these 99 kinases were required for the negative effects of CSNK-1 on innate immunity. Together our 100 results suggest that CSNK-1 CSNK1G integrates animal's responses to oxidative stress and 101 pathogen infections.

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To understand the tissue-specific functions of csnk-1, we first examined the activity of the 154 csnk-1 promoter. A GFP transgene driven by the promoter was broadly expressed when 155 injected into wildtype animals (Fig. 1E). The expression was obvious in the pharynx, head 156 neurons, intestine, ventral cord, vulval muscles, body-wall muscles, dorsal cord and 157 ventrodorsal commissures (Fig. 1F-J). Expression was also observed in the epidermis 158 (hypodermis) (Fig. 1F and H). 159

csnk-1 is a negative regulator of innate immunity gene expression and animal survival 261 in the pathogenic PA14 262
To understand how csnk-1 affects global gene expression, we analyzed the transcriptome of 263 csnk-1(lf) mutant L4 larva (Fig. 3A, heat map). In total, 968 genes exhibited more than 1.5-fold 264 changes in expression (up-regulated or down-regulated) in csnk-1(lf) mutants (Table S4A), 265 among which 579 exhibited more than 2-fold changes (398 up and 181 down) (Fig. 3B, Table  266 S4B and S4C). Gene Ontology (GO) analyses of genes with more than 2-fold changes 267 identified "defense response to other organisms" and "innate immune response" among the 268 most significantly affected biological processes ( Fig. S7A and Table S4D). The KEGG 269 analyses identified "drug metabolism-cytochrome P450" and "metabolism of xenobiotics by 270 cytochrome P450" as the most significantly affected pathways ( Fig. S7A and Table S4D). 271

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Based on the transcriptome, the expression levels of genes in the "defense response to other 273 organism" term were variable (Fig. S7B, 30 up and 4 down) and all 21 genes in the "innate 274 immune response" term were included in this group (Fig. S7B and Table S4G, genes in blue, 275 20 up and 1 down). We therefore examined whether up-regulated genes and down-regulated 276 genes might carry out different functions. The results indicated that "defense response to other 277 organisms" and "innate immune response" terms were primarily affected by up-regulated 278 genes (Fig. 3C, Table S4E and S4F), while the "drug metabolism-cytochrome P450" pathway 279 was affected by both up-regulated and down-regulated genes (Table S4E and S4F).

NSY-1 and LIN-45 mediate the negative effects of CSNK-1 on animal survival in PA14 290
As a kinase, the effects of CSNK-1 might be mediated by downstream phosphorylation signals. 291 To identify protein phosphorylation affected by CSNK-1, we performed mass spectrometry-292 based phosphoproteomic analyses on csnk-1(lf) mutant L4 larva. 293

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We performed KEGG pathway analyses on these proteins (Tables S5D and S5E). Decreased 304 phosphorylation primarily affected proteins in calcium signaling, MAPK signaling, ErbB 305 signaling, and ABC transporters pathways ( Fig. S8B and Table S5D), while increased 306 phosphorylation primarily affected proteins in MAPK signaling, FoxO signaling and calcium 307 signaling pathways ( Fig. S8B and Table S5E). These proteins also affected multiple GO terms 308 (Table S5D and S5E). 309

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Finally we compared the differentially expressed genes in csnk-1(lf) mutants (Table S4)  In this study, we found that CSNK-1 interacts with the BLI-3/TSP-15/DOXA-1 dual oxidase 386 complex to negatively regulate animal survival in an oxidative stressor. CSNK-1 also 387 negatively regulates the expression of innate immunity genes and innate immune response, 388 which appears to be mediated by NSY-1 and LIN-45. Furthermore, CSNK-1 and SKN-1 might 389 function in parallel to regulate oxidative stress responses. Together, our results suggest that Consistent with these studies, we found that eggs laid by csnk-1(lf) mutants failed to hatch.  (Table S2) SKN-1 is a key activator of stress responses in C. elegans (Blackwell et al., 2015). We 498 previously found that skn-1(gf) and a bli-3 missense mutation (mac40) that affects the oxidase 499 domain together can cause animal survival in a higher concentration of iodide (50 mM NaI), 500 while either single mutation failed to do so (Xu et al., 2018). In addition, bli-3(lf); skn-1(lf) 501 double mutants could not survive in 5 mM NaI, while bli-3(lf) single mutants can (Xu et al., 502 2015(Xu et al., 502 , 2018. 503

Transmembrane transporters with decreased phosphorylation in csnk-1(lf) mutants 513
GO analyses found that a prominent group of proteins with decreased phosphorylation in csnk-514 1(lf) mutants were transmembrane transporters, including solute carriers and ABC transporters 515 ( Fig. S8C and S8D, Table S5D and S5G). A detailed analysis of the relationship between 516 CSNK-1 and these transporters is beyond the scope of our current study. Nevertheless, it 517 could be interesting to explore along this line, considering that these transporters are important 518 for cellular homeostasis and that the functions of most of them are unclear (César-Razquin et 519 al., 2015). In addition, GO analyses on proteins with increased phosphorylation in csnk-1(lf) 520 mutants identified terms such as "cytoskeletal protein binding", "developmental process", 521 "supramolecular complex", etc., to be significantly affected (Table S5E). These changes imply 522 that CSNK-1 might have broad functions beyond its effects in oxidative stress and innate 523 immune responses.

Conclusions 540
Oxidative stress response is intimately related to innate and adaptive immunity. It is critical that 541 the activities of the regulators and pathways are coordinated to generate efficient and 542 balanced responses to oxidative stress and pathogens. We suggest that CSNK1G plays a key 543 role in integrating these processes.  Table 4