Evolutionary analysis of p38 stress-activated kinases in unicellular relatives of animals suggests an ancestral function in osmotic stress

Abstract

p38 kinases are key elements of the cellular stress response in animals. They mediate the cell response to a multitude of stress stimuli, from osmotic shock to inflammation and oncogenes. However, it is unknown how such diversity of function in stress evolved in this kinase subfamily. Here, we show that the p38 kinase was already present in a common ancestor of animals and fungi. Later, in animals, it diversified into three JNK kinases and four p38 kinases. Moreover, we identified a fifth p38 paralog in fishes and amphibians. Our analysis shows that each p38 paralog has specific amino acid substitutions around the hinge point, a region between the N-terminal and C-terminal protein domains. We showed that this region can be used to distinguish between individual paralogs and predict their specific. Finally, we showed that the response to hyperosmotic stress in Capsaspora owczarzaki, a close unicellular relative of animals, follows a typical for the p38 kinases pattern of phosphorylation-dephosphorylation. At the same time, Capsaspora cells upregulate the expression of GPD1 protein resembling an osmotic stress response in yeasts. Overall, our results show that the ancestral p38 stress pathway originated in the root of opisthokonts, most likely as a cells reaction to salinity change in the environment. In animals, the pathway became more complex and incorporated more stimuli and downstream targets due to the p38 sequence evolution in the docking and substrate binding sites around the hinge region. Overall, this study improves our understanding of the p38 evolution and opens new perspectives for the p38 research.

A stress-responsive p38 signaling axis in choanoflagellates

“…The environmental stress response is comprised of stress-activated kinases, including p38, that evolved before the divergence of animals and fungi (50). Recent work by Shabardina et al (2022) found that the S. rosetta p38 kinase we identified by mass spectrometry, Sr-p38, is closely related to metazoan p38 kinases that respond to diverse environmental stressors (52).…”

“…The environmental stress response is comprised of stress-activated kinases, including p38, that evolved before the divergence of animals and fungi (50). Recent work by Shabardina et al (2022) found that the S. rosetta p38 kinase we identified by mass spectrometry, Sr-p38, is closely related to metazoan p38 kinases that respond to diverse environmental stressors (52). Our approach of using small molecules to uncover Sr-p38's role in S. rosetta was useful, as p38 likely would have evaded genetic knockout due to its known essentiality in other organisms (49).…”

“…In animals and fungi, p38 kinases regulate cell proliferation and are activated in response to environmental stressors (49)(50)(51)(52).…”

“…Although previous studies have predicted multiple p38 kinases in S. rosetta (50,53), only the p38 recovered from the ActivX binding assay (EGD79605) has the diagnostic "TGY" motif that distinguishes p38 kinases from other related kinases in humans and yeast (Figure S7A) (54). Hereafter we refer to this protein as Sr-p38.…”

“…Choanoflagellates live in sun-lit water zones that undergo daily and yearly fluctuations in temperature and nutrients (57)(58)(59). Because environmental stressors activate p38 kinases in animals and fungi (49)(50)(51)(52)(60)(61)(62)(63), we wondered if stressors relevant to choanoflagellates would activate Sr-p38 kinase signaling. All 23 choanoflagellate species sequenced thus far (10,13,53) express at least one predicted p38 kinase with a conserved "TGY" motif (Figure S7B).…”