A stress-responsive p38 signaling axis in choanoflagellates
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Abstract
Animal kinases regulate cellular responses to environmental stimuli, including cell differentiation, migration, survival, and response to stress, but the ancestry of these functions is poorly understood. Choanoflagellates, the closest living relatives of animals, encode homologs of diverse animal kinases and have emerged as model organisms for reconstructing animal origins. However, efforts to identify key kinase regulators in choanoflagellates have been constrained by the limitations of currently available genetic tools. Here, we report on a framework that combines small molecule-driven kinase discovery with targeted genetics to reveal kinase function in choanoflagellates. To study the physiological roles of choanoflagellate kinases, we established two high-throughput platforms to screen the model choanoflagellate Salpingoeca rosetta with a curated library of human kinase inhibitors. We identified 95 diverse kinase inhibitors that disrupt S. rosetta cell proliferation. By focusing on one inhibitor, sorafenib, we identified a p38 kinase as a regulator of the heat shock response in S. rosetta . This finding reveals a conserved p38 function between choanoflagellates, animals, and fungi. Moreover, this study demonstrates that existing kinase inhibitors can serve as powerful tools to examine the ancestral roles of kinases that regulate modern animal development.
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A stress-responsive p38 signaling axis in choanoflagellates
Review coordinated by Life Science Editors Foundation Reviewed by: Dr. Angela Andersen, Life Science Editors Foundation & Life Science Editors. Potential Conflicts of Interest: None.
PUNCHLINE: A stress-responsive p38 signaling pathway in choanoflagellates reveals deep evolutionary conservation of cellular stress adaptation mechanisms—functionally linking unicellular and multicellular stress responses.
BACKGROUND: Cells across all domains of life must sense and respond to environmental stress, and kinase signaling pathways play a critical role in mediating these responses. In animals, p38 mitogen-activated protein kinase (MAPK) is a well-known regulator of stress responses, cell proliferation, and differentiation. However, its evolutionary origins remain unclear.
A stress-responsive p38 signaling axis in choanoflagellates
Review coordinated by Life Science Editors Foundation Reviewed by: Dr. Angela Andersen, Life Science Editors Foundation & Life Science Editors. Potential Conflicts of Interest: None.
PUNCHLINE: A stress-responsive p38 signaling pathway in choanoflagellates reveals deep evolutionary conservation of cellular stress adaptation mechanisms—functionally linking unicellular and multicellular stress responses.
BACKGROUND: Cells across all domains of life must sense and respond to environmental stress, and kinase signaling pathways play a critical role in mediating these responses. In animals, p38 mitogen-activated protein kinase (MAPK) is a well-known regulator of stress responses, cell proliferation, and differentiation. However, its evolutionary origins remain unclear. Choanoflagellates—the closest living relatives of animals—provide a unique window into the early evolution of signaling pathways before multicellularity. While previous studies have identified kinase homologs in choanoflagellates, their functional roles have been difficult to study due to limited genetic tools. This study uses high-throughput small-molecule screening and CRISPR-based gene editing in Salpingoeca rosetta to systematically dissect p38 kinase signaling in response to environmental stress.
Questions Addressed: How do kinases regulate stress responses in choanoflagellates? Can human kinase inhibitors be repurposed to probe kinase function in choanoflagellates? SUMMARY: This study functionally characterizes a stress-responsive p38 kinase pathway in choanoflagellates, demonstrating that kinase signaling in unicellular organisms plays a key role in environmental stress adaptation. Using a high-throughput screen of 1,255 human kinase inhibitors, the authors identified 95 compounds that disrupt S. rosetta proliferation. By focusing on sorafenib, a known human kinase inhibitor, they discovered that p38 kinase in S. rosetta is activated by heat shock and other stressors, revealing an ancient and conserved function for this pathway.
Key Results
Kinase Inhibitor Screening Identifies Regulators of S. rosetta Proliferation A comprehensive kinase inhibitor screen was conducted using 1,255 human kinase inhibitors. 95 inhibitors significantly affected S. rosetta cell growth, suggesting deep conservation of kinase function between choanoflagellates and animals. The library covered all major kinase families, and flow cytometry and imaging validated inhibitor effects.
Sorafenib Inhibits p38 Kinase and Blocks Stress-Induced Phosphorylation S. rosetta p38 kinase was identified as a sorafenib target, supporting its role in stress signaling. Heat shock increases p38 phosphorylation, but this activation is blocked by sorafenib, confirming a conserved stress-responsive pathway. p38 kinases in S. rosetta share critical catalytic residues with human p38, further supporting functional conservation.
p38 Activation is Stress-Specific and Not Required for Proliferation While p38 is activated by heat shock and oxidative stress, its inhibition does not prevent S. rosetta proliferation. CRISPR knockout of p38 (Sr-p38¹⁻¹⁵) confirmed that p38 activation is required for stress response but not cell division.
p38 Kinase Function Precedes Multicellularity The study reveals that p38’s role in stress adaptation predates animals, suggesting that stress responses were critical for early eukaryotic evolution. p38 homologs are present across choanoflagellates, reinforcing its ancient function.
STRENGTHS: Bridges a Functional Gap in Evolutionary Biology. This study moves beyond comparative genomics by functionally testing kinase signaling in a unicellular organism, shedding light on the ancestral origins of stress pathways.
High-Throughput Chemical Genetics as a Tool for Evolutionary Biology. Using human kinase inhibitors to probe choanoflagellate signaling is an innovative approach that extends the power of small-molecule screening beyond traditional model organisms.
p38 MAPK as a Conserved Stress Sensor. The discovery that choanoflagellates use p38 signaling to respond to stress suggests that stress adaptation mechanisms evolved before multicellularity—a key insight into early eukaryotic evolution.
Biomedical and Biotechnological Implications. Understanding how stress signaling evolved could have implications for drug targeting in diseases like cancer and neurodegeneration, where kinase dysregulation plays a role.
FUTURE WORK: • Does This Apply to Other Kinases? • How Does p38 Interact with Other Stress Pathways? • Do other unicellular relatives of animals use p38 for stress signaling? • How does p38 respond to other environmental stressors (e.g., salinity, bacterial signals)?
FINAL TAKEAWAY: This study functionally validates a stress-responsive p38 signaling pathway in choanoflagellates, providing compelling evidence that key elements of stress adaptation predate multicellularity. Beyond evolutionary implications, this work pioneers the use of kinase inhibitors to probe non-model organisms, opening up new avenues for studying the origins of complex cellular regulation.
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Sr-p38 binds to sorafenib, is activated by environmental stressors, and regulates S. rosetta cell proliferation.(A) Sorafenib binds to Sr-p38. The ActivX ATP probe was used to pull down kinases from S. rosetta lysates that were pretreated with either DMSO or the ATP-competitive inhibitor sorafenib. We found that pretreatment with sorafenib reduced the level of Sr-p38 recovered using the ActivX ATP probe, indicating that sorafenib and Sr-p38 interact and outcompete ActivX ATP probe binding. Kinases plotted are only those that were identified in both vehicle and sorafenib pre-treatments. For full kinase enrichment list, see Table S2, and for alignment of Sr-p38 with those from animals and fungi, see Fig. S7.(B-C) Sr-p38 kinase is activated by heat shock and oxidative stress. S. rosetta cells, normally cultured at 22°C were incubated at …
Sr-p38 binds to sorafenib, is activated by environmental stressors, and regulates S. rosetta cell proliferation.(A) Sorafenib binds to Sr-p38. The ActivX ATP probe was used to pull down kinases from S. rosetta lysates that were pretreated with either DMSO or the ATP-competitive inhibitor sorafenib. We found that pretreatment with sorafenib reduced the level of Sr-p38 recovered using the ActivX ATP probe, indicating that sorafenib and Sr-p38 interact and outcompete ActivX ATP probe binding. Kinases plotted are only those that were identified in both vehicle and sorafenib pre-treatments. For full kinase enrichment list, see Table S2, and for alignment of Sr-p38 with those from animals and fungi, see Fig. S7.(B-C) Sr-p38 kinase is activated by heat shock and oxidative stress. S. rosetta cells, normally cultured at 22°C were incubated at 37°C or treated with hydrogen peroxide for 10 min. or 30 min. Lysates from the treated cultures were analyzed by western blot with a p38 antibody specific for phosphorylated p38 kinase (phospho-p38) to identify if any changes in p38 phosphorylation occurred. (B) 30 minutes of heat shock was sufficient to induce p38 phosphorylation as was (C) 10 min of treatment with 0.5M H2O2. A 12% Bis-Tris SDS-PAGE gel was used to resolve the western bands observed. An anisomycin-treated human cell lysate was used as a positive control to validate the phospho-p38 antibody in Figure S7C. Raw blot images and details on western blot cropping are available at: https://doi.org/10.6084/m9.figshare.20669730.v1(D) Sr-p38 phosphorylation is inhibited by sorafenib, but not by the sorafenib analog APS6-46. S. rosetta cultures pretreated with 10 µM or 1 µM sorafenib for 30 minutes followed by 30 minutes of heat shock at 37°C had decreased p38 phosphorylation. APS6-46 treated cultures were not different from vehicle (DMSO) control. Data from all sorafenib analogs tested are shown in Figure S8A-B. Treatment growth curves, dose response, and tyrosine phosphorylation analysis with APS6-46 treated cultures are in Figure S8C-E. Raw blot image and details on western blot cropping are available at: https://doi.org/10.6084/m9.figshare.20669730.v1(E-F) Selective inhibition of Sr-p38 disrupts S. rosetta cell proliferation. S. rosetta cultures were treated with sorafenib or one of two p38-specific inhibitors, skepinone-L or BIRB 796, in 24-well plates over an 80-hour growth course. (E) At 40 hours, cells treated with 10 µM skepinone-L, BIRB 796 or sorafenib showed little evidence of cell proliferation in comparison to vehicle (DMSO) control (p-value <0.01). (F) Cells treated with 1 µM skepinone-L or BIRB 796 had reduced cell density in comparison to vehicle (DMSO) control (p-value <0.01) at 60 hours. Three biological replicates were conducted per experiment and significance was determined by determined by a two-way ANOVA multiple comparisons test. Movie S5 shows a timelapse of S. rosetta cells treated with skepinone-L.(G) Sr-p38 phosphorylation is not inhibited by the p38-specific inhibitors skepinone-L or BIRB 796. S. rosetta cultures pretreated with 10 µM of skepinone-L and BIRB 796 for 30 minutes followed by 30 minutes of heat shock at 37°C were not different from vehicle (DMSO) control. Raw blot image and details on western blot cropping are available at: https://doi.org/10.6084/m9.figshare.20669730.v1(H) Proposed mechanism for regulation of Sr-p38 by tyrosine kinases and the essentiality of Sr-p38 for S. rosetta cell proliferation. Sr-p38 kinase is phosphorylated by upstream tyrosine kinases and is necessary for cell proliferation. Sorafenib inhibits Sr-p38 phosphorylation by blocking the activity of upstream tyrosine kinases. p38 inhibitors that do not inhibit these upstream tyrosine kinases also do not reduce Sr-p38 phosphorylation but do block Sr-p38 kinase activity and thereby block S. rosetta cell proliferation.
It's awesome to identify the specific target of a kinase inhibitor! Such a clever experiment!
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The kinase inhibitor screen described here is useful to identify kinases that coordinate cell proliferation in choanoflagellates. This chemical biology approach would also be incredibly useful to identify kinases involved in cell proliferation in other protists. The use of the ActivX probe in the context of a competitive inhibition assay is a clever approach to identify a specific target of a kinase inhibitor. A few questions for the authors:
- How did you decide to focus your attention on kinases, given the vast diversity of enzymes in cell biology?
- Related to question 1, have you considered similar screens with GTPase inhibitors or with other specific enzyme inhibitors?
- Why did you choose to screen for cell proliferation versus other phenotypes?
- Have you screened for kinase inhibitors that disrupt rosette formation?
I imagine that this …
The kinase inhibitor screen described here is useful to identify kinases that coordinate cell proliferation in choanoflagellates. This chemical biology approach would also be incredibly useful to identify kinases involved in cell proliferation in other protists. The use of the ActivX probe in the context of a competitive inhibition assay is a clever approach to identify a specific target of a kinase inhibitor. A few questions for the authors:
- How did you decide to focus your attention on kinases, given the vast diversity of enzymes in cell biology?
- Related to question 1, have you considered similar screens with GTPase inhibitors or with other specific enzyme inhibitors?
- Why did you choose to screen for cell proliferation versus other phenotypes?
- Have you screened for kinase inhibitors that disrupt rosette formation?
I imagine that this technique will enable lots of researchers to perform similar chemical screens in other emerging research organisms.
-
Sr-p38 binds to sorafenib, is activated by environmental stressors, and regulates S. rosetta cell proliferation.(A) Sorafenib binds to Sr-p38. The ActivX ATP probe was used to pull down kinases from S. rosetta lysates that were pretreated with either DMSO or the ATP-competitive inhibitor sorafenib. We found that pretreatment with sorafenib reduced the level of Sr-p38 recovered using the ActivX ATP probe, indicating that sorafenib and Sr-p38 interact and outcompete ActivX ATP probe binding. Kinases plotted are only those that were identified in both vehicle and sorafenib pre-treatments. For full kinase enrichment list, see Table S2, and for alignment of Sr-p38 with those from animals and fungi, see Fig. S7.(B-C) Sr-p38 kinase is activated by heat shock and oxidative stress. S. rosetta cells, normally cultured at 22°C were incubated at …
Sr-p38 binds to sorafenib, is activated by environmental stressors, and regulates S. rosetta cell proliferation.(A) Sorafenib binds to Sr-p38. The ActivX ATP probe was used to pull down kinases from S. rosetta lysates that were pretreated with either DMSO or the ATP-competitive inhibitor sorafenib. We found that pretreatment with sorafenib reduced the level of Sr-p38 recovered using the ActivX ATP probe, indicating that sorafenib and Sr-p38 interact and outcompete ActivX ATP probe binding. Kinases plotted are only those that were identified in both vehicle and sorafenib pre-treatments. For full kinase enrichment list, see Table S2, and for alignment of Sr-p38 with those from animals and fungi, see Fig. S7.(B-C) Sr-p38 kinase is activated by heat shock and oxidative stress. S. rosetta cells, normally cultured at 22°C were incubated at 37°C or treated with hydrogen peroxide for 10 min. or 30 min. Lysates from the treated cultures were analyzed by western blot with a p38 antibody specific for phosphorylated p38 kinase (phospho-p38) to identify if any changes in p38 phosphorylation occurred. (B) 30 minutes of heat shock was sufficient to induce p38 phosphorylation as was (C) 10 min of treatment with 0.5M H2O2. A 12% Bis-Tris SDS-PAGE gel was used to resolve the western bands observed. An anisomycin-treated human cell lysate was used as a positive control to validate the phospho-p38 antibody in Figure S7C. Raw blot images and details on western blot cropping are available at: https://doi.org/10.6084/m9.figshare.20669730.v1(D) Sr-p38 phosphorylation is inhibited by sorafenib, but not by the sorafenib analog APS6-46. S. rosetta cultures pretreated with 10 µM or 1 µM sorafenib for 30 minutes followed by 30 minutes of heat shock at 37°C had decreased p38 phosphorylation. APS6-46 treated cultures were not different from vehicle (DMSO) control. Data from all sorafenib analogs tested are shown in Figure S8A-B. Treatment growth curves, dose response, and tyrosine phosphorylation analysis with APS6-46 treated cultures are in Figure S8C-E. Raw blot image and details on western blot cropping are available at: https://doi.org/10.6084/m9.figshare.20669730.v1(E-F) Selective inhibition of Sr-p38 disrupts S. rosetta cell proliferation. S. rosetta cultures were treated with sorafenib or one of two p38-specific inhibitors, skepinone-L or BIRB 796, in 24-well plates over an 80-hour growth course. (E) At 40 hours, cells treated with 10 µM skepinone-L, BIRB 796 or sorafenib showed little evidence of cell proliferation in comparison to vehicle (DMSO) control (p-value <0.01). (F) Cells treated with 1 µM skepinone-L or BIRB 796 had reduced cell density in comparison to vehicle (DMSO) control (p-value <0.01) at 60 hours. Three biological replicates were conducted per experiment and significance was determined by determined by a two-way ANOVA multiple comparisons test. Movie S5 shows a timelapse of S. rosetta cells treated with skepinone-L.(G) Sr-p38 phosphorylation is not inhibited by the p38-specific inhibitors skepinone-L or BIRB 796. S. rosetta cultures pretreated with 10 µM of skepinone-L and BIRB 796 for 30 minutes followed by 30 minutes of heat shock at 37°C were not different from vehicle (DMSO) control. Raw blot image and details on western blot cropping are available at: https://doi.org/10.6084/m9.figshare.20669730.v1(H) Proposed mechanism for regulation of Sr-p38 by tyrosine kinases and the essentiality of Sr-p38 for S. rosetta cell proliferation. Sr-p38 kinase is phosphorylated by upstream tyrosine kinases and is necessary for cell proliferation. Sorafenib inhibits Sr-p38 phosphorylation by blocking the activity of upstream tyrosine kinases. p38 inhibitors that do not inhibit these upstream tyrosine kinases also do not reduce Sr-p38 phosphorylation but do block Sr-p38 kinase activity and thereby block S. rosetta cell proliferation.
It's awesome to identify the specific target of a kinase inhibitor! Such a clever experiment!
-
The kinase inhibitor screen described here is useful to identify kinases that coordinate cell proliferation in choanoflagellates. This chemical biology approach would also be incredibly useful to identify kinases involved in cell proliferation in other protists. The use of the ActivX probe in the context of a competitive inhibition assay is a clever approach to identify a specific target of a kinase inhibitor. A few questions for the authors:
- How did you decide to focus your attention on kinases, given the vast diversity of enzymes in cell biology?
- Related to question 1, have you considered similar screens with GTPase inhibitors or with other specific enzyme inhibitors?
- Why did you choose to screen for cell proliferation versus other phenotypes?
- Have you screened for kinase inhibitors that disrupt rosette formation?
I imagine that this …
The kinase inhibitor screen described here is useful to identify kinases that coordinate cell proliferation in choanoflagellates. This chemical biology approach would also be incredibly useful to identify kinases involved in cell proliferation in other protists. The use of the ActivX probe in the context of a competitive inhibition assay is a clever approach to identify a specific target of a kinase inhibitor. A few questions for the authors:
- How did you decide to focus your attention on kinases, given the vast diversity of enzymes in cell biology?
- Related to question 1, have you considered similar screens with GTPase inhibitors or with other specific enzyme inhibitors?
- Why did you choose to screen for cell proliferation versus other phenotypes?
- Have you screened for kinase inhibitors that disrupt rosette formation?
I imagine that this technique will enable lots of researchers to perform similar chemical screens in other emerging research organisms.
-