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 study kinase signaling in choanoflagellates have been constrained by the limitations of currently available genetic tools. Here we demonstrate that small molecule approaches provide a complementary and scalable approach for studying 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 exploring structure-activity relationships of one inhibitor, sorafenib, we identified a p38 kinase as a regulator of heat and oxidative stress in S. rosetta . This finding indicates 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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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.
-