Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control
Abstract p38 mitogen-activated protein kinases (MAPKs) participate in autophagic signaling; and previous reports suggest that pyridinyl imidazole p38 MAPK inhibitors, including SB203580 and SB202190, induce cell death in some cancer cell-types through unrestrained autophagy. Subsequent studies, howe...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Nature Publishing Group
2024-01-01
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| Series: | Cell Death and Disease |
| Online Access: | https://doi.org/10.1038/s41419-024-06423-0 |
| _version_ | 1797276294397820928 |
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| author | Daric J. Wible Zalak Parikh Eun Jeong Cho Miao-Der Chen Collene R. Jeter Somshuvra Mukhopadhyay Kevin N. Dalby Shankar Varadarajan Shawn B. Bratton |
| author_facet | Daric J. Wible Zalak Parikh Eun Jeong Cho Miao-Der Chen Collene R. Jeter Somshuvra Mukhopadhyay Kevin N. Dalby Shankar Varadarajan Shawn B. Bratton |
| author_sort | Daric J. Wible |
| collection | DOAJ |
| description | Abstract p38 mitogen-activated protein kinases (MAPKs) participate in autophagic signaling; and previous reports suggest that pyridinyl imidazole p38 MAPK inhibitors, including SB203580 and SB202190, induce cell death in some cancer cell-types through unrestrained autophagy. Subsequent studies, however, have suggested that the associated cytoplasmic vacuolation resulted from off-target inhibition of an unidentified enzyme. Herein, we report that SB203580-induced vacuolation is rapid, reversible, and relies on the class III phosphatidylinositol 3-kinase (PIK3C3) complex and the production of phosphatidylinositol 3-phosphate [PI(3)P] but not on autophagy per se. Rather, vacuolation resulted from the accumulation of Rab7 on late endosome and lysosome (LEL) membranes, combined with an osmotic imbalance that triggered severe swelling in these organelles. Inhibition of PIKfyve, the lipid kinase that converts PI(3)P to PI(3,5)P2 on LEL membranes, produced a similar phenotype in cells; therefore, we performed in vitro kinase assays and discovered that both SB203580 and SB202190 directly inhibited recombinant PIKfyve. Cancer cells treated with either drug likewise displayed significant reductions in the endogenous levels of PI(3,5)P2. Despite these results, SB203580-induced vacuolation was not entirely due to off-target inhibition of PIKfyve, as a drug-resistant p38α mutant suppressed vacuolation; and combined genetic deletion of both p38α and p38β dramatically sensitized cells to established PIKfyve inhibitors, including YM201636 and apilimod. The rate of vacuole dissolution (i.e., LEL fission), following the removal of apilimod, was also significantly reduced in cells treated with BIRB-796, a structurally unrelated p38 MAPK inhibitor. Thus, our studies indicate that pyridinyl imidazole p38 MAPK inhibitors induce cytoplasmic vacuolation through the combined inhibition of both PIKfyve and p38 MAPKs, and more generally, that p38 MAPKs act epistatically to PIKfyve, most likely to promote LEL fission. |
| first_indexed | 2024-03-07T15:26:11Z |
| format | Article |
| id | doaj.art-d44578e962124c259e90c2981820c4c4 |
| institution | Directory Open Access Journal |
| issn | 2041-4889 |
| language | English |
| last_indexed | 2024-03-07T15:26:11Z |
| publishDate | 2024-01-01 |
| publisher | Nature Publishing Group |
| record_format | Article |
| series | Cell Death and Disease |
| spelling | doaj.art-d44578e962124c259e90c2981820c4c42024-03-05T17:07:11ZengNature Publishing GroupCell Death and Disease2041-48892024-01-0115111410.1038/s41419-024-06423-0Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume controlDaric J. Wible0Zalak Parikh1Eun Jeong Cho2Miao-Der Chen3Collene R. Jeter4Somshuvra Mukhopadhyay5Kevin N. Dalby6Shankar Varadarajan7Shawn B. Bratton8Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer CenterDepartment of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer CenterTargeted Therapeutic Drug Discovery and Development Program, Division of Chemical Biology & Medicinal Chemistry, College of Pharmacy, The University of Texas at AustinDepartment of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer CenterDepartment of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer CenterDivision of Pharmacology & Toxicology, College of Pharmacy, The University of Texas at AustinTargeted Therapeutic Drug Discovery and Development Program, Division of Chemical Biology & Medicinal Chemistry, College of Pharmacy, The University of Texas at AustinInstitute of Translational Medicine, University of LiverpoolDepartment of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer CenterAbstract p38 mitogen-activated protein kinases (MAPKs) participate in autophagic signaling; and previous reports suggest that pyridinyl imidazole p38 MAPK inhibitors, including SB203580 and SB202190, induce cell death in some cancer cell-types through unrestrained autophagy. Subsequent studies, however, have suggested that the associated cytoplasmic vacuolation resulted from off-target inhibition of an unidentified enzyme. Herein, we report that SB203580-induced vacuolation is rapid, reversible, and relies on the class III phosphatidylinositol 3-kinase (PIK3C3) complex and the production of phosphatidylinositol 3-phosphate [PI(3)P] but not on autophagy per se. Rather, vacuolation resulted from the accumulation of Rab7 on late endosome and lysosome (LEL) membranes, combined with an osmotic imbalance that triggered severe swelling in these organelles. Inhibition of PIKfyve, the lipid kinase that converts PI(3)P to PI(3,5)P2 on LEL membranes, produced a similar phenotype in cells; therefore, we performed in vitro kinase assays and discovered that both SB203580 and SB202190 directly inhibited recombinant PIKfyve. Cancer cells treated with either drug likewise displayed significant reductions in the endogenous levels of PI(3,5)P2. Despite these results, SB203580-induced vacuolation was not entirely due to off-target inhibition of PIKfyve, as a drug-resistant p38α mutant suppressed vacuolation; and combined genetic deletion of both p38α and p38β dramatically sensitized cells to established PIKfyve inhibitors, including YM201636 and apilimod. The rate of vacuole dissolution (i.e., LEL fission), following the removal of apilimod, was also significantly reduced in cells treated with BIRB-796, a structurally unrelated p38 MAPK inhibitor. Thus, our studies indicate that pyridinyl imidazole p38 MAPK inhibitors induce cytoplasmic vacuolation through the combined inhibition of both PIKfyve and p38 MAPKs, and more generally, that p38 MAPKs act epistatically to PIKfyve, most likely to promote LEL fission.https://doi.org/10.1038/s41419-024-06423-0 |
| spellingShingle | Daric J. Wible Zalak Parikh Eun Jeong Cho Miao-Der Chen Collene R. Jeter Somshuvra Mukhopadhyay Kevin N. Dalby Shankar Varadarajan Shawn B. Bratton Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control Cell Death and Disease |
| title | Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control |
| title_full | Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control |
| title_fullStr | Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control |
| title_full_unstemmed | Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control |
| title_short | Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control |
| title_sort | unexpected inhibition of the lipid kinase pikfyve reveals an epistatic role for p38 mapks in endolysosomal fission and volume control |
| url | https://doi.org/10.1038/s41419-024-06423-0 |
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