The phosphorylation of sorting nexin 5 at serine 226 regulates retrograde transport and macropinocytosis.
Sorting nexin 5 (SNX5), a member of sorting nexin family, plays an important role in membrane trafficking, including the retrograde trafficking of the cation independent mannose 6-phosphate receptor (CI-M6PR) and macropinocytosis. Using ESI-LCMS/MS analysis, we confirmed that SNX5 serine 226 is phos...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2018-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC6231649?pdf=render |
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| author | Nao Itai Tsukasa Shimazu Takayuki Kimura Issei Ibe Ryo Yamashita Yasushi Kaburagi Taeko Dohi Takashi Tonozuka Toshifumi Takao Atsushi Nishikawa |
| author_facet | Nao Itai Tsukasa Shimazu Takayuki Kimura Issei Ibe Ryo Yamashita Yasushi Kaburagi Taeko Dohi Takashi Tonozuka Toshifumi Takao Atsushi Nishikawa |
| author_sort | Nao Itai |
| collection | DOAJ |
| description | Sorting nexin 5 (SNX5), a member of sorting nexin family, plays an important role in membrane trafficking, including the retrograde trafficking of the cation independent mannose 6-phosphate receptor (CI-M6PR) and macropinocytosis. Using ESI-LCMS/MS analysis, we confirmed that SNX5 serine 226 is phosphorylated. Since SNX5 forms heterodimers with SNX1 or SNX2, we examined the effect of phosphorylation at S226 on the heterodimer formations. Wild-type and mutants of SNX5, in which S226 was mutated to a glutamic acid or an alanine, were expressed in 8505C cells. In pull-down assays using SNX5 as bait, only the S226E mutant failed to precipitate both SNX1 and SNX2. Confocal microscopy data indicated that the wild type and S226A mutant were colocalized with SNX1 and SNX2 in endosomes, but the S226E was not. SNX5 and SNX6 support each other's functions and are involved with CI-M6PR retrograde trafficking. In SNX5 and SNX6 double knockdown cells, CI-M6PR was dispersed and colocalized with the endosomal marker EEA1. In a rescue experiment using SNX5 mutants, the S226A rescued CI-M6PR localization, similar to control cells, but S226E did not. Furthermore, the decrease in the uptake of dextran by macropinocytosis in SNX5 knockdown cells was recovered by the expression of rescue-wild type or S226A mutant, but not by the rescue-S226E mutant. These observations indicate that SNX5 constitutive phosphorylation that mimics the mutant S226E decreases the active SNX5 in these cells. The phosphorylation of SNX5 regulates the dimerization with SNX1 or SNX2, and this suggests that it controls membrane trafficking and protein sorting. |
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| id | doaj.art-e301724a7b6c446b90100a50673b2c2a |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-13T03:24:34Z |
| publishDate | 2018-01-01 |
| publisher | Public Library of Science (PLoS) |
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| spelling | doaj.art-e301724a7b6c446b90100a50673b2c2a2022-12-22T03:04:42ZengPublic Library of Science (PLoS)PLoS ONE1932-62032018-01-011311e020720510.1371/journal.pone.0207205The phosphorylation of sorting nexin 5 at serine 226 regulates retrograde transport and macropinocytosis.Nao ItaiTsukasa ShimazuTakayuki KimuraIssei IbeRyo YamashitaYasushi KaburagiTaeko DohiTakashi TonozukaToshifumi TakaoAtsushi NishikawaSorting nexin 5 (SNX5), a member of sorting nexin family, plays an important role in membrane trafficking, including the retrograde trafficking of the cation independent mannose 6-phosphate receptor (CI-M6PR) and macropinocytosis. Using ESI-LCMS/MS analysis, we confirmed that SNX5 serine 226 is phosphorylated. Since SNX5 forms heterodimers with SNX1 or SNX2, we examined the effect of phosphorylation at S226 on the heterodimer formations. Wild-type and mutants of SNX5, in which S226 was mutated to a glutamic acid or an alanine, were expressed in 8505C cells. In pull-down assays using SNX5 as bait, only the S226E mutant failed to precipitate both SNX1 and SNX2. Confocal microscopy data indicated that the wild type and S226A mutant were colocalized with SNX1 and SNX2 in endosomes, but the S226E was not. SNX5 and SNX6 support each other's functions and are involved with CI-M6PR retrograde trafficking. In SNX5 and SNX6 double knockdown cells, CI-M6PR was dispersed and colocalized with the endosomal marker EEA1. In a rescue experiment using SNX5 mutants, the S226A rescued CI-M6PR localization, similar to control cells, but S226E did not. Furthermore, the decrease in the uptake of dextran by macropinocytosis in SNX5 knockdown cells was recovered by the expression of rescue-wild type or S226A mutant, but not by the rescue-S226E mutant. These observations indicate that SNX5 constitutive phosphorylation that mimics the mutant S226E decreases the active SNX5 in these cells. The phosphorylation of SNX5 regulates the dimerization with SNX1 or SNX2, and this suggests that it controls membrane trafficking and protein sorting.http://europepmc.org/articles/PMC6231649?pdf=render |
| spellingShingle | Nao Itai Tsukasa Shimazu Takayuki Kimura Issei Ibe Ryo Yamashita Yasushi Kaburagi Taeko Dohi Takashi Tonozuka Toshifumi Takao Atsushi Nishikawa The phosphorylation of sorting nexin 5 at serine 226 regulates retrograde transport and macropinocytosis. PLoS ONE |
| title | The phosphorylation of sorting nexin 5 at serine 226 regulates retrograde transport and macropinocytosis. |
| title_full | The phosphorylation of sorting nexin 5 at serine 226 regulates retrograde transport and macropinocytosis. |
| title_fullStr | The phosphorylation of sorting nexin 5 at serine 226 regulates retrograde transport and macropinocytosis. |
| title_full_unstemmed | The phosphorylation of sorting nexin 5 at serine 226 regulates retrograde transport and macropinocytosis. |
| title_short | The phosphorylation of sorting nexin 5 at serine 226 regulates retrograde transport and macropinocytosis. |
| title_sort | phosphorylation of sorting nexin 5 at serine 226 regulates retrograde transport and macropinocytosis |
| url | http://europepmc.org/articles/PMC6231649?pdf=render |
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