The Drosophila MCPH1-B isoform is a substrate of the APCCdh1 E3 ubiquitin ligase complex
The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that coordinates progression through the cell cycle by temporally and spatially promoting the degradation of key proteins. Many of these targeted proteins have been shown to play important roles in regulating orderly progres...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
The Company of Biologists
2014-06-01
|
| Series: | Biology Open |
| Subjects: | |
| Online Access: | http://bio.biologists.org/content/3/7/669 |
| _version_ | 1830302706054463488 |
|---|---|
| author | Sarah G. Hainline Jamie L. Rickmyre Leif R. Neitzel Laura A. Lee Ethan Lee |
| author_facet | Sarah G. Hainline Jamie L. Rickmyre Leif R. Neitzel Laura A. Lee Ethan Lee |
| author_sort | Sarah G. Hainline |
| collection | DOAJ |
| description | The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that coordinates progression through the cell cycle by temporally and spatially promoting the degradation of key proteins. Many of these targeted proteins have been shown to play important roles in regulating orderly progression through the cell cycle. Using a previously described Drosophila in vitro expression cloning approach, we screened for new substrates of the APC in Xenopus egg extract and identified Drosophila MCPH1 (dMCPH1), a protein encoded by the homolog of a causative gene for autosomal recessive primary microcephaly in humans. The dMCPH1-B splice form, but not the dMCPH1-C splice form, undergoes robust degradation in Xenopus interphase egg extract in a Cdh1-dependent manner. Degradation of dMCPH1-B is controlled by an N-terminal destruction box (D-box) motif as its deletion or mutation blocks dMCPH1-B degradation. dMCPH1 levels are increased in Drosophila morula (APC2) mutant embryos, consistent with dMCPH1 being an APC substrate in vivo. Using a purified, reconstituted system, we show that dMCPH1-B is ubiquitinated by APCCdh1, indicating that the effect of APC on dMCPH1-B ubiquitination and degradation is direct. Full-length human MCPH1 (hMCPH1) has been predicted to be an APC substrate based on its interaction with the APC subunit Cdc27. We were not able to detect changes in hMCPH1 levels during the cell cycle in cultured human cells. Overexpression of hMCPH1 (or dMCPH1-B) in developing Xenopus embryos, however, disrupts cell division, suggesting that proper regulation of hMCPH1 and dMCPH1-B activity plays a critical role in proper cell-cycle progression. |
| first_indexed | 2024-12-19T09:11:04Z |
| format | Article |
| id | doaj.art-fd92eda84eeb4745b088e018b126717c |
| institution | Directory Open Access Journal |
| issn | 2046-6390 |
| language | English |
| last_indexed | 2024-12-19T09:11:04Z |
| publishDate | 2014-06-01 |
| publisher | The Company of Biologists |
| record_format | Article |
| series | Biology Open |
| spelling | doaj.art-fd92eda84eeb4745b088e018b126717c2022-12-21T20:28:11ZengThe Company of BiologistsBiology Open2046-63902014-06-013766967610.1242/bio.2014831820148318The Drosophila MCPH1-B isoform is a substrate of the APCCdh1 E3 ubiquitin ligase complexSarah G. HainlineJamie L. Rickmyre0Leif R. NeitzelLaura A. LeeEthan Lee Present address: Sarah Cannon Research Institute, Nashville, TN 37203, USA. The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that coordinates progression through the cell cycle by temporally and spatially promoting the degradation of key proteins. Many of these targeted proteins have been shown to play important roles in regulating orderly progression through the cell cycle. Using a previously described Drosophila in vitro expression cloning approach, we screened for new substrates of the APC in Xenopus egg extract and identified Drosophila MCPH1 (dMCPH1), a protein encoded by the homolog of a causative gene for autosomal recessive primary microcephaly in humans. The dMCPH1-B splice form, but not the dMCPH1-C splice form, undergoes robust degradation in Xenopus interphase egg extract in a Cdh1-dependent manner. Degradation of dMCPH1-B is controlled by an N-terminal destruction box (D-box) motif as its deletion or mutation blocks dMCPH1-B degradation. dMCPH1 levels are increased in Drosophila morula (APC2) mutant embryos, consistent with dMCPH1 being an APC substrate in vivo. Using a purified, reconstituted system, we show that dMCPH1-B is ubiquitinated by APCCdh1, indicating that the effect of APC on dMCPH1-B ubiquitination and degradation is direct. Full-length human MCPH1 (hMCPH1) has been predicted to be an APC substrate based on its interaction with the APC subunit Cdc27. We were not able to detect changes in hMCPH1 levels during the cell cycle in cultured human cells. Overexpression of hMCPH1 (or dMCPH1-B) in developing Xenopus embryos, however, disrupts cell division, suggesting that proper regulation of hMCPH1 and dMCPH1-B activity plays a critical role in proper cell-cycle progression.http://bio.biologists.org/content/3/7/669Anaphase-Promoting ComplexDrosophilaMCPH1UbiquitinationXenopus egg extract |
| spellingShingle | Sarah G. Hainline Jamie L. Rickmyre Leif R. Neitzel Laura A. Lee Ethan Lee The Drosophila MCPH1-B isoform is a substrate of the APCCdh1 E3 ubiquitin ligase complex Biology Open Anaphase-Promoting Complex Drosophila MCPH1 Ubiquitination Xenopus egg extract |
| title | The Drosophila MCPH1-B isoform is a substrate of the APCCdh1 E3 ubiquitin ligase complex |
| title_full | The Drosophila MCPH1-B isoform is a substrate of the APCCdh1 E3 ubiquitin ligase complex |
| title_fullStr | The Drosophila MCPH1-B isoform is a substrate of the APCCdh1 E3 ubiquitin ligase complex |
| title_full_unstemmed | The Drosophila MCPH1-B isoform is a substrate of the APCCdh1 E3 ubiquitin ligase complex |
| title_short | The Drosophila MCPH1-B isoform is a substrate of the APCCdh1 E3 ubiquitin ligase complex |
| title_sort | drosophila mcph1 b isoform is a substrate of the apccdh1 e3 ubiquitin ligase complex |
| topic | Anaphase-Promoting Complex Drosophila MCPH1 Ubiquitination Xenopus egg extract |
| url | http://bio.biologists.org/content/3/7/669 |
| work_keys_str_mv | AT sarahghainline thedrosophilamcph1bisoformisasubstrateoftheapccdh1e3ubiquitinligasecomplex AT jamielrickmyre thedrosophilamcph1bisoformisasubstrateoftheapccdh1e3ubiquitinligasecomplex AT leifrneitzel thedrosophilamcph1bisoformisasubstrateoftheapccdh1e3ubiquitinligasecomplex AT lauraalee thedrosophilamcph1bisoformisasubstrateoftheapccdh1e3ubiquitinligasecomplex AT ethanlee thedrosophilamcph1bisoformisasubstrateoftheapccdh1e3ubiquitinligasecomplex AT sarahghainline drosophilamcph1bisoformisasubstrateoftheapccdh1e3ubiquitinligasecomplex AT jamielrickmyre drosophilamcph1bisoformisasubstrateoftheapccdh1e3ubiquitinligasecomplex AT leifrneitzel drosophilamcph1bisoformisasubstrateoftheapccdh1e3ubiquitinligasecomplex AT lauraalee drosophilamcph1bisoformisasubstrateoftheapccdh1e3ubiquitinligasecomplex AT ethanlee drosophilamcph1bisoformisasubstrateoftheapccdh1e3ubiquitinligasecomplex |