Abstract
ABSTRACTThe 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.
Highlights
The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that catalyzes ubiquitin-mediated proteasomal degradation of target proteins
In the primary screen that involved the use of radiolabeled protein pools, both candidates were stable in the presence of Xenopus buffer (XB) and CycBD90, but they degraded in Xenopus egg extract supplemented with Cdh1, suggesting that they are substrates of APCCdh1 and not APCCdc20
We show that Drosophila MCPH1 (dMCPH1)-B undergoes Cdh1dependent degradation in Xenopus egg extract and not Cdc20dependent degradation
Summary
The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that catalyzes ubiquitin-mediated proteasomal degradation of target proteins. Cdc associates with the APC in early mitosis, leading to the destruction of proteins that control the onset of anaphase, whereas Cdh promotes degradation of APC substrates that control late mitosis and the following G1 phase. These co-activators provide APC substrate specificity by facilitating the recognition of specific destruction motifs (e.g. degrons) such as the D-box (RxxLxxxxN) or KEN box (Lys–Glu–Asn) (King et al, 1996; Min and Lindon, 2012; Pfleger and Kirschner, 2000). Mutations of these motifs block the recognition of the protein by the APC, preventing their APC-mediated destruction
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