The Anaphase Promoting Complex Induces Substrate Degradation during Neuronal Differentiation

Dympna Harmey,Scott Simanski,Anthony Smith,Carole Zaki Moussa,Nagi G Ayad

doi:10.1074/jbc.m804944200

Dympna Harmey, Scott Simanski + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m804944200

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Abstract

The anaphase promoting complex (APC) is an E3 ubiquitin ligase required for the metaphase-to-anaphase transition and mitotic exit. However, APC also plays roles in G(1), where it is regulated by Cdh1, and APC activity has also been detected in differentiated and non-proliferating cells, suggesting that it may play roles outside the cell cycle. Here, we report that disrupting APC(Cdh1) activity inhibits neurite outgrowth of both PC12 pheochromocytoma cells and primary cerebellar granule cells. APC(Cdh1) activity dramatically increases as PC12 cells differentiate in response to nerve growth factor. Furthermore, a key target degraded by APC(Cdh1) following nerve growth factor treatment is the F-box protein Skp2, and APC(Cdh1)-mediated destruction of Skp2 is essential for proper terminal differentiation of neuronal precursors.

Highlights

The essential role of APCCdc20 in initiating mitotic transitions is firmly established [1]
TGF-␤ stimulation of epithelial cells induces hyperactivation of anaphase promoting complex (APC) [12, 13], and these changes occur within minutes, suggesting that APC activation is an immediate response to TGF-␤ or is a cue to withdraw from the cell cycle
We report here that the nerve growth factor (NGF) signaling pathway rapidly induces APCCdh1 activity and that NGF induces the rapid degradation of APC substrates, including cyclin B1 and the F-box protein Skp2, which is necessary for degradation of the cyclin-dependent kinase inhibitor p27Kip1 [14]

Summary

EXPERIMENTAL PROCEDURES

Degradation and Ubiquitinylation Assays—In vitro degradation assays were performed essentially as described [15]. PC12 cells were treated for various times with NGF, and cell extracts were prepared after hypotonic swelling and repeated freeze-thaw cycles. Extracts were supplemented with ubiquitin (0.1 ␮g/ml), cycloheximide (1 ␮g/ml), and an energy-regenerating system. In vitro translated (35S-labeled) cyclin B, ⌬dboxcyclin B, Skp, or ⌬dbox-Skp proteins were incubated in 20 ␮l of cell extract for the indicated times at room temperature, and the reactions were stopped by adding SDS-containing sample buffer. In vitro ubiquitination assays were performed as described [10]. PC12 cells were incubated in the presence or absence of FEBRUARY 13, 2009 VOLUME 284 NUMBER 7

APC Induces Substrate Degradation

RESULTS

Methods