Mono-ubiquitination Drives Nuclear Export of the Human DCN1-like Protein hDCNL1

Kenneth Wu,Hua Yan,Lei Fang,Xinjiang Wang,Cathie Pfleger,Xuejun Jiang,Lan Huang,Zhen-Qiang Pan

doi:10.1074/jbc.m111.273045

Abstract

Conjugation of Nedd8 to a cullin protein, termed neddylation, is an evolutionarily conserved process that functions to activate the cullin-RING family E3 ubiquitin ligases, leading to increased proteasomal degradation of a wide range of substrate proteins. Recent emerging evidence demonstrates that cellular neddylation requires the action of Dcn1, which, in humans, consists of five homologues designated as hDCNL1-5. Here we revealed a previously unknown mechanism that regulates hDCNL1. In cultured mammalian cells ectopically expressed hDCNL1 was mono-ubiquitinated predominantly at K143, K149, and K171. Using a classical chromatographic purification strategy, we identified Nedd4-1 as an E3 ligase that can catalyze mono-ubiquitination of hDCNL1 in a reconstituted ubiquitination system. In addition, the hDCNL1 N-terminal ubiquitin-binding domain is necessary and sufficient to mediate mono-ubiquitination. Finally, fluorescence microscopic and subcellular fractionation analyses revealed a role for mono-ubiquitination in driving nuclear export of hDCNL1. Taken together, these results suggest a mono-ubiquitination-mediated mechanism that governs nuclear-cytoplasmic trafficking of hDCNL1, thereby regulating hDCNL1-dependent activation of the cullin-RING E3 ubiquitin ligases in selected cellular compartments.

Highlights

To assess the effects of purified hDCNL1 in neddylation, we employed the reconstituted neddylation assay that consists of purified Nedd8, APP-BP1/Uba3, Ubc12, and ROC1-CUL1324–776
His-hDCNL1 activated cullin 1 (CUL1) neddylation as well. These results demonstrate that hDCNL1 activates CUL1 neddylation directly, with the most pronounced effect seen in the presence of low concentration of Ubc12
We suggest that the mono-ubiquitination of hDCNL1 by Nedd4-1 may proceed in a mechanism independent of WW domain-mediated interactions

Summary

Introduction

Nuclear Export of hDCNL1 by Mono-ubiquitination this interaction appears critical for stimulating cullin neddylation in vitro [16]. To conjugate 32P-Nedd8 to CUL1, 32P-Nedd8 (1 ␮l) was incubated in a reaction (30 ␮l) that contained 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 2 mM ATP, 0.6 mM DTT, 0.1 mg/ml BSA, APP-BP1/Uba3 (3.3 nM), Ubc12 (3 nM), and ROC1-CUL1324–776 (0.25 ␮M), in the presence or absence of GST-hDCNL1 (1.2 ␮M or as indicated).

Results

Conclusion