Regulation of Cellular Signaling by CUL5 is Dependent on Its Neddylation Status

CUL5 acts as the scaffold protein of the E3 ligase complex in ubiquitin-dependent protein degradation pathways. Overexpression of CUL5 inhibits cellular proliferation, whereas inhibition of CUL5 expression induces proliferation and may contribute to clinical disorders including cancer. The effects of CUL5 depend on its post-translational modification by NEDD8 (neddylation), a process that represents a potential therapeutic target. This study explores the structure-function relationship between CUL5 and its neddylation status in vitro.

CUL5 was mutated at the putative neddylation site Lys (K) 724 to Arg (K724RCUL5) and at three potential neddylation sites K724, K727, and K728 (K724R/K727R/K728RCUL5). Because mutation of the PKA phosphorylation site Ser (S) 730 (S730ACUL5) was previously shown to induce neddylation, a K724R/S730ACUL5 mutant was also generated. Mutant and wild-type constructs were expressed in rat endothelial cells (RAMEC), T47D cancer cells, and COS-1 cells. Cellular proliferation, MAPK phosphorylation, ERα expression, and CUL5 neddylation status were analyzed, including treatment with the neddylation inhibitor MLN4924.

Expression of wild-type CUL5 attenuated cell growth in RAMEC, T47D, and COS-1 cells. In contrast, expression of K724RCUL5 and K724R/S730ACUL5 mutants induced cellular growth, whereas the K724R/K727R/K728RCUL5 mutant had no significant effect on proliferation. In T47D cells, MAPK phosphorylation and estrogen receptor (ERα) expression were directly correlated with the neddylation status of CUL5. Western blot analysis of COS-1 cells treated with MLN4924 demonstrated that CUL5 remained neddylated in all mutant cell lines.

These findings suggest that modification of CUL5 by NEDD8 may occur at multiple lysine residues and that multi-site neddylation may contribute to the diverse regulatory effects of CUL5 on cellular signaling and proliferation.