Evidence that the transcriptional repressor ICER is regulated via the N-end rule for ubiquitination

ICER is a transcriptional repressor that is mono-or poly-ubiquitinated. This either causes ICER to be translocated from the nucleus, or degraded via the proteasome, respectively. In order to further studies the proteins involved in ICER regulation mass spectrometry analysis was performed to identify potential candidates. We identified twenty eight ICER-interacting proteins in human melanoma cells, Sk-Mel-24. In this study we focus on two proteins with potential roles in ICER proteasomal degradation in response to the N-end rule for ubiquitination: the N-alpha-acetyltransferase 15 (NAA15) and the E3 ubiquitin-protein ligase UBR4. Using an HA-tag on the N- or C-terminus of ICER ((N)HAICER or ICER(C)HA) it was found that the N-terminus of ICER is important for its interaction to UBR4, whereas NARG1 interaction is independent of HA-tag position. Silencing RNA experiments show that both NAA15 and UBR4 up-regulates ICER levels and that ICER's N-terminus is important for this regulation. The N-terminus of ICER was found to have dire consequences on its regulation by ubiquitination and cellular functions. The half-life of (N)HAICER was found to be about twice as long as ICER(C)HA. Polyubiquitination of ICER was found to be dependent on its N-terminus and mediated by UBR4. This data strongly suggests that ICER is ubiquitinated as a response to the N-end rule that governs protein degradation rate through recognition of the N-terminal residue of proteins. Furthermore, we found that (N)HAICER inhibits transcription two times more efficiently than ICER(C)HA, and causes apoptosis 5 times more efficiently than ICER(C)HA. As forced expression of ICER has been shown before to block cells in mitosis, our data represent a potentially novel mechanism for apoptosis of cells in mitotic arrest.