Ubiquitin-dependent proteolysis of the microtubule end-binding protein 1, EB1, is controlled by the COP9 signalosome: Possible consequences for microtubule filament stability

The CON signalosome (CSN) is a regulatory particle of the ubiquitin (Ub) proteasome system (UPS) consisting of eight subunits (CSN1-CSN8). We show that the CSN stabilizes the microtubule end-binding protein 1 (EB1) towards degradation by the UPS. EB1, the master regulator of microtubule plus ends, controls microtubute growth and dynamics. Therefore, regulation of EB1 stability by the CSN has consequences for microtubule function. EB1 binds the CSN via subunit CSN5. The C terminus of EB1 is sufficient for interaction with the CSN. Dimerization of EB1 is a prerequisite for complex association and subsequent CSN-mediated phosphorylation, as revealed by studies with the EB1I224A mutant, which is unable to dimerize. In cells, EB1 and CSN co-localize to the centrosome, as demonstrated by confocal fluorescence microscopy. EBI is ubiquitinated and its proteolysis can be inhibited by MG132, demonstrating that it is a substrate of the UPS. Its degradation is accelerated by inhibition of CSNassociated kinases. HeLa cells permanently expressing siRNAs against CSNI (siCSNI) or CSN3 (siCSN3) exhibit reduced levels of the CSN complex accompanied by lower steady-state concentrations of EB1. In siCSNI cells, EB1 is less phosphorylated as compared with control cells, demonstrating that the protein is most likely protected towards the UPS by CSN-mediated phosphorylation. The CSN-dependent EBI stabilization is not due to the CSN-associated deubiquitinating enzyme USP15. Treatment with nocodazole revealed a significantly increased sensitivity of siCSN1 and siCSN3 cells towards the microtubule depolymerizing drug accompanied by a collapse of microtubule filaments. A nocodazole-induced cellcycle arrest was partially rescued by CSN1 or EB1. These data demonstrate that the CSN-dependent protection of EBI is important for microtubule function. (c) 2007 Elsevier Ltd. All rights reserved.