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J Mol Biol. 2007 Apr 27;368(2):550-63. Epub 2007 Feb 22.
Ubiquitin-dependent proteolysis of the microtubule end-binding protein 1, EB1, is controlled by the COP9 signalosome: possible consequences for microtubule filament stability.
The
COP9 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 microtubule 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. EB1 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 CSN-associated kinases. HeLa cells
permanently expressing siRNAs against CSN1 (siCSN1) or CSN3 (siCSN3)
exhibit reduced levels of the CSN complex accompanied by lower
steady-state concentrations of EB1. In siCSN1 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 EB1 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 cell-cycle
arrest was partially rescued by CSN1 or EB1. These data demonstrate that
the CSN-dependent protection of EB1 is important for microtubule
function.
- PMID:
- 17350042
- DOI:
- 10.1016/j.jmb.2007.02.052
- [Indexed for MEDLINE]
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