Abstract
Purpose: :
It is well known that the cell cycle is controlled by the ubiquitin system through the timed destruction of cell cycle regulatory proteins. We previously showed that the ubiquitin conjugating enzyme, UbcH7 plays a role in controlling the entry into- and exit from- S phase of the cell cycle in lens epithelial and other cell types (Whitcomb et. al 2009 Mol. Biol. Cell v20:1-9). Depletion of UbcH7 increases the length of time it takes to traverse S phase and decreases cell proliferation, while over expression of UbcH7 delays the G1 to S transition. The purpose of this study was to understand the mechanism by which UbcH7 exerts its influence on the cell cycle.
Methods: :
We depleted UbcH7 in cells using siRNA in a lens epithelial cell line as well as other cell types. We over expressed UbcH7 using plasmid expression vectors. Subsequently, we examined the levels of cell cycle regulatory proteins using immunoblots. We used a scratch assay to determine the rate of cell migration after UbcH7 depletion in a lens cell line. We complemented these cellular assays with in vitro ubiquitination and degradation assays using 35S radiolabeled proteins expressed in E. coli.
Results: :
We found that UbcH7 is involved in the ubiquitination and degradation of the cyclin dependent kinase inhibitor, p27 in a novel manner. Surprisingly we observed that UbcH7 inhibits the ubiquitination and degradation of p27. Additionally, UbcH7 appears to affect the level of p27 primarily in the cytosol. Since cytosolic p27 is involved in cell migration, we asked whether depletion of UbcH7 affected the rate at which lens epithelial cells moved to close a wound in a scratch assay. We observed that depletion of UbcH7 decreased the migration of lens cells in the wound healing assay.
Conclusions: :
We demonstrated that UbcH7 inhibits the degradation and ubiquitination of the cell cycle regulatory protein p27. This was surprising as ubiquitination enzymes generally promote degradation. Additionally, we show that UbcH7 depletion decreases the rate of cell migration. Given that secondary cataracts are due to the proliferation and migration of lens epithelial cells remaining after removal of the cataractous lens, these data suggest that inhibition of UbcH7 might be an attractive target for therapy to decrease the incidence of secondary cataract.
Keywords: cataract • cell survival • proliferation