Abstract
Purpose: :
Timely degradation of cell cycle regulatory proteins by the ubiquitin dependent proteolytic pathway (UPP) is an established paradigm of cell cycle regulation during the G2/M and G1/S transitions. Far less is known about the rules for UPP regulation during S phase. Here we present novel evidence that dynamic cell-cycle dependent regulation of levels of the ubiquitin-conjugating enzyme (Ubc) H7, regulates both entrance into S phase as well as timely progression through S phase to G2.
Methods: :
We used human lens and HeLa cell lines, RNAi to suppress UbcH7 and FAX analyses to monitor cell cycle.
Results: :
We showed that UbcH7 protein levels are dramatically reduced in S phase but are fully restored by G2. Knockdown of UbcH7 increases the proportion of cells in S phase and decreases the proportion in G1. Consistently, UbcH7 over expression reduces the proportion of S phase cells while increasing the proportion of G1 cells. UbcH7 depletion doubles the time to complete S phase in synchronized cells, suggesting a role for UbcH7 targets in the completion of S phase and entry into G2. Notably, these effects of UbcH7 knockdown were coincident with elevated levels of the checkpoint kinase Chk1 and P-280 Chk1 (but not Chk2) and with attenuated levels of phosphorylated PTEN. These results argue that UbcH7 promotes S phase progression to G2 by modulating the intra-S phase checkpoint mediated by Chk1. Furthermore, UbcH7 levels appear to be regulated by post-translational mechanisms involving the UPP.
Conclusions: :
Together these data identify UbcH7 as a regulator of S phase entry and progression and suggest a new role for UbcH7 and the UPP in cell proliferation in health and disease.
Keywords: proliferation • proteolysis • cell survival