Ubiquitin-conjugating enzyme 3 delays human lens epithelial cells in metaphase.
OBJECTIVE
Ubc3/Cdc34 is a ubiquitin-conjugating enzyme (Ubc) with well established functions in the G1-to-S-phase transition. Expecting to find similar effects in human lens epithelial cells (HLECs), the authors explored roles for this ubiquitin-conjugating enzyme in regulation of the HLEC cycle.


METHODS
Catalytically incompetent Ubc3 (C88S, L97S), wild-type (wt)Ubc3, and mutant (mt)Ubc2 (C93A) were expressed in HLECs, by using an adenoviral vector, and cell cycle progression was assessed.


RESULTS
Expression of mt- and wtUbc3, but not empty virus or mtUbc2, delayed the cell cycle in metaphase, rather than the expected G1 phase. Expression of both Ubc3s also stabilized M-phase regulators, cyclin A, cyclin B, and securin. Thus, it appeared that the Ubc3 enzymes were playing roles different from canonical proteolytic functions in targeting G1/S regulators for degradation. We also directly investigated the effect of inhibiting the proteasome on the cell cycle of HLECs. When the proteasome inhibitor was added to S-phase cells, the M-phase regulators were stabilized, and the cells were arrested in the G2/M phase. In contrast, if the proteasome inhibitor was added before the cells entered the S phase, stabilization of the G1 kinase inhibitors p21WAF and p27KIP was observed and the cells were arrested in the G1 phase.


CONCLUSIONS
The ubiquitin-proteasome pathway is involved in regulation of transitions between all phases of the HLEC cycle. However, in contrast with previously described roles for Ubc3 in governing G1/S transitions, expression of Ubc3 delays the HLEC cycle in metaphase. The data suggest novel roles for Ubc3 that do not involve the transfer of ubiquitin in the M phase in the HLEC cell cycle.
Liu
Qing
Q
Laboratory for Nutrition and Vision Research, JMUSDA-HNRCA at Tufts University, Boston, Massachusetts 02111, USA.
Shang
Fu
F
Whitcomb
Elizabeth
E
Guo
Weimin
W
Li
Wei
W
Taylor
Allen
A
D000256:Adenoviridae / Q000235:genetics; D064173:Anaphase-Promoting Complex-Cyclosome; D015153:Blotting, Western; D018929:Cell Culture Techniques; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology*; D008677:Metaphase / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes / Q000502:physiology*
C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; C577447:CDC34 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D015739:Nocodazole