Ubiquitin-conjugating enzyme 3 delays human lens epithelial cells in metaphase

Qing Liu, Fu Shang, Elizabeth Whitcomb, Weimin Guo, Wei Li, Allen Taylor

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

PURPOSE. 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.

Original languageEnglish
Pages (from-to)1302-1309
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume47
Issue number4
DOIs
StatePublished - Apr 1 2006

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Ubiquitin-Conjugating Enzymes
Metaphase
Lenses
Cell Cycle
Epithelial Cells
Cell Division
S Phase
Proteasome Inhibitors
G1 Phase
Proteasome Endopeptidase Complex
Ubiquitin
Securin
Cyclin B
Cyclin A
G2 Phase
Phase Transition
Phosphotransferases
Viruses
Enzymes

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Ubiquitin-conjugating enzyme 3 delays human lens epithelial cells in metaphase. / Liu, Qing; Shang, Fu; Whitcomb, Elizabeth; Guo, Weimin; Li, Wei; Taylor, Allen.

In: Investigative Ophthalmology and Visual Science, Vol. 47, No. 4, 01.04.2006, p. 1302-1309.

Research output: Contribution to journalArticle

Liu, Qing ; Shang, Fu ; Whitcomb, Elizabeth ; Guo, Weimin ; Li, Wei ; Taylor, Allen. / Ubiquitin-conjugating enzyme 3 delays human lens epithelial cells in metaphase. In: Investigative Ophthalmology and Visual Science. 2006 ; Vol. 47, No. 4. pp. 1302-1309.
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abstract = "PURPOSE. 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.",
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AU - Taylor, Allen

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N2 - PURPOSE. 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.

AB - PURPOSE. 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.

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