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. 2009 Jan;20(1):1-9.
doi: 10.1091/mbc.e08-01-0036. Epub 2008 Oct 22.

Novel control of S phase of the cell cycle by ubiquitin-conjugating enzyme H7

Elizabeth A Whitcomb  1 , Edward J Dudek, Qing Liu, Allen Taylor
Affiliations

Novel control of S phase of the cell cycle by ubiquitin-conjugating enzyme H7

Elizabeth A Whitcomb et al. Mol Biol Cell. 2009 Jan.

Abstract

Timely degradation of regulatory proteins by the ubiquitin proteolytic pathway (UPP) is an established paradigm of cell cycle regulation during the G2/M and G1/S transitions. Less is known about roles for the UPP during S phase. Here we present evidence that dynamic cell cycle-dependent changes in levels of UbcH7 regulate entrance into and progression through S phase. In diverse cell lines, 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 doubles the time to traverse S phase, whereas UbcH7 overexpression reduces the proportion of cells in S phase. These data suggest a role for UbcH7 targets in the completion of S phase and entry into G2. Notably, UbcH7 knockdown was coincident with elevated levels of the checkpoint kinase Chk1 but not Chk2. 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 a UPP. Together the data identify novel roles for the UPP, specifically UbcH7 in the regulation of S phase transit time as well as in cell proliferation.

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Figures

Figure 1.
Figure 1.
UbcH7 is regulated in a cell cycle–dependent manner. (A) Cell cycle profile of cells synchronized by treatment with 2 mM HU. HeLa cells were treated for 18 h, HU-containing medium was washed out, and cells were allowed to resume cycling in medium without drugs. Mean fluorescence intensity relating to DNA content is plotted on the X axis, and cell number is plotted on the Y axis. (B) UbcH7 levels are low in S phase and rise in G2/M. HeLa (left panel) or HLE (right panel) cells were treated with HU for 18 h and then released from drug treatment to resume cycle. Lysates, prepared from samples at times after drug release, were blotted using α-UbcH7, α-UbcH10, α-Ubc3, α-Ubc2, and α-E1, the latter being used as a loading control. We and others have demonstrated no alterations in E1 levels during different phases of the cell cycle (Stephen et al., 1996; Liu et al., 2004). Because E2 levels from the same samples were assessed on different immunoblots, E1 controls are shown for each blot. The majority of cells (≥75%) were in the cell cycle phase indicated. Bottom, quantitation of gels shown above. (C) UbcH7 is high in mitotic cells. Immunofluorescent staining with α-UbcH7 (left), α-tubulin (middle), or isotype matched control antibody (right) on cells treated for 18 h with nocodazole. Mitotic cells are noted with a white arrow in all panels.
Figure 2.
Figure 2.
Levels of UbcH7 levels determine the duration of S phase of the cell cycle. (A–C) Asynchronous HeLa cells were treated with siRNA specific for UbcH7 or a nonspecific siRNA for 48, 72, or 96 h as indicated. (A) Cell lysates were blotted for α-UbcH7 or α-E1 as a loading control (top panels). Cells were analyzed for DNA content (bottom panels). (B) Average ratio of S phase during increased time of knockdown. The percentage of cells in S phase in UbcH7-depleted samples was compared with the percentage in NS siRNA-treated cells. Average of three to seven experiments; *p < 0.01. (C) Depletion of UbcH7 results in an increase in S phase in HEK-293 and HLE cells. Cells were treated with siRNA for 72 h. (D) COS cells were transfected with UbcH7 to increase levels of the enzyme or empty plasmid for 48 h. The cell cycle profile was determined by FACS analysis, and the ratio of G1 or S phase cells from cells expressing UbcH7 compared with the empty vector was averaged from three independent experiments. Right panel, Western blot of lysates from cells expressing UbcH7 or empty plasmid.
Figure 3.
Figure 3.
UbcH7 knockdown delays progression from S to G2 phase. HeLa cells were treated for 48 h with siRNA as indicated. Cells were then treated overnight with 2 mM HU for synchronization. (A) Immunoblot showing knockdown of UbcH7. (B) Cell cycle profile of cells at times after drug release. Cell cycle numbers are from duplicate samples. Similar delays in S phase progression to G2 were also observed with another UbcH7 siRNA sequence. (C) Top, cyclin A and E1 levels from samples treated with NS siRNA or UbcH7-specific siRNA immediately after drug release or 12 h after release. Bottom panel, quantitation of cyclin A levels normalized to E1 levels. Normalized cyclin A levels were compared between UbcH7 siRNA-treated cells and NS siRNA-treated cells at each time point. (D) Knockdown of UbcH7 decreases cell proliferation. Comparison of MTS OD between NS siRNA and UbcH7 siRNA at 72 and 96 h of knockdown.
Figure 4.
Figure 4.
UbcH7 is a substrate of the ubiquitin proteasome pathway: (A) HeLa cells were treated for 4 h with cycloheximide in the presence or absence of MG132 as indicated (top). Levels of UbcH7 were normalized to E1 (bottom). (B) Ubiquitination reactions were performed with 125I-UbcH7, reticulocyte lysate, Ubc4, and ubiquitin as indicated. Bands for monoubiquitinated (bottom panel, short exposure) and polyubiquitinated (top panel, long exposure) UbcH7 are noted. A nonspecific band is indicated by an asterisk. As noted at the top of the gel in lane 2, some low background level of UbcH7 oligomerization or ubiquitination is occurring in the presence of lysate and Ubc4. (C) 125I-UbcH7 was added to reticulocyte lysates containing ATP, Ubc4, and MG132 as indicated. Degradation was calculated by comparing the TCA-soluble counts to total radioactivity after 2 h. (D) HeLa cells synchronized using HU as in Figure 1A were lysed, and the resulting extracts were used for degradation of 125I-UbcH7 in the presence of Ubc4, ATP, and ubiquitin. The data shown are the average of five experiments with independent extracts. *p < 0.05 S versus G2/M.
Figure 5.
Figure 5.
UbcH7 knockdown increases Chk1 levels and decreases phosphorylated PTEN specifically. Asynchronously growing cells were treated with UbcH7 siRNA or NS siRNA for 72 h and were blotted for proteins indicated. All results were observed in at least two experiments each run in duplicate. (A) Samples from cells depleted of UbcH7 were blotted with α-Chk1, α-Chk2, α-E1, or α-UbcH7 as indicated. The E1 loading control is shown for each immunoblot. (B) Samples were blotted with α-E1, α-E6-AP, α-PTEN, α-UbcH10, or α-UbcH7 as indicated. (C) Left, HeLa cells were treated with siRNA specific for UbcH7 for 72 h and stained for α-Chk1 (top) or α-UbcH7 (bottom). Right, HeLa cells were treated with UbcH7 siRNA or NS siRNA for 72 or 96 h. Percentage of cells containing Chk1 foci were quantified from three separate experiments. *p < 0.05. (D) Samples were blotted with α-P-Ser 280 Chk1, α-P-Ser 380 PTEN (αP-PTEN), α-Akt, α-P-Ser 380/Thr 382/383 PTEN (αP3-PTEN), or α-E1 as indicated. (E) HeLa cells were synchronized at the G1/S boundary by treatment with 2 mM HU for 18 h. Cells were released from drug synchronization, and samples were obtained immediately after treatment (G1), 4 h after release into drug-free media (S phase), or 8 h after release (G2/M) and blotted with α-E1, α-P-Thr 382/383 PTEN (αP3-PTEN) or α-P-Ser 280 Chk1as indicated.
Figure 6.
Figure 6.
Model for UbcH7 regulation of Chk1 and PTEN. Dashed lines indicate proposed roles for UbcH7.
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