The cell cycle is tightly controlled by the timed destruction of cell cycle regulatory proteins via the UPP. Here we present novel evidence that UbcH7 controls the entry into and duration of S phase. The level of UbcH7 protein naturally varies in a cell cycle–dependent manner. When the level of UbcH7 is decreased, an increase in the S phase population is observed, due to a delay in the completion of S phase and progression to G2/M. Conversely, when UbcH7 is over expressed, there is a decrease in the proportion of cells in S phase with an apparent block in S phase entry. The regulation of UbcH7 levels appears to be through UPP-dependent proteolysis in S phase. Inverse relationships between levels of Chk1 and UbcH7 and direct relationships between levels of UbcH7 and P3-PTEN are consistent with UbcH7 mediating its control via the Akt pathway.
Cell cycle differences in UbcH7 protein levels were first noted using a lens epithelial cell line, where an increase in UbcH7 was observed as cells progress from S to G2/M (Liu et al., 2004
). The present study clarifies and generalizes the relationship between UbcH7 levels and cell cycle progression. We observed that alteration of UbcH7 levels affects cell cycle progression in four different cell types, indicating that the role of UbcH7 in regulating the duration of S phase of the cell cycle is generalizable. Specifically, in HU-synchronized cells we noted a striking decrease in UbcH7 levels when all the cells were in S phase when compared with UbcH7 levels observed when the cells were at the G1/S boundary (B). The UbcH7 levels recovered as the cells progressed into G2. The absence of a concerted change between UbcH7 protein levels and UbcH7 mRNA levels coupled with in vitro observations that UbcH7 is ubiquitinated and that in vivo degradation of UbcH7 in cells is stabilized by the proteasome inhibitor MG132 () suggest that the cell cycle–related modulation in UbcH7 protein levels are regulated via a UPP-dependent pathway. This is supported by the observation that degradation of UbcH7 in vitro is markedly increased in the presence of ubiquitin and Ubc4 and requires ATP. Additionally, these data suggest that Ubc4 is an E2 that can participate in the degradation of UbcH7. Furthermore, the inability of inactive UbcH7 to be degraded is consistent with a catalytic role for UbcH7 in its own degradation. Taken together, these data suggest that UbcH7 is unique with respect to other E2s (Rape and Kirschner, 2004
) in that its activity is required for its own degradation and that degradation is enhanced by additional E2 activity.
We also sought mechanistic information about how UbcH7 depletion results in a delay in progression from S to G2 phase. Our discoveries that knockdown of UbcH7 results in an increase in Chk1 but not Chk2 (A) and that this increase is coupled to an increase in P-280 Chk1 suggests involvement of the Akt pathway (). A role for the Akt pathway in regulating P-280 Chk1 is corroborated by the marked decrease in P3-PTEN when UbcH7 is decreased (see D and ). In addition, our observation of increased P-280 Chk1 appears more consistent with modulation of Chk1 by the Akt pathway (Shtivelman et al., 2002
; Puc et al., 2005
) because phosphorylation via ATR would result in decreased Chk1 levels (Zhang et al., 2005
). This is further supported by our observation of increased perinuclear cytoplasmic staining of Chk1 in UbcH7-depleted cells (C). Chk1 relocalizes to the cytoplasm in PTEN-depleted cells (Puc et al., 2005
), and PTEN modulates Chk1 via the Akt pathway. Furthermore, phosphorylation by Akt precludes phosphorylation by ATR, and therefore Chk1 is stabilized rather than targeted for degradation. Taken together the data indicate that the increase in Chk1 is due, at least in part, to an increase in signaling through Akt. Additionally, UbcH7 can catalyze the ubiquitination of Chk1 in vitro (Y.-W. Zhang, personal communication; Zhang et al., 2005
) suggesting that Chk1 may be a direct UbcH7 target (see ).
It was recently demonstrated that UbcH7 can participate in the ubiquitination of PTEN in conjunction with NEDD 4.1 in cell free assays (Wang et al., 2007
). However, it has also been shown that NEDD 4.1 is dispensable for the ubiquitination of PTEN (Fouladkou et al., 2008
). Thus, the involvement of UbcH7 in controlling PTEN levels requires further investigation.
UbcH7 interacts with several E3 ligases, including Parkin (Shimura et al., 2000
), Cbl (Yokouchi et al., 1999
; Zheng et al., 2000
), E6-AP (Nuber et al., 1998
; Huang et al., 1999
), NK-lytic–associated molecule (Fortier and Kornbluth, 2006
), NEDD4 (Anan et al., 1998
; Wang et al., 2007
), TRIAD-1 (Marteijn et al., 2005
), Smurf2 (Ogunjimi et al., 2005
), TRAF6 (Geetha et al., 2005
), and components of the SCF complex (Staropoli et al., 2003
; Oh et al., 2004
). Additionally UbcH7 can bind to the E3 ligase BRCA-1 but does not catalyze its self-ubiquitinating activity (Brzovic et al., 2003
). Although UbcH7 working with one or more of these E3s may account for the S phase phenotype, at this point we cannot ascertain which of these associations explain our observations.
Only one other report related UbcH7 to cell proliferation. Our results differ from those of Pringa et al. (2000)
. They used a chronic knockdown model and found no relationship between cell cycle or proliferation and levels of UbcH7. However, it is difficult to determine whether their chronic knockdown could have allowed the outgrowth of cells with compensatory changes in gene expression that mask the function of UbcH7 as had been observed for acute versus chronic knockdown of the retinoblastoma (Rb) gene product (Sage et al., 2003
). We have consistently seen that after 4 d (96 h) of UbcH7 depletion, there is an increase in the percentage of cells in S phase (B and Supplementary Figure S3). After depletion for 8 d, an increase in S phase is still observed (Supplementary Figure S3). However, after 12 d of siRNA treatment, the level of UbcH7 recovers and the cell cycle profile normalizes. Thus, we conclude that the decrease in UbcH7 levels controls the increase in S phase percentage during this interval. We would predict that a longer knockdown of UbcH7 may result in the normalization of the cell cycle profile with compensatory changes in expression of other E2s or UbcH7 substrates.
In this report we provide a new paradigm for cell cycle control via the ubiquitin system describing a new role for UbcH7. We hypothesize that levels of UbcH7 must decrease in order for S phase to commence and rise for S phase to be completed. UbcH7 may be involved in controlling the levels of both phosphorylated and nonphosphorylated forms of Chk1 and phosphorylated PTEN. Additional understanding of how UbcH7 is activated and degraded will provide further insight into how this E2 is exerting control over cell cycle progression and may allow for more intelligent design of drugs to modulate proliferation for control of cancer, secondary cataract, and other proliferative diseases.