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. 2019 Jan;33(1):1235-1247.
doi: 10.1096/fj.201800960R. Epub 2018 Aug 16.

Stabilization of p27Kip1/CDKN1B by UBCH7/UBE2L3 catalyzed ubiquitinylation: a new paradigm in cell-cycle control

Elizabeth A Whitcomb  1 , Yien Che Tsai  2 , Johnvesly Basappa  1 , Ke Liu  1 , Aurélie K Le Feuvre  1 , Allan M Weissman  2 , Allen Taylor  1
Affiliations

Stabilization of p27Kip1/CDKN1B by UBCH7/UBE2L3 catalyzed ubiquitinylation: a new paradigm in cell-cycle control

Elizabeth A Whitcomb et al. FASEB J. 2019 Jan.

Abstract

Ubiquitinylation drives many cellular processes by targeting proteins for proteasomal degradation. Ubiquitin conjugation enzymes promote ubiquitinylation and, thus, degradation of protein substrates. Ubiquitinylation is a well-known posttranslational modification controlling cell-cycle transitions and levels or/and activation levels of ubiquitin-conjugating enzymes change during development and cell cycle. Progression through the cell cycle is tightly controlled by CDK inhibitors such as p27Kip1. Here we show that, in contrast to promoting its degradation, the ubiquitin-conjugating enzyme UBCH7/UBE2L3 specifically protects p27Kip1 from degradation. Overexpression of UBCH7/UBE2L3 stabilizes p27Kip1 and delays the G1-to-S transition, while depletion of UBCH7/UBE2L3 increases turnover of p27Kip1. Levels of p21Cip1/Waf1, p57Kip2, cyclin A and cyclin E, all of which are also involved in regulating the G1/S transition are not affected by UBCH7/UBE2L3 depletion. The effect of UBCH7/UBE2L3 on p27Kip1 is not due to alteration of the levels of any of the ubiquitin ligases known to ubiquitinylate p27Kip1. Rather, UBCH7/UBE2L3 catalyzes the conjugation of heterotypic ubiquitin chains on p27Kip1 that are proteolytically incompetent. These data reveal new controls and concepts about the ubiquitin proteasome system in which a ubiquitin-conjugating enzyme selectively inhibits and may even protect, rather than promote degradation of a crucial cell-cycle regulatory molecule.-Whitcomb, E. A., Tsai, Y. C., Basappa, J., Liu, K., Le Feuvre, A. K., Weissman, A. M., Taylor, A. Stabilization of p27Kip1/CDKN1B by UBCH7/UBE2L3 catalyzed ubiquitinylation: a new paradigm in cell-cycle control.

Keywords: CDKI; cell division; proteasome; ubiquitin-conjugating enzyme.

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Conflict of interest statement

Funding for this work was provided by U.S. National Institutes of Health (NIH) National Eye Institute Grants RO1 EY 13250, RO1 EY21212, and RO1 EY26979, U.S. Department of Agriculture contract 1950-510000-060-03A (to A.T.), and the NIH Center for Cancer Research–National Cancer Institute Intramural Research Program (to Y.C.T. and A.M.W.). The authors thank E. Bejarano-Fernandez (Tufts University) for critical review and W. Yang (Brandeis University, Waltham, MA, USA) for expert technical assistance. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Expression of UBCH7/UBE2L3 impairs G1-to-S transition via posttranslational stabilization of p27Kip1. A) HeLa cells were transfected with empty vector or vector expressing UBCH7/UBE2L3 (GFP-UBCH7) for 48 h. Cells were synchronized with 2 mM HU for 18 h. Drug was washed out, and cells were allowed to progress through cell cycle and were collected at times indicated. DNA content was measured using propidium iodide and fluorescence-activated cell sorting analysis gated on transfected cells. Data are average of 3 independent experiments. Significance values were calculated by paired Student’s t test. B) HEK-293 cells were transfected with vector expressing FLAG-p27Kip1 and vectors expressing WT UBCH7/UBE2L3 (UBCH7) or active site mutant of UBCH7/UBE2L3 (C89S UBCH7). After 48 h of expression, cells were collected and probed for anti-E1 (loading control) anti FLAG-p27 or anti UBCH7/UBE2L3, as indicated. C) HeLa cells were treated with siRNA for UBCH7/UBE2L3 or control siRNA for 72 h. mRNA was collected and subject to real-time quantitative PCR. Signals for UBCH7/UBE2L3 and p27Kip1 were compared to GAPDH levels and then normalized to NS siRNA signal. Data are presented as means ± sem, n = 3. D) WT, but not mutant UBCH7/UBE2L3, stabilizes p27Kip1 in cell-free assays. All reactions contain ATP-generating system, cell lysate, ubiquitin, and [35S]-p27Kip1. Degradation of p27Kip1 without addition of E2 was set as 100%. Average of 10 independent experiments ± sem.
Figure 2
Figure 2
UBCH7/UBE2L3 depletion increases proteasomal turnover of p27Kip1 but not p21cip1/waf1. Cells were depleted of UBCH7/UBE2L3 for 72 h using siRNA. Lysates from treated cell lines were blotted for E1, UBCH7/UBE2L3 (UBCH7), p21, or p27 as indicated. A) UBCH7/UBE2L3 depletion results are evident decreased p27 levels. Right: quantification of 11 experiments from 3 cell lines ± sem. Significance value calculated by paired Student’s t test. B) p27, but not p21, is diminished after UBCH7/UBE2L3 depletion. Right: quantification from 5 independent experiments ± sem. Significance value was calculated by paired Student’s t test. C) UBCH7/UBE2L3 depletion increases proteasomal turnover of p27Kip1. After 72 h depletion, HeLa cells were treated with cycloheximide (CHX) or MG132, as indicated, for up to 8 h. Cytosolic and nuclear fractions were separated and subjected to immunoblot as indicated. Quantification from 4 independent experiments. *P < 0.05.
Figure 3
Figure 3
UBCH7/UBE2L3 depletion does not alter levels of E3s known to target p27Kip1 for proteasomal degradation. HeLa and HEK-293 cells were treated with siRNA specific for UBCH7/UBE2L3 or control siRNA for 72 h. Cells were lysed and probed for p27Kip1, UBCH7/UBE2L3, and E1 as loading control. A) Lysates from HeLa and HEK-293 cells were probed in 3 separate Western blots for E3s. B) HeLa lysates were probed for DDB1, p27Kip1, UBCH7/UBE2L3, and E1 as noted.
Figure 4
Figure 4
UBCH7/UBE2L3 catalyzes ubiquitinylation of p27Kip1. In vitro ubiquitinylation assay using HELC G0 lysate and His6-p27Kip1. Ubiquitinylation was carried out in presence of 500 nM Cdc34/Ubc3/UBE2R1 (Ubc3) or UBCH7/UBE2L3 (UBCH7) as indicated; ATP and ubiquitin were added (+) in lanes as noted. Immunoblots are for total ubiquitin. Brackets identify high mass conjugates in stacking gel. A) Ubiquitinylation with WT ubiquitin. Left: total ubiquitinylation; right, after Ni-NTA agarose purification. First 4 lanes in each blot contained His6-p27Kip1; lanes 5–8 had no His6-p27Kip1. B) p27Kip1 was isolated as in (A), and ubiquitinylation was performed with K-only mutant ubiquitins: K11O, K29O, K63O, K48R or K48O. C) Quantification from 3 (K48R) or 4 (K48O) independent experiments.
Figure 5
Figure 5
UBCH7/UBE2L3 catalyzes attachment of nonproteasome targeting chains on p27Kip1. In vitro ubiquitinylation assays were performed in presence and absence of MG132. A) Left: total ubiquitination. Right: p27Kip1 specific ubiquitinylation. B) Quantification of conjugates on p27Kip1 in absence of MG132, normalized to amount of conjugation (6, 12), formed in absence of additional E2. C) Fold change of ubiquitin conjugates on p27Kip1 in presence compared to absence of MG132. B, C) Average of 8 to 10 independent experiments. Significance values calculated by paired Student’s t test; data presented as means ± sem. D) Ubiquitinylation of p27Kip1 in cells depleted of UBCH7/UBE2L3. Left: lysate or anti FLAG immunoprecipitates probed for E1 (loading control) UBCH7/UBE2L3 (UBCH7) or FLAG (p27Kip1), as indicated. Middle, immunoprecipitates probed with anti-ubiquitin. Right, immunoprecipitates probed with anti-K48 ubiquitin. Representative of 4 independent experiments. E) K29 and K63 ubiquitin chains protect p27Kip1 from degradation in UBCH7/UBE2L3-dependent manner. HEK-293 cells were transfected with UBCH7/UBE2L3 or empty vector (EV) and FLAG-p27Kip1 with WT or K-to-R mutant ubiquitin. Cells were treated for 4 h with cycloheximide (CHX) and probed with anti-FLAG. Luciferase was cotransfected and used as loading and transfection control. Representative of 3 independent experiments.
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