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. 2014 Apr 1;74(7):2006-14.
doi: 10.1158/0008-5472.CAN-13-1263. Epub 2014 Feb 7.

"V体育ios版" PP2A-B55β antagonizes cyclin E1 proteolysis and promotes its dysregulation in cancer

Yingmeei Tan  1 , Dahui Sun, Weijian Jiang, Kathleen Klotz-Noack, Ajay A Vashisht, James Wohlschlegel, Martin Widschwendter, Charles Spruck
Affiliations

PP2A-B55β antagonizes cyclin E1 proteolysis and promotes its dysregulation in cancer

Yingmeei Tan et al. Cancer Res. .

Abstract

Cyclin E1 regulates the initiation of S-phase in cellular division VSports手机版. However, in many cancers, cyclin E1 is aberrantly overexpressed and this molecular phenotype correlates with increased tumor aggressiveness and poor patient survival. The molecular cause(s) of cyclin E1 abnormalities in cancers is poorly understood. Here, we show that cyclin E1 overexpression in cancer is promoted by dysregulation of the protein phosphatase PP2A-B55β. PP2A-B55β targets the N- and C-terminal phosphodegrons of cyclin E1 for dephosphorylation, thus protecting it from degradation mediated by the SCF(Fbxw7) ubiquitin ligase. Augmented B55β expression stabilizes cyclin E1 and promotes its overexpression in cancer-derived cell lines and breast tumors. Conversely, B55β ablation enforces the degradation of cyclin E1 and inhibits cancer cell proliferation in vitro and tumor formation in vivo. Therefore, PP2A-B55β promotes cyclin E1 overexpression by antagonizing its degradation and its inhibition could represent a therapeutic mechanism for abrogating cyclin E1 function in cancers. .

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Conflict of interest statement

Conflicts of interest: None

Figures

Figure 1
Figure 1. PP2A dephosphorylates cyclin E1 and antagonizes its ubiquitylation by SCFFbxw7
A, Cyclin E1 co-IPs with PP2A subunits Cα (left) and Aα (right) expressed in HEK293T cells. WCE, whole cell extract. B, Endogenous cyclin E1 co-IPs with endogenous Aα and Cα from HeLa cells. C, Increased phosphorylation of the N- and C-terminal phosphodegrons (p-Thr77, p-Thr395) of cyclin E1 in HeLa cells transfected with Cα siRNA. Actin is shown as a loading control. MG132 (10 µM) was added to the cells 1 hr prior to harvesting to stabilize phosphorylated forms of cyclin E1. D, PP2A dephosphorylates cyclin E1 phosphodegrons in vitro. Immunoblot of p-Thr77, p-Thr395, and p-Thr399 of recombinant cyclin E1-Cdk2 incubated with recombinant PP2A core enzyme (A/C subunits). PP2A inhibitor okadaic acid (OA) and non-specific phosphatase λPPase are shown as controls. E, PP2A protects cyclin E1 from ubiquitylation in vivo. HEK293T cells were transfected with the indicated expression plasmids and siRNAs, Myc-cyclin E1 or mutant Myc-cyclin E1 (Thr77Ala/Thr395Ala) immunoprecipitated, and ubiquitylated forms detected by immunoblotting with anti-HA antibodies. Exposure below demonstrates IP efficiency of Myc-cyclin E1 and Myc-cyclin E1 (Thr77Ala/Thr395Ala). F, PP2A reduces E1 ubiquitylation by SCFFbxw7α in vitro. Recombinant cyclin E1-Cdk2 complexes were incubated with recombinant SCFFbxw7α. PP2A core enzyme was added to the indicated reaction. SCF core is SCFFbxw7α without substrate recognition component Fbxw7α.
Figure 2
Figure 2. PP2A-B55β regulates cyclin E1 phosphorylation
A, Cyclin E1 co-IPs with B-regulatory subunits B55β, B55δ, B56β, and B56δ in HEK293T cells. Asterisk indicates IgG heavy chain. B, Knockdown of B55β, B56β and B56δ expression in MDA-MB-231 cells decreases cyclin E1 protein levels. Ku86 is shown as a loading control. C, Endogenous cyclin E1 co-IPs with Flag-B55β expressed in HeLa cells. D, B55β is localized to the cytoplasm and nucleus in cells. HeLa cells were transfected with a plasmid that expresses Flag-B55β and cytoplasmic and nuclear fractions isolated. Exclusive nuclear protein PARP is shown as verification of fractionation efficiency. E, B55β knockdown-induced decrease of cyclin E1 in MDA-MB-231 cells is proteasome-dependent. B55β expression was knocked-down for 48 hrs and proteasome inhibitor Mg132 added 4 hrs prior to harvesting cells. F, B55β knockdown-induced decrease of cyclin E1 is phosphorylation-dependent. Cells were treated with Cdk2 and GSK3 inhibitors for 4 hrs prior to harvesting. G, PP2A-B55β dephosphorylates cyclin E1 in vitro. PP2A-B55β complexes were assembled in HEK293T cells and used in in vitro phosphatase reactions with recombinant cyclin E1-Cdk2 as substrate.
Figure 3
Figure 3. B55β regulates cyclin E1 stability and determines its level in cell lines and cell division cycles
A, B55β stabilizes cyclin E1. HEK293T cells were co-transfected with plasmids that express Myc-cyclin E1, Cdk2, Flag-Fbxw7α, and Flag-B55β or empty vector, protein synthesis halted by cyclohexamide (CHX) treatment, and Myc-cyclin E1 stability analyzed by immunoblotting. B, B55β is required for proper accumulation of cyclin E1 at the G1/S transition. HeLa cells were transfected with control or B55β siRNA, the cell cycle blocked at the G1/S boundary, and then released from block and cell cycle proteins analyzed by immunoblotting. qPCR data for expression of B55β, B56β and B56δ is shown below. C, B55β determines cyclin E1 levels in MDA-MB-231 breast cancer cells.
Figure 4
Figure 4. B55β expression correlates with cyclin E1 levels in breast cancers and its knockdown inhibits breast cancer cell proliferation
A & B, B55β expression correlates with cyclin E1 protein level in breast cancers. B55β and CCNE1 expression were quantified by qPCR and cyclin E1 protein by Western blot analysis. A, All tumors. B, Breast cancer specimens that express CCNE1 at ≥50% above the mean. C, B55β expression knockdown reduces cyclin E1 levels in a panel of breast cancer cell lines with functional SCFFbxw7. qPCR data showing B55β knockdown efficiency is shown below. Cell line SUM149PT contains defective FBXW7 alleles. D, B55β knockdown reduces cyclin E1-associated kinase activity in breast cancer cells. Cyclin E1-Cdk2 was immunoprecipitated from extracts and used in in vitro kinase assays with human recombinant pRb as substrate. E, B55β knockdown inhibits breast cancer cell proliferation. TNBC cell lines MDA-MB-231 and MDA-MB-157 were transduced with control or B55β shRNA-expressing lentiviruses and cells counted. Experiments were performed in triplicate.
Figure 5
Figure 5. B55β knockdown inhibits breast cancer formation in nude mice and model of PP2A-B55β regulation of cyclin E1 in cellular division and cancer
A, Growth curves of control and B55β shRNA-expressing breast cancers. MDA-MB-231-luc cells were transduced with control or B55β shRNA-expressing lentiviruses and then implanted into the mammary fat pads of nude mice. Twelve mice were analyzed for each cell line. B, Representative luminescent images of tumors 6 weeks post-implantation. Examples of excised tumors at 8 weeks post-implantation are shown on right. C, B55β knockdown inhibits metastasis progression. Luminescent images of mice 8 weeks post-implantation showing bronchial metastases of a control cell tumor but not B55β shRNA-expressing tumor. D, Model of B55β regulation of cyclin E1 in cellular division and cancer. Details explained in text.
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