Hippo signaling is intrinsically regulated during cell cycle progression by APC/CCdh1

Abstract

The Hippo-YAP/TAZ signaling pathway plays a pivotal role in growth control during development and regeneration and its dysregulation is widely implicated in various cancers. To further understand the cellular and molecular mechanisms underlying Hippo signaling regulation, we have found that activities of core Hippo signaling components, large tumor suppressor (LATS) kinases and YAP/TAZ transcription factors, oscillate during mitotic cell cycle. We further identified that the anaphase-promoting complex/cyclosome (APC/C)^Cdh1 E3 ubiquitin ligase complex, which plays a key role governing eukaryotic cell cycle progression, intrinsically regulates Hippo signaling activities. CDH1 recognizes LATS kinases to promote their degradation and, hence, YAP/TAZ regulation by LATS phosphorylation is under cell cycle control. As a result, YAP/TAZ activities peak in G1 phase. Furthermore, we show in Drosophila eye and wing development that Cdh1 is required in vivo to regulate the LATS homolog Warts with a conserved mechanism. Cdh1 reduction increased Warts levels, which resulted in reduction of the eye and wing sizes in a Yorkie dependent manner. Therefore, LATS degradation by APC/C^Cdh1 represents a previously unappreciated and evolutionarily conserved layer of Hippo signaling regulation.

Keywords: APC/CCdh1; Hippo signaling; LATS1/2; YAP/TAZ; mitotic cell cycle.

Fig. 1.

APC/C^Cdh1 is required for YAP/TAZ activities. (A) HeLa cells were synchronized by double thymidine (2 mM) treatment and then released. Whole-cell lysates were subjected to Western blot analysis at indicated times after release. Western blot analysis was performed using the indicated antibodies. Asy, asynchronous. (B) HeLa cells were transduced with the Premo FUCCI cell cycle sensor and incubated overnight for expression of Geminin-GFP (S/G2/M phase) and Cdt1-RFP (G1 phase). FUCCI-expressing HeLa cells were fixed with 4% fresh PFA and stained with anti-YAP/TAZ antibodies (purple). Nuclei were stained with DAPI. (Original magnification, 600×.) YAP/TAZ nuclear localization were quantified as percentage of cells with YAP/TAZ nuclear localization in the total cells of a specific cell cycle phase. A schematic diagram of FUCCI is shown (Lower Left). (C) Asynchronized HeLa cells were transfected with control siGFP, two siCDH1s or siCDC20s. Levels of TAZ, p-YAP (S127), YAP, CDH1, CDC20, and GAPDH (loading control) were determined by Western blot analysis. (D) YAP/TAZ reporter activities (3xSd-Luc) in asynchronized cells by transfection with the indicated plasmids in the presence of siGFP or siCDH1. n = 3 independent experiments. Error bars represent ±SD. (E) Cdh1^+/+ WT or Cdh1^−/− KO MEFs lysates were subjected to Western blotting analysis with indicated antibodies. (F) Quantitative real-time PCR analysis for the expression of YAP/TAZ-target genes (CTGF and ANKRD1) after knockdown of CDH1 or YAP by siRNA in asynchronized HEK293T cells. The quantities of indicated mRNA were normalized by GAPDH. n = 3 independent experiments. (G) Western blot analysis of CTGF, p-YAP(S127), YAP, and CDH1 in nonsynchronized HeLa cells transfected with the indicated siRNAs. (H) HeLa cells were transfected with control siGFP and siAPC10. Cell lysates were subjected to Western blot analysis with indicated antibodies. In all quantified Western blotting results, representative blots are shown. Data are means ± SD of three biological replicates. **P < 0.01 (two-tailed Student’s t test). ns, not significant.

Fig. 2.

APC/C^Cdh1 promotes LATS1/2 kinases degradation. (A) HeLa cells were first synchronized by DTB, and then released for the tindicated time. Western blot analysis was performed using the indicated antibodies. (B) HeLa cells were synchronized by thymidine-nocodazole block, and cell lysates were collected at the indicated time point after release. Western blot analysis was performed using the indicated antibodies. (C) Cdh1^+/+ WT or Cdh1^−/− KO MEFs lysates were subjected to Western blot analysis with indicated antibodies. (D) Loss of CDH1 significantly prolongs half-life of LATS1/2. Western blotting analysis of LATS1 and LATS2 proteins in asynchronized HeLa cells treated with cyclohexamide (CHX) for the indicated time (Upper). The line graphs show quantified LATS1 and LATS2 levels at indicated time (Lower). n = 4 independent experiments. Error bars represent ±SD, **P < 0.01 (two-tailed Student’s t test). (E) Myc-CDH1 was transfected in asynchronized HeLa cells, which were incubated with 20 μM MG132 for 8 h. Cell lysates were subjected to Western blot analysis with the indicated antibodies; **P < 0.01 (two-tailed Student’s t test). (F and G) HA-Ubiquitin was transfected with the indicated plasmids in the asynchronized HEK293T cells, 8 h after 20 μM MG132 treatment, LATS proteins were immunoprecipitated from the cell lysates and analyzed by Western blotting. (H) Reconstituted LATS1 expression in the LATS1/2^−/− HeLa cells rescued Hippo signaling defects. Cell lysates were subjected to Western blot analysis with indicated antibodies. (I) siGFP or siCDH1 was transfected into control and LATS1/2^−/− HeLa cells for 72 h. Cell lysates were subjected to Western blot analysis with indicated antibodies.

Fig. 3.

LATS interacts with WD40 repeats of CDH1. (A) Flag-LATS1 was transfected with or without Myc-CDH1 into the HEK293T cells. Cell lysates were subjected to co-IP and coprecipitated LATS1 or CDH1 was detected by Western blot analysis. (B) Endogenous interaction between LATS1 and CDH1 detected by co-IP followed by Western blot analysis. (C) Cells were stained with rabbit anti-CDH1 antibody and/or goat anti-LATS1 antibody, and in situ interaction between LATS1 and CDH1 (red dots) was detected with secondary proximity probes as described in Materials and Methods. (Scale bars, 10 μm.) (D) WD40 repeats of CDH1 is required to interact with LATS1. Flag-LATS1 and the indicated Myc-Cdh1 constructs (Right) were expressed in HEK293 T. Twenty-four hours posttransfection, the cells were pretreated with 20 μM MG132 for 8 h before collecting for co-IP and Western blotting assays (Left). WCL, whole-cell lysates.

Fig. 4.

APC/C^Cdh1 requires both evolutionarily conserved A-box and D-box for LATS degradation. (A) Sequence alignment of four putative D-box motifs evolutionary conserved in LATS1 and LATS2 kinases. (B) Myc-CDH1 were transfected with either wild-type Flag- LATS1 or indicated mutant constructs, and then cell lysates were subjected to immunoprecipitation assay. (C) LATS1 mutated in all of four D-box motifs is still degraded by ectopic CDH1 expression. Flag wild-type or mutant LATS1 was transfected into HEK293T cells with or without Myc-CDH1. Cell lysates were subjected to Western blot analysis with the indicated antibodies. Representative blots are shown; error bars represent ±SD of three biological replicates. **P < 0.01 (two-tailed Student’s t test). (D) Western blot analysis of cell lysates and immunoprecipitation derived from HEK293T cells transfected with wild-type LATS1 or truncation mutants with Myc-CDH1 construct. Twenty-four hours posttransfection, the cells were pretreated with 20 μM MG132 for 8 h before collecting (Left). Mapping studies from serial N- or C-terminal deletion reveals that LATS kinase contains two different interacting regions in CDH1. A schematic diagram showed LATS1 deletion mutants used in immunoprecipitation analysis (Right). (E) Schematic illustration of LATS wild-type and D-box, A-box LATS mutants. (F) Myc-CDH1 were transfected with either wild-type Flag-LATS1 or indicated mutant construct, and then cell lysates were subjected to immunoprecipitation assay. (G and H) Both the D-box and A-box are required for the degradation of LATS kinase by APC/C^Cdh1. Myc-Cdh1 or siCDH1 were transfected with wild-type Flag-LATS or indicated mutant constructs, and then subjected to Western blot analysis with indicated antibodies. Representative blots are shown; data are mean D-box and A-box significantly prolonged half-life of LATS1. (I) Western blotting analysis of LATS1 proteins in HeLa cells treated with cyclohexamide (CHX) for indicated time (Upper). The line graphs show quantified LATS1 levels at indicated time (Lower). n = 3 independent experiments. Error bars represent ±SD. *P < 0.05; **P < 0.01 (two-tailed Student’s t test). (J) Mutations in LATS1 disrupted its kinase activity. LATS1 mutants with D-box and/or A-box mutations were transfected in to the LATS1/2 null mutant HeLa cells. LATS1 and YAP phosphorylation were analyzed by Western blotting.

Fig. 5.

YAP regulates E2F1 transcription. (A) Five putative TEAD binding elements (TBE) are located ∼3 kb upstream of the TSS of E2F1 (Upper). TEAD family transcription factors associate with the indicated motif (underlined, Lower). YAP recognizes and binds consensus sequence (GGAATG) through TEAD. (B) ChIP-qPCR assay was performed at the indicated TEAD biding sites in the promoter of E2F1, and HBB or CTGF was used as a negative or positive control for YAP binding, respectively. n = 3 independent experiments. Error bars represent ±SEM, *P < 0.05, **P < 0.01 (two-tailed Student’s t test). (C) qPCR analysis of E2F1 or YAP/TAZ-target genes (CTGF and CYR61) in cells-depleted YAP/TAZ. n = 3 independent experiments. Error bars represent ±SEM **P < 0.01 (two-tailed Student’s t test). (D) Western blot analysis of endogenous proteins of HEK293T or HeLa cells in the presence of the indicated siRNAs. (E) Heat-map of RNA-seq data showing expression of E2F family genes in indicated mice. (n = 3 mice per genotype). (F) qPCR (Left) or Western blotting analysis (Right) of E2F1 expression in the livers of indicated mice. n = 3 independent experiments. Error bars represent ±SEM, **P < 0.01 (two-tailed Student’s t test). (G) Heterozygous removal of Yap in the livers of Mst1/2 DKOmice restores E2F1 expression. n = 3 independent experiments. Error bars represent ±SEM, **P < 0.01 (two-tailed Student’s t test).

Fig. 6.

Cdh1 regulates organ size via the Hippo pathway in Drosophila. (A) Eye discs expressing UAS-Myc-Wts and UAS-GFP (internal control) with or without UAS-Cdh1-RNAi under the control of GMR-Gal4 were immunostained with anti-GFP and anti-Myc antibodies. (B) Extracts from eye discs expressing UAS-Myc-Wts with or without UAS-Cdh1-RNAi under the control of GMR-Gal4 were subjected to Western blot analysis with anti-Myc antibody to detect Myc-Wts. UAS-GFP was coexpressed as an internal control. (C) Eye discs expressing Diap1-GFP with or without UAS-Cdh1-RNAi under the control of GMR-Gal4 were immunostained with anti-GFP and anti-Tubulin antibodies. (D) Extracts from eye discs Diap1-GFP with or without UAS-Cdh1-RNAi under the control of GMR-Gal4 were subjected to Western blot analysis with anti-GFP and anti-Tubulin antibodies. (E) Cdh1 inhibits Wts in organ size control. Side views of a control adult fly eye (GMR > Gal4) or eyes expressing UAS-Cdh1-RNAi, UAS-Wts, or both UAS-Cdh1-RNAi and UAS-Myc-Wts under the control of GMR-Gal4. (F–H) Side views of a control adult fly eye (GMR > Gal4) or eyes expressing both UAS-Cdh1-RNAi and UAS-Wts-RNAi (F) UAS-Yki (G) or UAS-Yki3SA (H) under the control of GMR-Gal4. (I) Wing discs expressing GFP-Wts with or without UAS-Cdh-RNAi under the control of hh-Gal4 were immunostained with anti-GFP and anti-Ci antibodies. Ci marks the anterior compartment. (J) Adult wing expressing UAS-Cdh-RNAi under the control of hh-Gal4 exhibited reduced posterior wing size. Eye or wing surface areas were measured by ImageJ. Error bars represent ±SD, **P < 0.01 n = 4 for each genotype. (Magnification: A, C, and I, 30×; E–H, 10×; J, 5×.) A, anterior wing compartment; P, posterior wing compartment.