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. 2007 Nov 1;21(21):2747-61.
doi: 10.1101/gad.1602907.

Inactivation of YAP Oncoprotein by the Hippo Pathway Is Involved in Cell Contact Inhibition and Tissue Growth Control

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Free PMC article

Inactivation of YAP Oncoprotein by the Hippo Pathway Is Involved in Cell Contact Inhibition and Tissue Growth Control

Bin Zhao et al. Genes Dev. .
Free PMC article

Abstract

The Hippo pathway plays a key role in organ size control by regulating cell proliferation and apoptosis in Drosophila. Although recent genetic studies have shown that the Hippo pathway is regulated by the NF2 and Fat tumor suppressors, the physiological regulations of this pathway are unknown. Here we show that in mammalian cells, the transcription coactivator YAP (Yes-associated protein), is inhibited by cell density via the Hippo pathway. Phosphorylation by the Lats tumor suppressor kinase leads to cytoplasmic translocation and inactivation of the YAP oncoprotein. Furthermore, attenuation of this phosphorylation of YAP or Yorkie (Yki), the Drosophila homolog of YAP, potentiates their growth-promoting function in vivo. Moreover, YAP overexpression regulates gene expression in a manner opposite to cell density, and is able to overcome cell contact inhibition. Inhibition of YAP function restores contact inhibition in a human cancer cell line bearing deletion of Salvador (Sav), a Hippo pathway component. Interestingly, we observed that YAP protein is elevated and nuclear localized in some human liver and prostate cancers. Our observations demonstrate that YAP plays a key role in the Hippo pathway to control cell proliferation in response to cell contact.

Figures

Figure 1.
Figure 1.
YAP localization and phosphorylation are regulated by cell density. (A) YAP localization is affected by cell density. NIH-3T3 and MCF10A cells were cultured sparsely or to confluence. YAP was stained with anti-YAP antibody. (B) MCF10A cells at the edge of a large colony have high nuclear YAP. YAP was stained with anti-YAP antibody. (C) High cell density induces YAP phosphorylation. NIH-3T3 cell lysates from cells at different densities were probed with anti-YAP antibody. λ phosphatase treatment is indicated.
Figure 2.
Figure 2.
The Hippo pathway regulates YAP phosphorylation, activity, and localization. (A) Coexpression of Mst2 and Lats2 decreases YAP2 mobility. Flag-YAP2 was cotransfected with indicated plasmids into HEK293 cells. Western blot was performed as indicated. (B) In vitro phosphorylation of YAP2 by Lats2. HA-Lats2 was immunoprecipitated from transfected HEK293 cells. In vitro kinase assay was performed using purified GST-YAP2 as a substrate in the presence of [32P]ATP. GST-Sin1 was used as a negative control. (KR) Kinase-inactive mutant. (C) YAP2 activity is inhibited by Mst2 and Lats2. Indicated plasmids were cotransfected with a 5× UAS-luciferase reporter and a CMV-β-gal construct into 293T cells. Luciferase activity was measured and normalized to β-galactosidase activity. (D) YAP2 activity is inhibited by Merlin and Expanded. Experiments are similar to those in C. The Ex used is human FRMD6. (E) Activation of the Hippo pathway causes YAP cytoplasmic localization. HeLa cells were transfected with indicated plasmids. Endogenous YAP2 was stained to visualize the localization. (F) Cell density-induced YAP translocation is Merlin dependent. RT4-D6-P2T Schwannoma cell lines with empty vector, inducible wild-type Merlin, or a Merlin-L64P mutant were cultured to confluence. Merlin expression was induced by doxycycline for 2 d. (Left panel) Expression of Merlin was determined by Western blot. Endogenous YAP was stained and YAP localization was quantified.
Figure 3.
Figure 3.
Lats inhibits YAP by phosphorylating HXRXXS motifs. (A) YAP2 contains five HXRXXS motifs. The yeast Dbf2 optimal target sequence was aligned with the five HXRXXS motifs of human YAP2. (B) Ser127 is the major phosphorylation site in YAP2. Wild-type or mutant Flag-YAP2 was cotransfected with HA-Mst2 and HA-Lats2 as indicated. YAP2 mobility shift was determined by anti-Flag Western blot. (C) Lats2 directly phosphorylates YAP2 on HXRXXS motifs. In vitro phosphorylation of YAP2 mutants with immunoprecipitated HA-Lats2 was performed. Phosphorylation of GST-YAP2 was detected by either 32P incorporation or anti-phospho-YAP (S127) Western blot. (Bottom panel) GST-YAP2 input was shown by Coomassie Blue staining. (D) YAP2 phosphorylation-defective mutants S127A and 5SA are resistant to inhibition by Mst2 and Lats2. The reporter assay is similar to those in Figure 2C. The fold activity inhibition of each mutant by Mst2/Lats2 is indicated at the top of this panel. (E) Coexpression of Mst2 and Lats2 increases YAP2 S127 phosphorylation. Flag-YAP2 was cotransfected with HA-Lats2 and Flag-Mst2 into HEK293 cells as indicated. Flag-YAP2 was immunoprecipitated and phosphorylation of S127 was detected by pYAP (S127) antibody. (F) Knockdown of Lats decreases endogenous YAP S127 phosphorylation. HeLa cells were transfected twice with small interfering RNA for Lats1 and Lats2 as indicated. Phosphorylation and protein levels of endogenous YAP were determined by Western blot. Knockdown of Lats was verified by the anti-Lats antibody, which recognizes both Lats1 and Lats2. (G) YAP S127 phosphorylation increases with cell density. NIH-3T3 and MEF cells were harvested at different densities, and YAP phosphorylation was assayed. (H) Lats2 kinase activity increases with cell density. NIH-3T3 cells were harvested at different densities. Endogenous Lats2 was immunoprecipitated and used in an in vitro kinase assay. Phosphorylation of GST-YAP2 was detected by anti-phospho-YAP (S127) Western blot. Rheb IP was included as a negative control.
Figure 4.
Figure 4.
Phosphorylation promotes YAP cytoplasmic localization and inhibits transcription factor binding. (A) Ser127 is required for YAP2 cytoplasmic localization induced by Lats2. Flag-YAP2 wild type or mutants were transfected alone or together with HA-Lats2 into HeLa cells. Cells were stained with Flag and HA antibodies. (B) Phosphorylation is required for cell density-induced YAP2 cytoplasmic translocation. MCF10A cells stably expressing Myc-YAP2 or Myc-YAP2-5SA were cultured at low or high density. Myc-YAP2 was stained with anti-Myc antibody. (C) Lats and Mst decrease YAP2/TEAD4 interaction in vivo in a S127-dependent manner. Indicated plasmids were transfected into HEK293 cells. Flag-YAP2 was immunoprecipitated, and coprecipitated Myc-TEAD4 was detected by Western blot. (D) YAP2 dephosphorylation does not affect its interaction with TEAD4 in vitro. Flag-YAP2 (cotransfected with Mst2 and lats2) immunoprecipitated from HEK293 cells were treated with λ phosphatase as indicated and then used in an in vitro TEAD pull-down assay. Myc-TEAD4 was prepared from transfected HEK293 cells. The final products were analyzed by Western blot.
Figure 5.
Figure 5.
S127 phosphorylation regulates YAP and 14–3–3 interaction. (A) Dephosphorylation abolishes the interaction between YAP2 and 14–3–3 in vitro. Flag-YAP2 immunoprecipitated from transfected HEK293 cells was treated with λ phosphatase as indicated and then used to pull down endogenous 14–3–3 from HEK293 cell lysate. The products were analyzed by Western blot. (B) Lats2 but not Akt enhances YAP2 and 14–3–3 interaction. Flag-YAP2 plasmids were cotransfected with Myc-14–3–3 and other indicated plasmids into HEK293 cells. Myc-14–3–3 was immunoprecipitated and coimmunoprecipitated Flag-YAP2 was detected. (C) Mutation of H122 but not P129 decreases YAP2 S127 phosphorylation by Lats2. In vitro phosphorylation of YAP2 mutants by immunoprecipitated HA-Lats2 was performed. Phosphorylation of GST-YAP2 was detected by 32P incorporation. (Bottom panel) GST-YAP2 input was shown by Coomassie Blue staining. 4SA (S127) denotes that four of the five Lats phosphorylation sites were mutated to alanine except Ser127. (D) Mutation of His122 in YAP2 impairs Ser127 phosphorylation and 14–3–3 binding. Indicated plasmids were transfected into HEK293 cells. Flag-YAP2 was immunoprecipitated, and the immunoprecipitates were probed as indicated. (E) Pro129 of YAP2 is required for 14–3–3 binding. Experiments were similar to those in D.
Figure 6.
Figure 6.
S127 phosphorylation regulates YAP and Yki biological function in vivo. (A) Alignment of the Homo sapiens YAP2 and the Drosophila melanogaster Yki wild-type and Dbo mutant proteins around the S127 (YAP2) residue. Mutated residues are shown in green. (B) Dominant active yorkie mutations around the phosphorylation site S168 mimic hippo loss-of-function phenotypes. (Panel a) Wild-type wing. (Panel b) Hpo overexpression driven by nubbin-Gal4. (Panel c) nubbin-Gal4 UAS-Hpo, ykiDbo/+. (Panel d) ykiDbo/+. (Panel e) A fly with an eye mosaic for a mutation in the white gene. Clones were induced using the eye-specific FLP driver (eyFLP), and a cell-lethal mutation on the homologous (w+) chromosome was used to eliminate twin spot clones, which increased the area of the w cell clones. (Panel f) A fly with a mosaic eye induced by the same method as in e. However, this fly carries a ykiDbo mutation on the w chromosome. (Panels g,h) Eye imaginal discs from third instar larvae containing wt and ykiDbo mutant clones that were marked by the absence of GFP (gray). (Panels i–l) ykiDbo mutant clones marked by the absence of GFP. (Panel i) Eye imaginal disc containing ykiDbo mutant clones and labeled for BrdU incorporation (red in panel i, and grayscale in panel i′). Asterisks indicate the morphogenetic furrow, arrows indicate the second mitotic wave, and arrowheads point to ectopic cell proliferation in ykiDbo mutant clones posterior to the second mitotic wave. (Panle j) Mid-pupal retina stained with Discs large (Dlg) antibodies to visualize cell outlines (red in panel j, and grayscale in panel j′). ykiDbo mutant clones showed extra interommatidial cells (arrowhead). (Panel k) ykiDbo mutant clones showed up-regulated expression of Cyclin E (arrowheads) (red in panel k, and grayscale in panel k′), most conspicuously behind the second mitotic wave (arrows). (Panel l) ykiDbo mutant clones showed increased Ex (red in panel l, and grayscale panel l′) levels in the eye imaginal disc. (C) The phosphorylation-defective YAP2-S127A is more active in promoting tissue growth in Drosophila. (Panels a–d) Third instar larval eye discs were analyzed for the transcriptional activities of diap1-lacZ reporter genes. Anterior is to the left. Red arrows indicate the morphogenetic furrow. (Panels e–h) Mid-pupal eye discs were stained with Discs large (Dlg) antibody to outline cells. SEM (scanning electron microscopy) images of fly adult eyes are presented in panels i–l. Genotypes of the fly tissues are GMR-Gal4/+; diap1-lacZ/+ (panel a), GMR-Gal4/UAS-Flag-YAP2; diap1-lacZ/+ (panel b), GMR-Gal4/UAS-Flag-YAP2S127A; diap1-lacZ/+ (panel c), GMR-Gal4/UAS-yki-V5; diap1-lacZ/+ (panel d), wild-type (Canton S) (panels e,i), GMR-Gal4/UAS-Flag-YAP2 (panels f,j), GMR-Gal4/UAS-Flag-YAP2S127A, (panels g,k), and GMR-Gal4/UAS-yki-V5 (panels h,l).
Figure 7.
Figure 7.
YAP regulates density-dependent gene expression and alteration of YAP activity affects cell contact inhibition. (A) High cell density and YAP affect gene expression in opposite manners. YAP-regulated genes were revealed by microarray analyses of control and YAP-overexpressing NIH-3T3 cells. Density-regulated genes were also identified by microarray analysis of sparse and confluent cells. Genes that show more than twofold differences were used in the comparison. P values were calculated by Fisher exact test. (B) Quantitative RT–PCR confirmation of YAP and cell density-regulated genes. Total RNA isolated from NIH-3T3 cells stably expressing YAP2 or vector control (top chart) and from low- or high-density cultures (bottom chart) were analyzed by quantitative RT–PCR and normalized to HPRT (hypoxanthine phosphoribosyltransferase 1). (C) Correlation of cell proliferation and nuclear YAP localization. Confluent MCF10A culture was scratched. Six hours later, cells were fixed and stained for YAP and Ki67. (D) YAP promotes cell growth and elevates saturation density. Growth curves of NIH-3T3 cells stably expressing YAP2 or vector were determined. Confluent density is indicated. (E) YAP promotes proliferation of confluent cells. Vector and YAP-overexpressing NIH-3T3 cells were cultured to confluence. Cells at a similar density were pulse-labeled with BrdU followed by staining with anti-BrdU and 7-AAD (a fluorescent dye for total DNA) for flow cytometric analysis. (F) Dominant-negative YAP restores contact inhibition in ACHN cancer cells. ACHN cells stably expressing vector or Myc-YAP2-5SA-ΔC were cultured to low density or confluence. Cell morphologies are shown in the left panels. The loss of contact inhibition in ACHN cells is evidence that cells pile on top of each other. Myc-YAP2-5SA-ΔC expression level is shown by Western blot in the right panels.
Figure 8.
Figure 8.
(A) Elevated YAP protein and nuclear localization in human cancers. Tissue microarrays of liver and prostate cancer were stained with anti-YAP antibody (brown). Cell nuclei were counterstained with Hematoxylin (blue). (B) Nuclear YAP protein is significantly elevated in human cancers. Samples were scored based on median nuclear staining intensity, ranging from 0 to 6 (0 for negative, and 6 for very strong staining). Strong staining was considered a score of 2 or higher for liver and 3 or higher for prostate. P values (Fisher exact test) indicate the differences in the proportions of strong YAP staining between cancer and normal specimens. (C) A model for YAP regulation by cell contact via the Hippo pathway.

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