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. 2012 Oct 1;26(19):2138-43.
doi: 10.1101/gad.197582.112. Epub 2012 Sep 12.

Regulation of the Hippo-YAP pathway by protease-activated receptors (PARs)

Jung-Soon Mo  1 , Fa-Xing Yu, Rui Gong, Joan Heller Brown, Kun-Liang Guan
Affiliations

Regulation of the Hippo-YAP pathway by protease-activated receptors (PARs)

Jung-Soon Mo et al. Genes Dev. .

Abstract

The Hippo signaling pathway plays a crucial role in tissue growth and tumorigenesis. Core components of the Hippo pathway include the MST1/2 and Lats1/2 kinases. Acting downstream from the Hippo pathway are the YAP/TAZ transcription coactivators, which are inhibited through phosphorylation by Lats. However, upstream signals that regulate the Hippo pathway have not been well delineated. Here we report that stimulation of protease-activated receptors (PARs) activates YAP/TAZ by decreasing phosphorylation and increasing nuclear localization. PAR1 acts through G(12/13) and Rho GTPase to inhibit the Lats1/2 kinase. Our observations establish thrombin as a physiological signal for the Hippo pathway and implicate Hippo-YAP as a key downstream signaling branch of PAR activation.

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Figures

Figure 1.
Figure 1.
Agonists of PARs induce dephosphorylation and activation of YAP and TAZ. (A) Thrombin induces YAP dephosphorylation. HEK293A cells were serum-starved for 24 h and then stimulated with thrombin at various doses (units per milliliter) for 30 min. YAP phosphorylation was determined by phos-tag. Thrombin also induced TAZ mobility shift. The numbers on the left of each panel denote molecular weight markers. (B,D) HEK293A cells were serum-starved for 12 h and then stimulated with TRAP6 or SLIGRL for the indicated durations. Cells were lysed and subjected to immunoblotting with the indicated antibodies. (C,E). HEK293A cells were stimulated with different concentrations of TRAP6 or SLIGRL for 1 h. (F) YAP subcellular localization was determined by immunofluorescence staining for endogenous YAP (red); DAPI (blue) was used for cell nuclei. Cells were stimulated with TRAP6 and SLIGRL for 1 h. The images shown represent typical staining pattern of the majority cells. Quantifications are shown in the bottom panel. (G) TRAP6 and SLIGRL stimulate YAP interaction with TEAD1. Cell lysates were subjected to immunoprecipitation with control IgG or YAP antibody. The coimmunoprecipitated TEAD1 and 14-3-3 were detected by immunoblotting.
Figure 2.
Figure 2.
TRAP6 activates YAP/TAZ via G12/13. (A) HEK293A cells were transfected with the indicated siRNAs. YAP phosphorylation was determined by both phos-tag, which detects phosphorylation by enhancing mobility shift, and phospho-YAP (S127) antibody. TAZ phosphorylation can be visualized by mobility shift. The knockdown efficiency of Gq/11 and G12/13 is indicated by immunoblotting. (B) MCF10A cells were transfected with siG12/13 as indicated. Cells were treated with 2 μM TRAP6 for 1 h before fixation. YAP subcellular localization was determined by immunofluorescence staining for endogenous YAP (red) and F-actin (green); DAPI (blue) was used for cell nuclei.
Figure 3.
Figure 3.
TRAP6 activates YAP/TAZ through Rho and cytoskeletons. (A) HEK293A cells were transiently cotransfected with the indicated plasmids and were treated with 2 μM TRAP6 for 1 h. The cells were then lysed and subjected to immunoblotting analysis with the indicated antibodies. (B) HEK293A cells were pretreated with 2 μg/mL C3 for 4 h and then stimulated with TRAP6 for the indicated durations. The presence of FBS is indicated. (C) MCF10A cells were pretreated with 2 μg/mL C3 for 4 h and then incubated with TRAP6 for 1 h. After fixation, YAP subcellular localization was determined by immunofluorescence staining for endogenous YAP (red), F-actin (green); DAPI (blue) was used for cell nuclei. (D) MCF10A cells were pretreated with Latrunculin B for 10 min and then stimulated with TRAP6 for 1 h. Immunofluorescence staining is similar to that shown in C.
Figure 4.
Figure 4.
TRAP6 inhibits Lats kinase activity. (A) HEK293A cells were treated with 2 μM TRAP6 for 1 h. The activity of immunoprecipitated MST1 was determined by in vitro kinase assay using GST-Mob as a substrate. P32 incoporation into GST-Mob was detected by autoradiograph. (B) HEK293A cells were cotransfected with the indicated plasmids. After transfection, cells were treated with 2 μM TRAP6 for 1 h. YAP phosphorylation was determined. (C) HEK293A cells were pretreated with 2 μg/mL C3 for 4 h and then incubated with TRAP6 for the indicated times. The presence of FBS is indicated. Lats1 immunoprecipitated from the cell lysates was subjected to in vitro kinase assays using GST-YAP as a substrate. YAP phosphorylation was detected by phospho-YAP (S127) antibody. (D) Stimulation conditions were similar to those in C. Lats1 was immunoprecipitated with anti-Lats1 antibody from the cell lysates and then subjected to immunoblotting as indicated. (E) Flag-YAP was cotransfected with wild-type or a kinase-dead (KR) mutant of HA-Lats2. After transfection, cells were treated with 2 μM TRAP6 for 1 h. (B,E) Phosphorylation of Flag-YAP was determined by phos-tag gel.
Figure 5.
Figure 5.
YAP/TAZ mediate physiological functions of PARs in gene induction, cell migration, and invasion. (A) YAP/TAZ knockdown by siRNAs in MCF10A cells (left) and MDA-MB-231 cells (right). Cells were transfected with the indicated siRNAs. The protein levels were determined by immunoblotting. (B,C) MDA-MB-231 cells were transfected with the indicated siRNAs and serum-starved for 12 h. (B) After treating with 2 μM TRAP6 for 4 h, mRNA levels of CTGF, Cyr61, CXCL1, END1, FSTL, INHBA, and PTGS were measured by quantitative PCR. (C) Protein levels of YAP/TAZ, CTGF, and Cyr61 were determined by immunoblotting. (D) MCF10A cells were transiently transfected with the indicated siRNAs. Cell migration was determined by transwell cell migration assay. Cells were stained with crystal violet (top panel) and quantified (bottom panel). (E) MDA-MB-231 cells were transiently transfected with the indicated siRNAs. Cell invasion was determined by Matrigel invasion assay.
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References

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