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, 23 (4), 790-9

Tyrosine Phosphorylation Controls Runx2-mediated Subnuclear Targeting of YAP to Repress Transcription

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Tyrosine Phosphorylation Controls Runx2-mediated Subnuclear Targeting of YAP to Repress Transcription

Sayyed K Zaidi et al. EMBO J.

Abstract

Src/Yes tyrosine kinase signaling contributes to the regulation of bone homeostasis and inhibits osteoblast activity. Here we show that the endogenous Yes-associated protein (YAP), a mediator of Src/Yes signaling, interacts with the native Runx2 protein, an osteoblast-related transcription factor, and suppresses Runx2 transcriptional activity in a dose-dependent manner. Runx2, through its PY motif, recruits YAP to subnuclear domains in situ and to the osteocalcin (OC) gene promoter in vivo. Inhibition of Src/Yes kinase blocks tyrosine phosphorylation of YAP and dissociates endogenous Runx2-YAP complexes. Consequently, recruitment of the YAP co-repressor to subnuclear domains is abrogated and expression of the endogenous OC gene is induced. Our results suggest that Src/Yes signals are integrated through organization of Runx2-YAP transcriptional complexes at subnuclear sites to attenuate skeletal gene expression.

Figures

Figure 1
Figure 1
Endogenous YAP and Runx2 proteins interact in vivo and co-localize in situ in osseous cells. (A) Endogenous Runx2 was immunoprecipitated from ROS 17/2.8 cells with a rabbit polyclonal antibody (1:2000) raised against the Runx2 C-terminus (Zhang et al, 2000). A rabbit polyclonal antibody was used to detect endogenous YAP (top panel). Normal goat IgG was used as a control. The middle panel shows efficient immunoprecipitation of endogenous Runx2. The bottom panel shows the expression of endogenous YAP in ROS 17/2.8 cells (20% of total input). (B) In situ immunofluorescence of whole cell (WC) and nuclear matrix-intermediate filament (NM-IF) preparations was performed to assess the nucleo-cytoplasmic distribution and subnuclear localization of endogenous YAP in nonosseous (HeLa) and osseous (ROS 17/2.8) cells. YAP is predominantly nuclear in both HeLa and ROS 17/2.8 cells (top panels), but is only associated with the nuclear matrix ROS 17/2.8 cells (bottom panels). (C) Same as (B), using deconvoluted images. The merged image reveals that endogenous YAP resides in Runx2 containing subnuclear foci in ROS 17/2.8 cells (bar=10 μm).
Figure 2
Figure 2
The PY motif of Runx2 is required for interaction with YAP and its recruitment to subnuclear sites as well as target gene promoters in vivo. (A) ROS 17/2.8 cells were cotransfected with Xpress-YAP- and HA-tagged wild-type or the Y433A mutant of Runx2 (numbering according to the mouse MASNS/Runx2 isoform). After 24 h of transfection, proteins were precipitated using a monoclonal antibody against the HA tag (2 μg) and separated by SDS–PAGE. A mouse monoclonal antibody (1:5000) was used to detect XPR-YAP. The blots were stripped and were incubated with HA antibody (1:3000) to assess the expression of Runx2 (wild type or mutant) proteins or with XPR antibody to assess the expression of exogenous YAP. (B) HeLa cells, transfected with XPR-YAP (0.5 μg), were processed for WC and NM-IF preparations. YAP is present both in the cytoplasm and nucleus (right panel), but neither exogenous nor endogenous YAP is found in the nuclear matrix of HeLa cells (left panel). (C) Runx2 does not alter the subcellular localization of YAP in HeLa cells cotransfected with XPR-YAP and HA-Runx2 (wild type and Y433A) (WC, top panels). YAP is associated with the nuclear matrix when co-expressed with wild-type Runx2 (middle panel), but not if the interaction of Runx2 with YAP is disrupted (Y433A mutant, bottom panel) (bar=10 μm). (D) Chromatin immunoprecipitation assay was performed using ROS 17/2.8 cells transfected with indicated tagged expression constructs. Purified immunoprecipitated DNA was amplified with primers spanning the Runx-binding sites B and C in rat osteocalcin promoter as described. OC-derived DNA was observed in chromatin immunoprecipitations with the HA and Xpress antibodies from cells expressing both HA-tagged Runx2 and Xpress-tagged YAP after 21 cycles of PCR amplification. Control lanes (−) from untransfected cells show that antibodies against HA or XPR tags do not nonspecifically precipitate chromatin (lanes 2 and 3). A low level of OC-derived DNA was detected from cells expressing YAP alone after 25 cycles of PCR amplification due to endogenous Runx2 (data not shown). The unrelated myogenin promoter does not exhibit signals (bottom panel).
Figure 3
Figure 3
YAP suppresses Runx2-mediated activation of the rat osteocalcin promoter. (A) The rOC −208 CAT reporter was transfected in HeLa cells along with the indicated Runx2 constructs (250 ng) and increasing concentrations (0 (first bar of each group), 250, 500 ng and 1 μg) of full-length YAP. Expression of YAP alone does not affect basal promoter activity. Runx2 activates osteocalcin promoter activity, which is suppressed in a dose-dependent manner by increasing concentrations of YAP. The Runx2 Y433A mutant, that does not interact with YAP, shows a 3–4-fold higher activity compared to wild-type Runx2. (B) YAP suppresses the activity of Runx2 in osteoblastic ROS 17/2.8 cells. In contrast to HeLa cells, wild-type YAP slightly enhances basal promoter activity (first group), and the Runx2 Y433A mutant moderately activates rOC −208 due to endogenous Runx2 in ROS 17/2.8 cells. The graphs represent at least three independent experiments (n=6 each); error bars=standard error of mean. (C) The rOC −208/CAT reporter was transfected with 250 ng each of Runx2 and YAP. Runx2 activates osteocalcin promoter activity, which is suppressed by YAP in osteoblastic MC3T3 cells, premyoblast C2C12 cells and fibroblast NIH3T3 cells. The Runx2 Y433A mutant which does not interact with YAP shows higher activity compared to wild-type Runx2 in C2C12 and NIH 3T3 cells. This effect, however, is not apparent in MC3T3 cells, probably because of higher levels of endogenous Runx2 protein. (D) Runx2–YAP complex regulates the activity of multiple Runx target promoters. ROS 17/2.8 cells were transfected with the indicated reporter and expression constructs. Cells were harvested 30 h after the transfection and subjected to luciferase reporter assay. The luciferase activity is expressed as fold activation (6 × OSE-Luc and TGFβRI-Luc promoter) or fold suppression (in case of Runx2-Luc and p21-Luc promoters). The open squares in promoter constructs represent functional Runx sites.
Figure 4
Figure 4
A DN inhibitor of Src tyrosine kinase relieves YAP-mediated suppression of Runx2 activity. ROS 17/2.8 cells were transfected with 1 μg rOC −208/CAT reporter along with 250 ng of Runx2 and/or YAP expression constructs. Two different concentrations of Src DN construct (200 and 800 ng) were used (bars 2 and 3, respectively, of each group). Cells were harvested 24–30 h after transfection and were subjected to CAT reporter assay. The graph represents results obtained from three independent experiments (n=6 each).
Figure 5
Figure 5
Activated Src family kinases contribute to the YAP–Runx2 interaction. (A) ROS 17/2.8 cells, transiently transfected with XPR-YAP and antibodies, were used to immunoprecipitate endogenous Yes or Src tyrosine kinases. Association of YAP with these kinases was assessed by western blotting using HRP-conjugated anti-XPR antibody. (B) ROS 17/2.8 cells, expressing indicated constructs, were treated with 5 μM of PP2 for 1 h and processed for immunoprecipitation. The Runx2–YAP interaction is completely abrogated in the presence of PP2 (panel 1). Panel 2 shows the efficiency of Runx2 immunoprecipitation, while panel 3 shows comparable levels of YAP overexpression in all lanes. (C) As in (B), but antibodies were used to immunoprecipitate endogenous Runx2. The effect of Src DN on the YAP–Runx2 interaction was assessed by western blotting using the HRP-conjugated anti-XPR antibody (panel 1). A mouse monoclonal antibody against Runx2 was used to assess the efficiency of immunoprecipitation of the endogenous Runx2 protein (panel 2). Expression of Src DN and XPR-YAP was confirmed with Src and HRP-Xpress antibodies, respectively (panels 3 and 4). In each immunoprecipitation, appropriate normal IgG was used as a control.
Figure 6
Figure 6
Inhibition of endogenous Src signaling or YAP activity induces endogenous osteocalcin gene transcription. (A) Total cellular RNA from ROS 17/2.8 cells was analyzed by northern blotting and treated with indicated concentrations of PP2 for 1 h. Endogenous osteocalcin gene transcription is induced in a dose-dependent manner when the activity of Src family members is inhibited by PP2 (upper panel). 18S RNA was used as a loading control (bottom panel). (B) Activity of Src tyrosine kinase or YAP was selectively inhibited by overexpressing DN inhibitors (Src DN for Src tyrosine kinase and YAP DN for YAP). Total cellular RNA was subjected to northern blot analysis from ROS 17/2.8 cells overexpressing the indicated plasmids. As shown in the top panel, the DN inhibitor of Src (lane 2) or YAP (lane 4) induces endogenous osteocalcin gene transcription. (C) Densitometric analysis reveals the extent to which endogenous osteocalcin is induced under each experimental condition analyzed. (The bar graph represents the ratio of densitometric units of osteocalcin and 18S transcripts.)
Figure 7
Figure 7
Tyrosine phosphorylation of YAP regulates its interaction with Runx2 and subsequent subnuclear trafficking. (A) HeLa cells, co-expressing YAP and Runx2, were transfected with Src DN or treated with PP2 (5 μM) for 1 h and WC or NM-IF preparations and in situ immunofluorescence microscopy. Inhibition of either Src tyrosine kinase (+Src DN) or its family members (+PP2) does not alter subcellular localization of YAP. Subnuclear trafficking of YAP is severely compromised when the kinase activity of Src alone or its family members is inhibited. (B) To directly assess the effects of Src DN or PP2 on tyrosine phosphorylation of endogenous YAP and its interaction with native Runx2, endogenous YAP was immunoprecipitated from ROS 17/2.8 cells. The immunoprecipitates were resolved by SDS–PAGE and subjected to western blot analysis. The tyrosine phosphorylation of YAP was determined by a mouse monoclonal phospho-specific antibody (Py; 1:2000) raised against phospho-tyrosine (top panel). The blot was stripped and re-probed with a mouse monoclonal antibody against Runx2 to assess the presence of endogenous Runx2 in the immunoprecipitates (middle panel). The efficiency of YAP immunoprecipitation was assessed by a YAP rabbit polyclonal antibody (bottom panel).

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