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, 284 (21), 14347-58

TEADs Mediate Nuclear Retention of TAZ to Promote Oncogenic Transformation

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TEADs Mediate Nuclear Retention of TAZ to Promote Oncogenic Transformation

Siew Wee Chan et al. J Biol Chem.

Abstract

The transcriptional coactivators YAP and TAZ are downstream targets inhibited by the Hippo tumor suppressor pathway. The expression level of TAZ is recently shown to be elevated in invasive breast cancer cells and some primary breast cancers. TAZ is important for breast cancer cell migration, invasion, and tumorigenesis, but the underlying mechanism is not defined. In this study, we show that TAZ interacts with TEAD transcriptional factors. Knockdown of TEADs suppresses TAZ-mediated oncogenic transformation of MCF10A cells. Uncoupling TAZ from Hippo regulation by S89A mutation enhances its transforming ability. Several residues located in the N-terminal region of TAZ are identified to be important for interaction with TEADs, and these same residues are equally important for TAZ to transform MCF10A cells. Mechanistically, TAZ mutants defective in interaction with TEADs fail to accumulate in the nucleus. Live cell imaging of enhanced green fluorescent protein-TAZ and its mutant defective in TEAD interaction suggests that TEAD interaction mediates nuclear retention. These results reveal a novel mechanism for TEADs to regulate nuclear retention and thus the transforming ability of TAZ.

Figures

FIGURE 1.
FIGURE 1.
TAZ interacts with TEADs. A, retention of TAZ and YAP by immobilized C-terminal regions of TEAD1–4. Cell extracts derived from Hs578T cells were incubated with immobilized GST-TEAD1 (amino acids 96–411), GST-TEAD2 (amino acids 121–447), GST-TEAD3 (amino acids 115–435), GST-TEAD4 (amino acids 119–434), or GST. Proteins retained by the beads, together with the starting material (input), were resolved by SDS-PAGE followed by immunoblotting with antibodies that react with both TAZ and YAP. B, GST-TEADs and GST resolved by SDS-PAGE and stained with Coomassie Blue. C, coimmunoprecipitation of TAZ and TEADs. 293 cells were transfected to express FLAG-TAZ and HA-TEAD1–4. The cell lysates were immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting with anti-HA-horseradish peroxidase to detect coimmunoprecipitated HA-TEADs.
FIGURE 2.
FIGURE 2.
TEADs are important for TAZ-driven transformation of MCF10A cells. A, TAZ-S89A resistant to Hippo inhibition has increased ability to promote anchorage-independent cell growth. Soft agar assays were done using MCF10A cells transduced with expressing vector (vector) and vector for expressing FLAG-TAZ and FLAG-TAZ-S89A, and the colonies grown up in soft agar after 1 month were stained with thiazolyl blue tetrazolium bromide. B, lysates derived form the cells were analyzed by immunoblotting using anti-FLAG antibody. C, the colony number was quantified from three independent soft agar assays and presented as percentage relative to S89A, which was arbitrarily set as 100%. D, S89A-driven anchorage-independent growth is suppressed by shRNA-mediated knockdown of TEADs. S89A-expressing MCF10A cells (S89A) transduced with vector or TEAD-targeting shRNAs (KD-1 or KD-2) were grown in soft agar for a month, and the colonies were stained. E, the relative expression levels of TEAD1–4 were assessed by real time PCR showing preferential silence of TEAD4. The average level of TEADs in vector cells was arbitrarily set at 1, and the standard deviations of three independent assays were indicated. F, quantification of soft agar colonies of S89A cells expressing TEADs shRNAs. The average from three independent experiments is presented. The average colony number from S89A cells expressing no shRNA was arbitrarily set at 100%.
FIGURE 3.
FIGURE 3.
Identification of residues in the N-terminal region of TAZ which are important for interaction with TEAD4. A, alignment of amino acid sequences of N-terminal regions of TAZ and YAP from different species, highlighting conserved residues that are mutated in small stretches (bars on top of the alignment). B, Whole cells lysates from MCF10A cells stably expressing HA-TEAD4 and different FLAG-S89A mutants were immunoprecipitated (IP) with anti-FLAG, and the immunoprecipitates were probed with anti-HA to detect coimmunoprecipitated HA-TEAD4. Mutations of residues L31F32 (M4) (lane 4), W43R44 (M7) (lane 7), L48P49 (M8) (lane 8), and F52F53 (M9) (lane 9) of S89A disrupted interaction with TEAD4.
FIGURE 4.
FIGURE 4.
Mutants of S89A defective in interaction with TEAD4 are no longer able to drive anchorage-independent growth. A, MCF10A cells expressing S89A-M4 and S89A-M9, defective in binding to TEAD4, are not able to induce anchorage-independent growth on soft agar, whereas TEAD interaction competent S89A-M1, S89A-M2, and S89A-M3 (to a less extent), like S89A, are transforming. B, quantification of colony numbers for all the S89A mutants in comparison with vector-, TAZ-, and S89A-transduced cells. The average colony number from three independent experiments was presented with standard deviations. The average number derived from S89A-expressing cells was arbitrarily set at 100%. C, whole cell lysates derived form cells stably expressing TAZ, S89A, and its mutants were analyzed by immunoblotting using anti-FLAG and anti-actin antibodies. D, fusion of TEAD4 to the C terminus of S89A-M9 restored the ability to drive anchorage-independent growth. Soft agar assays were done on cells expressing S89A, TEAD4, S89A-M9, and S89A-M9-TEAD4. E, quantification of colony numbers for various proteins in comparison with vector and S89A cells. The average colony number from two independent experiments was presented with standard deviations. The average number derived from S89A-expressing cells was arbitrarily set at 100%. F, lysates derived from cells expressing the indicated proteins were analyzed by immunoblotting with anti-FLAG and anti-TEAD4 antibodies.
FIGURE 5.
FIGURE 5.
S89A mutants defective in interaction with TEAD4 failed to accumulate in the nucleus. A, cells expressing TAZ, S89A, S89A-M1, S89A-M4, S89A-M5, and S89A-M9 were processed for indirect immunofluorescence microscopy to reveal their subcellular distribution. B, nuclear accumulation of S89A-M9 is restored after its fusing with TEAD4. The cells expressing S89A-M9-TEAD4 were processed for immunofluorescence microscopy to reveal the distribution of S89A-M9-TEAD4, whereas the nucleus was revealed by 4′,6-diamidino-2-phenylindole (DAPI) staining.
FIGURE 6.
FIGURE 6.
GFP-TAZ-M9 mutant shows more rapid rates of nuclear export compared with wild type GFP-TAZ, indicating that nuclear retention is compromised. A, normalized fluorescence recovery kinetics of GFP-TAZ following repeated photobleaching of the nucleus as highlighted in yellow. B, normalized fluorescence recovery kinetics of GFP-TAZ following repeated photobleaching of the cytoplasm as marked in yellow. C, normalized fluorescence recovery kinetics of GFP-TAZ-M9 following repeated photobleaching of the nucleus as highlighted in yellow. D, normalized fluorescence recovery kinetics of GFP-TAZ-M9 following repeated photobleaching of the cytoplasm as indicated in yellow. Gray scale images show points iv as indicated in each FRAP curve, respectively. E, scatter plot of half-life of GFP-TAZ and GFP-TAZ-M9 nuclear import. GFP-TAZ τ½, means ± S.E. = 0.67 ± 0.06 min, GFP-TAZ-M9 τ½, means ± S.E. = 0.65 ± 0.08 min. F, scatter plot of half-life of GFP-TAZ and GFP-TAZ-M9 nuclear export. GFP-TAZ τ½, means ± S.E. = 1.52 ± 0.07 min, GFP-TAZ-M9 τ½, means ± S.E. = 0.57 ± 0.07 min, p < 0.0001. Each point is the average half-life of 2 FRAP curves from the same cell, each experiment was performed in at least five different cells, and similar results were obtained. G, a model to depict two major mechanisms regulating the distribution and function activity of TAZ. Hippo pathway and 14-3-3 proteins mediate cytoplasmic sequestration, whereas TEAD interaction drives nuclear retention of TAZ.

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