Structural basis of YAP recognition by TEAD4 in the hippo pathway

Abstract

The Hippo signaling pathway controls cell growth, proliferation, and apoptosis by regulating the expression of target genes that execute these processes. Acting downstream from this pathway is the YAP transcriptional coactivator, whose biological function is mediated by the conserved TEAD family transcription factors. The interaction of YAP with TEADs is critical to regulate Hippo pathway-responsive genes. Here, we describe the crystal structure of the YAP-interacting C-terminal domain of TEAD4 in complex with the TEAD-interacting N-terminal domain of YAP. The structure reveals that the N-terminal region of YAP is folded into two short helices with an extended loop containing the PXXPhiP motif in between, while the C-terminal domain of TEAD4 has an immunoglobulin-like fold. YAP interacts with TEAD4 mainly through the two short helices. Point mutations of TEAD4 indicate that the residues important for YAP interaction are required for its transforming activity. Mutagenesis reveals that the PXXPhiP motif of YAP, although making few contacts with TEAD4, is important for TEAD4 interaction as well as for the transforming activity.

Figure 1.

The overall structure of the TEAD4–YAP complex. (A) Domain organization of TEAD4 and YAP showing the TEA domain (orange), the YAP-binding domain of mTEAD4 (green), and the TEAD-binding domain (pink) and the WW domains (yellow) of mYAP. (B) A ribbon diagram of the TEAD4–YAP complex. TEAD4 and YAP are shown in green and pink, respectively, and the PXXΦP motif in YAP is colored in cyan. Secondary structure for both TEAD4 and YAP are marked. (C) Surface view of TEAD4 (green) with bound YAP in ribbon diagram (pink, with the PXXΦP motif in cyan). The view is as in B. (D) Superposition of the structures of TEAD4 (green) and PDEδ (light blue).

Figure 2.

Sequence alignment of TEAD and YAP. (A) Alignment of amino acid sequences of the YAP-binding domains of TEAD1–4 and Sd. (B) Alignment of amino acid sequences of the TEAD-binding domains of YAP/Yorki/TAZ. Secondary structures for mTEAD4 and mYAP are indicated at the top of each alignment. Residues mutated in hTEAD4 and hYAP involved in the TEAD4–YAP interface are marked with the number sign (#) and an asterisk (*), respectively, while those mutated in hTAZ are marked with a dot (•). The PXXΦP motif unique to YAP/Yorkie is marked with a cyan solid line.

Figure 3.

The TEAD4–YAP interface. (A) The first interaction site involving helices α3 and α4 of TEAD4 and α1 of YAP. (B) The second interaction site involving the PXXΦP motif of YAP and the α4–β10 loop and the β6–β7 loop of TEAD4. (C) The third interaction site containing residues 71–85 of YAP and β4, β10, β11, β12 of TEAD4. TEAD4 and YAP are shown in green and pink, respectively. Residues in TEAD4 and those in YAP involved in the interface are shown in stick models. Hydrogen bonds are shown in black broken lines.

Figure 4.

Residues in TEAD4 involved in interactions with YAP are important for anchorage-independent cell growth. (A) Whole-cell lysates from 293 cells transiently expressing Flag-YAP, HA-TEAD4-WT, and different HA-TEAD4 mutants were immunoprecipitated with anti-Flag, and the immunoprecipitates were probed with anti-HA to detect coimmunoprecipitated HA-TEAD4 and its mutants. The blots were stripped and reprobed with anti-Flag-HRP to detect immunoprecipitated Flag-YAP. Mutations of residues W299 (lane 4), Y429 (lane 11), and K297 (lane 12) of hTEAD4 disrupted interaction with hYAP. These three mutants are highlighted with asterisks. (B) Transformation ability assay for mutants of hTEAD4. Soft agar assays were done using MCF10A cells transduced with expressing vector (vector) and vectors for expressing TEAD4-WT and the indicated TEAD4 mutants. The colonies grown up in soft agar after 1 mo were stained with thiazolyl blue tetrazolium bromide. (C) Quantification of the colony number from three independent soft agar assays. The colony numbers were obtained after subtracting those of vector control and presented as percentage relative to TEAD4-WT, which was arbitrarily set as 100%.

Figure 5.

Importance of the PXXΦP loop in interaction with TEAD4 and the transforming activity. (A) Whole-cell lysates from 293 cells transiently expressing HA-TEAD4, Flag-YAP, and Flag-YAP mutants were immunoprecipitated with anti-Flag, and the immunoprecipitates were probed with anti-HA to detect coimmunoprecipitated HA-TEAD4. The blots were stripped and reprobed with anti-Flag-HRP to detect immunoprecipitated Flag-YAP and its mutants. Single mutations of residues P81, V84, and P85 to Ala did not disrupt interaction with TEAD4, but mutation or deletion of the PXXΦP-containing loop in YAP disrupted interaction with TEAD4. (B) Transforming activities of YAP mutants. Soft agar assays were done as in Figure 4B, using MCF10A cells transduced with expressing vector, vectors for expressing Flag-YAP-WT, and the indicated mutants of YAP. (C) Quantification of the colony number from A. The detail is as described in the legend for Figure 4C.