Cell contact and Nf2/Merlin-dependent regulation of TEAD palmitoylation and activity Nam-Gyun Kima,b and Barry M. Gumbinera,b,c,1 aCenter for Developmental Biology and Regenerative Medicine, Seattle Children’s Research Institute, Seattle, WA 98101; bDepartment of Pediatrics, University of Washington School of Medicine, Seattle, WA 98195; and cDepartment of Biochemistry, University of Washington School of Medicine, Seattle, WA 98195 Edited by Joan S. Brugge, Harvard Medical School, Boston, MA, and approved April 10, 2019 (received for review November 19, 2018) The Hippo pathway is involved in regulating contact inhibition of S-palmitoylation is a posttranslational modification of proteins in proliferation and organ size control and responds to various physical which a saturated fatty acid, palmitate, is attached via thioester and biochemical stimuli. It is a kinase cascade that negatively linkage to cysteine residue and, less frequently, to serine and regulates the activity of cotranscription factors YAP and TAZ, which threonine residues of protein (16, 17). Palmitate is supplied ex- interact with DNA binding transcription factors including TEAD and ogenously by diet or de novo biosynthesized by fatty acid syn- activate the expression of target genes. In this study, we show that thase (FASN) (18), the only mammalian enzyme that catalyzes the palmitoylation of TEAD, which controls the activity and stability palmitate from acetyl CoA, malonyl-CoA, and NADPH. Unlike of TEAD proteins, is actively regulated by cell density independent of other lipid modifications, such as prenylation and myristoylation, Lats, the key kinase of the Hippo pathway. The expression of fatty the palmitoylation process is reversible and can be dynamically acid synthase and acetyl-CoA carboxylase involved in de novo modulated in response to cell stimulation (16). Some proteins, in- biosynthesis of palmitate is reduced by cell density in an Nf2/ cluding TEAD, undergo nonenzymatic autopalmitoylation by direct Merlin-dependent manner. Depalmitoylation of TEAD is mediated binding to palmitoyl-CoA (14). The protein palmitoylation process by depalmitoylases including APT2 and ABHD17A. Palmitoylation- can also be mediated by the palmitoyl-acyl-transferase (PAT) en- deficient TEAD4 mutant is unstable and degraded by proteasome zymes, which transfer palmitoyl group to -SH group on a target through the activity of the E3 ubiquitin ligase CHIP. These findings protein (17). The palmitate turnover of palmitoylated proteins is show that TEAD activity is tightly controlled through the regulation regulated by depalmitoylases, such as acyl-protein thioesterase CELL BIOLOGY of palmitoylation and stability via the orchestration of FASN, (APT), which remove palmitoyl group from target proteins (17). depalmitoylases, and E3 ubiquitin ligase in response to cell contact. Protein palmitoylation contributes to membrane association and, by enhancing hydrophobicity, exerts pleotropic effects on – Hippo signaling | TEAD | palmitoylation | fatty acid synthase | protein protein interaction, protein activity, aggregation, clus- depalmitoylase tering, and stability (16, 17). Approximately 2,000 proteins in- cluding EGFR, H/N-Ras, Cdc42, and PSD-95 are known to be palmitoylated in mammals (19). Recent studies have shown that he Hippo signaling pathway contributes to the regulation of the activity of TEAD1 and stability of TEAD2 protein are reg- contact inhibition of proliferation, cell differentiation, and T ulated by palmitoylation, the mutation of palmitoylated cysteine organ size control (1). The pathway is comprised of a highly residues preventing its binding to YAP and decreasing its sta- conserved kinase cascade that leads to the activation of Lats1/2 bility (14, 15). However, the regulatory mechanism that dynam- (Large tumor suppressor kinase 1/2), which regulates the activity ically controls TEAD palmitoylation and its biological roles has and subcellular localization of transcriptional coactivators YAP not yet been identified. In this study, we show that the cell contact (Yes-associated protein) and its paralogue TAZ (transcriptional or Nf2/merlin-dependent regulation of TEAD palmitoylation is coactivator with PDZ-binding motif). Mst1/2 (Mammalian Ste20- like kinases 1/2) and MAP4Ks (Mitogen-activated protein kinase Significance kinase kinase kinases) phosphorylate and activate the Lats1/2 ki- nases in response to a number of upstream signals (2–5). In a growth permissive state, YAP/TAZ are translocated into the nu- The Hippo signaling pathway regulates cell proliferation in re- cleus where they interact with various DNA binding transcription sponse to cell contact and a variety of other extracellular stimuli. It factors, especially TEADs (TEA domain family members), but also controls the activity and nuclear localization of cotranscriptional p73, Runx2, and ERBB4 (6). Because YAP/TAZ lack the DNA activator YAP, which interacts with DNA binding transcription binding domain, the transcription of their downstream target genes factor TEAD, for the expression of target genes involved in cell is determined by DNA binding transcription factors. proliferation. We show here that the expression level and tran- TEAD is required for YAP/TAZ to transcribe the target genes scriptional activity of TEAD are actively controlled by cell density involved in cell proliferation, oncogenic transformation, and epi- through the modulation of its palmitoylation status. TEAD palmi- thelial–mesenchymal transition (7). Mammals express four TEAD toylation is controlled via fatty acid synthase and depalmitoylases genes (TEAD1–4) with similar domain architectures: the N-terminal in response to cell density. Our study indicates that the regulation DNA binding domain, and the C-terminal protein interaction do- of palmitoylation status is a potential target for controlling TEAD- main, which binds to various transcriptional coactivators such as dependent processes, perhaps including cancer growth. YAP/TAZ and the Vestigial-like (VGLL) (8). Although they have identical DNA binding domains and recognize the same DNA se- Author contributions: N.-G.K. and B.M.G. designed research; N.-G.K. performed research; N.-G.K. contributed new reagents/analytic tools; N.-G.K. and B.M.G. analyzed data; and quence, each TEAD shows tissue-specific and developmental stage- N.-G.K. and B.M.G. wrote the paper. specific expression patterns (9, 10). This suggests a unique biological function for each TEAD protein. In addition, the activity and sub- The authors declare no conflict of interest. cellular localization of TEAD is regulated by biological conditions This article is a PNAS Direct Submission. and during development (8, 11–13). Published under the PNAS license. Recent studies suggest that palmitoylation of TEAD is im- 1To whom correspondence should be addressed. Email: [email protected]. portant for its stability and activity (14, 15). However, it is not This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. known whether the state of TEAD palmitoylation is regulated in 1073/pnas.1819400116/-/DCSupplemental. the cell or controlled by upstream signals that affect the pathway. Published online May 1, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1819400116 PNAS | May 14, 2019 | vol. 116 | no. 20 | 9877–9882 Downloaded by guest on September 27, 2021 controlled by the expression of FASN, depalmitoylases, and E3 YAP binding-deficient TEAD4 showed density-dependent re- ubiquitin ligase. ductions of TEAD4 protein (Fig. 1D). This implies that the density-dependent regulation of TEAD4 protein level is in- Results dependent from YAP binding. To identify the Hippo signaling Density and Nf2-Dependent Regulation of TEAD Activity Is Controlled component that contributes to the posttranslational regulation of in a Lats-Independent Manner. The phosphorylation and localiza- TEAD4 protein level, we depleted Hippo signaling components tion of YAP are controlled by cell density via the Hippo pathway. in HA-tagged TEAD4 expressing MCF-10A cells through CRISPR-mediated knockout (KO) of Lats1/2, the core kinase of siRNA transfection and examined the exogenous TEAD4 pro- the Hippo pathway, in an immortalized human mammary epi- tein expression. Depletion of Nf2/Merlin increased the level of thelial cell line, MCF-10A, decreased the phosphorylation of HA-tagged TEAD4 in MCF-10A cells at low and high densities YAP in sparse and dense cell cultures (Fig. 1A). However, the (Fig. 1E), while depletion of Lats or Mst kinases did not influ- expression of YAP target gene mRNAs was reduced by increases ence the exogenous TEAD4 protein expression level. Nf2 is a in cell density even in Lats KO MCF-10A cells (Fig. 1B). Ad- membrane associated protein that mediates contact inhibition of ditionally, the depletion of Lats1/2 or overexpression of domi- growth, in part through the regulation of the Hippo pathway nant negative Lats1 strongly increased the YAP-TEAD (20). These results suggest that cell density and Nf2 play im- transcriptional reporter activity in cells at low density; however, portant roles in the regulation of TEAD4 levels in a Lats- the reporter activity was significantly reduced in cells at high independent manner. density (Fig. 1C). This implies the presence of another regulatory pathway, in addition to the Lats-dependent regulation of YAP Palmitoylation of TEAD Is Decreased in Cells at High Density. TEAD activity, which inhibits the transcriptional activation of YAP- undergoes autopalmitoylation at evolutionarily conserved