Calmodulin activates the Hippo signaling pathway by promoting LATS1 kinase–mediated inhibitory phosphorylation of the transcriptional coactivator YAP

The Hippo signaling pathway regulates tissue growth and cell fate, and its dysregulation can induce tumorigenesis. When Hippo is activated by cell–cell contact, extracellular signals, or cell polarity among others, the large tumor suppressor 1 (LATS1) kinase catalyzes inhibitory phosphorylation of the transcriptional coactivator Yes-associated protein (YAP) to maintain YAP in the cytoplasm or promote its degradation. Separately, calmodulin is a Ca2+-dependent protein that modulates the activity of target proteins and regulates several signaling cascades; however, its potential role in the Hippo pathway has not been identified. Here, using diverse experimental approaches, including in vitro binding analyses, kinase assays, RT–PCR, and confocal microscopy, we reveal that calmodulin promotes Hippo signaling. We show that purified YAP and LATS1 bind directly to calmodulin and form a Ca2+-dependent ternary complex in vitro. Importantly, Ca2+/calmodulin directly stimulated the activity of LATS1 kinase. In cultured mammalian cells, we demonstrated that endogenous YAP and LATS1 coimmunoprecipitate with endogenous calmodulin. In cells with activated Hippo signaling, we show that calmodulin antagonism significantly (i) decreases YAP phosphorylation, (ii) increases expression of two Hippo target genes (connective tissue growth factor [CTGF] and cysteine-rich angiogenic inducer 61 [CYR61]) that regulate cell proliferation and tumor progression, and (iii) enhances the interaction of YAP with its major transcription factor, thereby facilitating transcription of target genes. Collectively, our data demonstrate that calmodulin activates the Hippo kinase cascade and inhibits YAP activity via a direct interaction with LATS1 and YAP, thereby uncovering previously unidentified crosstalk between the Ca2+/calmodulin and Hippo signaling pathways. The Hippo signaling pathway regulates tissue growth and cell fate, and its dysregulation can induce tumorigenesis. When Hippo is activated by cell–cell contact, extracellular signals, or cell polarity among others, the large tumor suppressor 1 (LATS1) kinase catalyzes inhibitory phosphorylation of the transcriptional coactivator Yes-associated protein (YAP) to maintain YAP in the cytoplasm or promote its degradation. Separately, calmodulin is a Ca2+-dependent protein that modulates the activity of target proteins and regulates several signaling cascades; however, its potential role in the Hippo pathway has not been identified. Here, using diverse experimental approaches, including in vitro binding analyses, kinase assays, RT–PCR, and confocal microscopy, we reveal that calmodulin promotes Hippo signaling. We show that purified YAP and LATS1 bind directly to calmodulin and form a Ca2+-dependent ternary complex in vitro. Importantly, Ca2+/calmodulin directly stimulated the activity of LATS1 kinase. In cultured mammalian cells, we demonstrated that endogenous YAP and LATS1 coimmunoprecipitate with endogenous calmodulin. In cells with activated Hippo signaling, we show that calmodulin antagonism significantly (i) decreases YAP phosphorylation, (ii) increases expression of two Hippo target genes (connective tissue growth factor [CTGF] and cysteine-rich angiogenic inducer 61 [CYR61]) that regulate cell proliferation and tumor progression, and (iii) enhances the interaction of YAP with its major transcription factor, thereby facilitating transcription of target genes. Collectively, our data demonstrate that calmodulin activates the Hippo kinase cascade and inhibits YAP activity via a direct interaction with LATS1 and YAP, thereby uncovering previously unidentified crosstalk between the Ca2+/calmodulin and Hippo signaling pathways. The Hippo pathway is a highly conserved signaling network that governs organ size and tissue homeostasis in higher-order vertebrates by restricting cell proliferation and promoting apoptosis (1Pan D. The hippo signaling pathway in development and cancer.Dev. Cell. 2010; 19: 491-505Abstract Full Text Full Text PDF PubMed Scopus (1614) Google Scholar, 2Halder G. Johnson R.L. Hippo signaling: Growth control and beyond.Development. 2011; 138: 9-22Crossref PubMed Scopus (756) Google Scholar). First identified in the fruit fly Drosophila melanogaster (3Harvey K.F. Pfleger C.M. Hariharan I.K. The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis.Cell. 2003; 114: 457-467Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar), this pathway consists of a serine/threonine kinase cascade and a downstream transcriptional module. In the core kinase cascade, when Hippo is activated, mammalian Ste20-like kinases 1 and 2 catalyze phosphorylation of large tumor suppressor 1 and 2 (LATS1/2) kinases (4Hergovich A. Schmitz D. Hemmings B.A. The human tumour suppressor LATS1 is activated by human MOB1 at the membrane.Biochem. Biophys. Res. Commun. 2006; 345: 50-58Crossref PubMed Scopus (132) Google Scholar, 5Chan E.H. Nousiainen M. Chalamalasetty R.B. Schäfer A. Nigg E.A. Silljé H.H. The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1.Oncogene. 2005; 24: 2076-2086Crossref PubMed Scopus (411) Google Scholar). Once phosphorylated, LATS1/2 become active and in turn catalyze phosphorylation of the transcriptional coactivators Yes-associated protein (YAP) and WW domain–containing transcription regulator protein 1 (also known as transcriptional coactivator with PDZ-binding motif [TAZ]) (6Huang J. Wu S. Barrera J. Matthews K. Pan D. The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the drosophila homolog of YAP.Cell. 2005; 122: 421-434Abstract Full Text Full Text PDF PubMed Scopus (1265) Google Scholar, 7Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. Zheng P. Ye K. Chinnaiyan A. Halder G. Lai Z.C. et al.Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (1946) Google Scholar, 8Hao Y. Chun A. Cheung K. Rashidi B. Yang X. Tumor suppressor LATS1 is a negative regulator of oncogene YAP.J. Biol. Chem. 2008; 283: 5496-5509Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 9Lei Q.Y. Zhang H. Zhao B. Zha Z.Y. Bai F. Pei X.H. Zhao S. Xiong Y. Guan K.L. TAZ promotes cell proliferation and epithelial-mesenchymal transition and is inhibited by the hippo pathway.Mol. Cell Biol. 2008; 28: 2426-2436Crossref PubMed Scopus (673) Google Scholar). Phosphorylation of YAP and TAZ on Ser127 and Ser89, respectively, triggers their association with 14-3-3 proteins, which leads to their retention in the cytoplasm (7Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. Zheng P. Ye K. Chinnaiyan A. Halder G. Lai Z.C. et al.Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (1946) Google Scholar, 9Lei Q.Y. Zhang H. Zhao B. Zha Z.Y. Bai F. Pei X.H. Zhao S. Xiong Y. Guan K.L. TAZ promotes cell proliferation and epithelial-mesenchymal transition and is inhibited by the hippo pathway.Mol. Cell Biol. 2008; 28: 2426-2436Crossref PubMed Scopus (673) Google Scholar). In contrast, when Hippo is inactive, unphosphorylated YAP and TAZ are translocated to the nucleus where they exert cotranscriptional activity by binding to transcription factors. The transcriptional enhanced associate domain (TEAD) family members are the major transcription factors to which YAP and TAZ bind, leading to expression of Hippo target genes involved in cell growth, proliferation, migration, and survival (10Zhao B. Ye X. Yu J. Li L. Li W. Li S. Yu J. Lin J.D. Wang C.Y. Chinnaiyan A.M. Lai Z.C. Guan K.L. TEAD mediates YAP-dependent gene induction and growth control.Genes Dev. 2008; 22: 1962-1971Crossref PubMed Scopus (1529) Google Scholar, 11Zhang H. Liu C.Y. Zha Z.Y. Zhao B. Yao J. Zhao S. Xiong Y. Lei Q.Y. Guan K.L. TEAD transcription factors mediate the function of TAZ in cell growth and epithelial-mesenchymal transition.J. Biol. Chem. 2009; 284: 13355-13362Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). In addition to regulating their subcellular localization, LATS1/2-mediated phosphorylation of YAP and TAZ, on Ser381 and Ser306, respectively, triggers their ubiquitination and subsequent degradation (12Zhao B. Li L. Tumaneng K. Wang C.Y. Guan K.L. A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP).Genes Dev. 2010; 24: 72-85Crossref PubMed Scopus (853) Google Scholar). Diverse signals, including mechanical cues, cell polarity, cell adhesion, extracellular soluble factors, and cellular stresses, modulate activation of the Hippo pathway (13Ma S. Meng Z. Chen R. Guan K.L. The hippo pathway: Biology and pathophysiology.Annu. Rev. Biochem. 2019; 88: 577-604Crossref PubMed Scopus (266) Google Scholar). Because of its key role in metazoan physiology and development, it is not surprising that dysregulation of the Hippo network may have severe consequences, including organomegaly and tumorigenesis, because of cell hyperproliferation and inhibition of apoptosis (14Barron D.A. Kagey J.D. The role of the Hippo pathway in human disease and tumorigenesis.Clin. Trans. Med. 2014; 3: 25Crossref PubMed Google Scholar). Ca2+ is a fundamental intracellular messenger that regulates a broad range of cellular functions. The effects of Ca2+ are conveyed via several Ca2+-binding proteins (15Clapham D.E. Calcium signaling.Cell. 2007; 131: 1047-1058Abstract Full Text Full Text PDF PubMed Scopus (2736) Google Scholar). The highly conserved and ubiquitous protein calmodulin is an archetypal example of Ca2+-binding protein. Binding of Ca2+ to its four Ca2+-binding sites induces a conformational change in calmodulin, facilitating association with numerous proteins (16Hoeflich K.P. Ikura M. Calmodulin in action: Diversity in target recognition and activation mechanisms.Cell. 2002; 108: 739-742Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). Through binding to its structurally and functionally diverse targets, calmodulin modulates their activity, thereby participating in a broad range of cellular processes, including cell cycle progression, cell proliferation, cyclic nucleotide metabolism, glycogen metabolism, cytoskeletal arrangement, and smooth muscle contraction (17Klee C.B. Vanaman T.C. Calmodulin.Adv. Protein Chem. 1982; 35: 213-321Crossref PubMed Scopus (731) Google Scholar). Calmodulin has been documented to regulate in a Ca2+-dependent manner several major signaling pathways, including the mitogen-activated protein kinase (18Agell N. Bachs O. Rocamora N. Villalonga P. Modulation of the Ras/Raf/MEK/ERK pathway by Ca2+, and calmodulin.Cell Signal. 2002; 14: 649-654Crossref PubMed Scopus (344) Google Scholar, 19Parvathaneni S. Li Z. Sacks D.B. Calmodulin influences MAPK signaling by binding KSR1.J. Biol. Chem. 2021; 296: 100577Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar) and PI3K/protein kinase B networks (20Deb T.B. Coticchia C.M. Dickson R.B. Calmodulin-mediated activation of Akt regulates survival of c-Myc-overexpressing mouse mammary carcinoma cells.J. Biol. Chem. 2004; 279: 38903-38911Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 21Joyal J.L. Burks D.J. Pons S. Matter W.F. Vlahos C.J. White M.F. Sacks D.B. Calmodulin activates phosphatidylinositol 3-kinase.J. Biol. Chem. 1997; 272: 28183-28186Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Nevertheless, despite reports that Ca2+ signaling influences the Hippo network (22Wei Y. Li W. Calcium, an emerging intracellular messenger for the Hippo pathway regulation.Front. Cell Dev. Biol. 2021; 9: 694828Crossref PubMed Scopus (1) Google Scholar), the possible involvement of calmodulin in modulating Hippo had not been investigated. Therefore, we tested the hypothesis that calmodulin regulates Hippo signaling. We observed that YAP and LATS1 interact with calmodulin both in cells and in vitro. Importantly, these interactions have functional consequences. In vitro, Ca2+/calmodulin directly stimulates LATS1 kinase activity. In cells, the highly selective cell-permeable calmodulin antagonist CGS9343B significantly reduces Hippo activation mediated by cell–cell contact or serum starvation. Altogether, our data identify for the first time a communication between the Ca2+/calmodulin and Hippo pathways and reveal that calmodulin is a previously unrecognized regulator of Hippo signaling. Reports that Ca2+ signaling modulates Hippo (22Wei Y. Li W. Calcium, an emerging intracellular messenger for the Hippo pathway regulation.Front. Cell Dev. Biol. 2021; 9: 694828Crossref PubMed Scopus (1) Google Scholar) prompted us to investigate whether the Ca2+-binding protein calmodulin, which regulates several signaling networks (18Agell N. Bachs O. Rocamora N. Villalonga P. Modulation of the Ras/Raf/MEK/ERK pathway by Ca2+, and calmodulin.Cell Signal. 2002; 14: 649-654Crossref PubMed Scopus (344) Google Scholar, 19Parvathaneni S. Li Z. Sacks D.B. Calmodulin influences MAPK signaling by binding KSR1.J. Biol. Chem. 2021; 296: 100577Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar, 20Deb T.B. Coticchia C.M. Dickson R.B. Calmodulin-mediated activation of Akt regulates survival of c-Myc-overexpressing mouse mammary carcinoma cells.J. Biol. Chem. 2004; 279: 38903-38911Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 21Joyal J.L. Burks D.J. Pons S. Matter W.F. Vlahos C.J. White M.F. Sacks D.B. Calmodulin activates phosphatidylinositol 3-kinase.J. Biol. Chem. 1997; 272: 28183-28186Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), influences Hippo activation. To do so, we first assessed whether calmodulin associates with the Hippo transcriptional coactivator YAP. Analysis was performed by immunoprecipitation. In order to stabilize transient protein–protein interactions, HeLa cells were exposed to dithiobis(succinimidyl propionate) (DSP) that reacts with primary amine groups and crosslinks bound proteins. Cells were then lysed, and endogenous YAP was immunoprecipitated with anti-YAP monoclonal antibody. Immunoglobulin G (IgG) was used as a negative control. Analysis of the immunoprecipitated samples by SDS-PAGE and Western blotting revealed that endogenous calmodulin coimmunoprecipitates with YAP (Fig. 1A). In order to ascertain if calmodulin also binds to other components of the Hippo pathway, we investigated whether it interacts with LATS1, the kinase that inhibits YAP. We immunoprecipitated endogenous LATS1 from HeLa cells. Western blotting demonstrated that calmodulin coimmunoprecipitated with LATS1 (Fig. 1B). YAP was also present in the immunoprecipitates. The absence of signal from the samples precipitated with IgG validates the specificity of the interactions. Together, our data imply that YAP and LATS1 interact with calmodulin in cells. Furthermore, coimmunoprecipitation of both calmodulin and YAP with LATS1 suggests that these proteins could form a ternary complex in cells. To validate these observations, we reciprocally immunoprecipitated calmodulin from HeLa cell lysates and probed the Western blot for YAP and LATS1. Both YAP and LATS1 coimmunoprecipitate with calmodulin (Fig. 1C). Again, the absence of signal from the samples precipitated with IgG validates the specificity of the interactions. These data confirm that endogenous YAP and LATS1 interact with endogenous calmodulin in cells and support the premise that the three proteins could form a complex in HeLa cells. To further characterize the binding of calmodulin to YAP and LATS1, we evaluated whether Ca2+ regulates their interaction. To do so, human embryonic kidney 293 (HEK293) cells were lysed in buffer containing 1 mM of either CaCl2 or of the Ca2+-chelator EGTA. Equal amounts of protein lysates were incubated with calmodulin-Sepharose, and samples were resolved by Western blotting. The amount of YAP bound to calmodulin was not significantly altered by the presence of Ca2+ or EGTA (Fig. 2, A and B). This result confirms that YAP binds to calmodulin, regardless of the presence/absence of Ca2+, in cell lysates. Similar analysis was performed to examine the possible effect of Ca2+ on the association of LATS1 with calmodulin. In contrast to YAP, the amount of LATS1 bound to calmodulin in the presence of Ca2+ is 11-fold greater than when Ca2+ is chelated with EGTA (Fig. 2, A and B). These data imply that the interaction of calmodulin with endogenous LATS1 is regulated by Ca2+. No YAP or LATS1 was detected in samples incubated with glutathione-S-transferase (GST)–Sepharose (Fig. 2A), demonstrating the specificity of binding of YAP and LATS1 to calmodulin. To determine whether YAP binds directly to calmodulin, we used pure proteins. Purified human recombinant YAP was incubated with calmodulin-Sepharose in the presence of either CaCl2 or EGTA. Western blotting reveals that pure YAP binds directly to pure calmodulin in the presence of Ca2+ (Fig. 2C). Importantly, chelation of Ca2+ by EGTA almost completely abolishes the calmodulin–YAP interaction (Fig. 2, C and D). This shows that YAP binds directly to calmodulin in a Ca2+-dependent manner. This further suggests that the interaction between YAP and calmodulin that was observed in the absence of Ca2+ in cell lysates is indirect and occurs in a complex with (an)other protein(s). Analogous binding assays were carried out with purified human recombinant LATS1. Similar to YAP, our data show that LATS1 binds directly to Ca2+/calmodulin (Fig. 2E). The binding of LATS1 to calmodulin is significantly reduced (by 70.9 ± 12.9%, mean ± SD), but not eliminated, by removal of Ca2+ with EGTA (Fig. 2, E and F). These data demonstrate that pure LATS1 binds directly to pure calmodulin in a Ca2+-regulated manner, similar to what was observed in cell lysates. The absence of YAP and LATS1 from the pull-downs with GST-Sepharose validates the specificity of their binding to calmodulin. Taken together, our data demonstrate direct binding of YAP and LATS1 to Ca2+/calmodulin. Coimmunoprecipitation of both YAP and calmodulin with LATS1, as well as YAP and LATS1 with calmodulin, suggests that the three proteins form a ternary complex in cells. To assess whether YAP, LATS1, and calmodulin can form a ternary complex in vitro, we simultaneously incubated purified YAP and LATS1 with calmodulin-Sepharose. Both YAP and LATS1 bind to calmodulin in the presence of Ca2+ (Fig. 3A). No binding was detected when Ca2+ was chelated with EGTA. These data support the concept that YAP, LATS1, and calmodulin form a Ca2+-dependent ternary complex in vitro. To further test this hypothesis, we incubated pure YAP, LATS1, and calmodulin, and then crosslinked the proteins with 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP). Samples were analyzed by Western blotting. LATS1 and YAP were detected at their expected size (Fig. 3B, upper panel, red and green bands, respectively). In addition, we observed bands that migrated at higher molecular mass; these comprise both YAP and LATS1 (Fig. 3, B, upper panel, yellow bands). These bands were not influenced by the presence or the absence of Ca2+. These data indicate the formation of Ca2+-independent YAP–LATS1 complexes. Importantly, probing the same membrane for calmodulin reveals that it is in the high molecular mass complex with YAP and LATS1, only when Ca2+ is present (Fig. 3B, lower panel). Together, these data demonstrate that pure YAP, LATS1, and calmodulin form a Ca2+-dependent complex in vitro. We next studied whether the binding of calmodulin to YAP and LATS1 affects Hippo signaling. Initially, we investigated whether calmodulin modulates phosphorylation of YAP on Ser127, which occurs when Hippo is activated (8Hao Y. Chun A. Cheung K. Rashidi B. Yang X. Tumor suppressor LATS1 is a negative regulator of oncogene YAP.J. Biol. Chem. 2008; 283: 5496-5509Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar). Serum starvation is known to activate the Hippo signaling network (23Yu F.X. Zhao B. Panupinthu N. Jewell J.L. Lian I. Wang L.H. Zhao J. Yuan H. Tumaneng K. Li H. Fu X.D. Mills G.B. Guan K.L. Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling.Cell. 2012; 150: 780-791Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar, 24Adler J.J. Johnson D.E. Heller B.L. Bringman L.R. Ranahan W.P. Conwell M.D. Sun Y. Hudmon A. Wells C.D. Serum deprivation inhibits the transcriptional co-activator YAP and cell growth via phosphorylation of the 130-kDa isoform of Angiomotin by the LATS1/2 protein kinases.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 17368-17373Crossref PubMed Scopus (99) Google Scholar). To validate this observation under our assay conditions, we quantified phosphorylated YAP (pYAP) as an indicator of Hippo activation. HeLa cells were grown in complete (10% fetal bovine serum [FBS]) or serum-free medium. Equal amounts of protein lysates from these cells were analyzed by Western blotting and probed for pYAP (Ser127) and total YAP. Starved cells display 2.1-fold more YAP phosphorylation than fed cells (Fig. 4A), confirming Hippo activation. To determine whether calmodulin influences Hippo activation by serum starvation, we used the highly selective cell-permeable calmodulin antagonist CGS9343B (25Norman J.A. Ansell J. Stone G.A. Wennogle L.P. Wasley J.W. CGS 9343B, a novel, potent, and selective inhibitor of calmodulin activity.Mol. Pharmacol. 1987; 31: 535-540PubMed Google Scholar). Starved HeLa cells were incubated with 40 μM CGS9343B. Dimethyl sulfoxide (DMSO) was the vehicle control. Western blotting of cell lysates reveals that YAP phosphorylation is significantly reduced by 74% by CGS9343B (Fig. 4A). The amount of total YAP was not significantly altered (Fig. 4A), validating that the decreased pYAP signal reflects diminished protein phosphorylation rather than protein degradation. Collectively, these data indicate that inhibition of calmodulin impairs the phosphorylation of YAP induced when Hippo is activated by serum starvation. In order to determine whether the effect of calmodulin is restricted to Hippo activation by serum starvation, we examined its potential role using a different signal to activate Hippo. Cells grown at low confluency have minimal activation of Hippo, whereas increased cell–cell contact at higher density activates signaling (7Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. Zheng P. Ye K. Chinnaiyan A. Halder G. Lai Z.C. et al.Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (1946) Google Scholar, 26Aragona M. Panciera T. Manfrin A. Giulitti S. Michielin F. Elvassore N. Dupont S. Piccolo S. A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors.Cell. 2013; 154: 1047-1059Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar). We initially validated these observations in our assay system by comparing the abundance of pYAP in HEK293 cells grown at low or high confluency (Fig. 4B). Consistent with published data (7Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. Zheng P. Ye K. Chinnaiyan A. Halder G. Lai Z.C. et al.Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (1946) Google Scholar, 26Aragona M. Panciera T. Manfrin A. Giulitti S. Michielin F. Elvassore N. Dupont S. Piccolo S. A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors.Cell. 2013; 154: 1047-1059Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar), we observed that cells at high confluency had 1.5-fold more YAP phosphorylation than those at low confluency (Fig. 4C). Inhibition of calmodulin with CGS9343B reduced by 12% the amount of pYAP in cells at high confluency (Fig. 4D), albeit without statistical significance. Taken together, our data demonstrate that calmodulin function is required for maximal YAP phosphorylation in cells when the Hippo kinase cascade is activated by cell starvation and, to a lesser extent, by cell–cell contact. When Hippo is active, LATS1/2-mediated phosphorylation of YAP on Ser127 hinders its translocation to the nucleus, hence inhibiting expression of Hippo target genes (7Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. Zheng P. Ye K. Chinnaiyan A. Halder G. Lai Z.C. et al.Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (1946) Google Scholar). Here, we assessed the level of expression of two endogenous Hippo target genes, connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (CYR61), in fed and serum-starved cells. Quantitative real-time PCR analysis revealed, as anticipated, that the expression level of CTGF and CYR61 is significantly lower in starved cells than in cells cultured with FBS (Fig. 5A). This result confirms Hippo activation by serum starvation. We used this assay to determine whether calmodulin antagonism modulates Hippo gene expression. HeLa cells cultured in serum-free medium were incubated with 40 μM CGS9343B or DMSO. Quantitative real-time PCR analysis shows that CGS9343B increases expression of CTGF and CYR61 by 2.6-fold and 4.0-fold, respectively (Fig. 5B). This result suggests that inhibiting calmodulin promotes YAP-mediated transcription when Hippo is activated by serum starvation. We also examined the effect of cell confluency on CTGF and CYR61 expression. Cells at high confluency have a significantly lower expression of CTGF and CYR61 than cells at low density (Fig. 5C), as expected. We investigated whether calmodulin antagonism influences YAP-mediated gene expression induced by cell–cell contact. CGS9343B significantly increases expression of both CTGF and CYR61 in cells at high density (Fig. 5D). Collectively, these data reveal that calmodulin impairs expression of at least two endogenous Hippo target genes when the pathway is activated by either cell–cell contact or cell starvation. When Hippo is inactive, YAP binding to TEAD transcription factors in the nucleus triggers expression of target genes. By contrast, Hippo activation prevents YAP from translocating to the nucleus and interacting with TEAD (10Zhao B. Ye X. Yu J. Li L. Li W. Li S. Yu J. Lin J.D. Wang C.Y. Chinnaiyan A.M. Lai Z.C. Guan K.L. TEAD mediates YAP-dependent gene induction and growth control.Genes Dev. 2008; 22: 1962-1971Crossref PubMed Scopus (1529) Google Scholar). To gain insight into the molecular mechanism by which calmodulin modulates expression of Hippo target genes, we measured the interaction of YAP with TEAD. We assessed the formation of YAP–TEAD complexes in the nucleus by proximity ligation assay (PLA). In this assay, fixed permeabilized cells are incubated with antibodies specific to YAP and TEAD1. A fluorescence signal is generated only when the two antibodies are in close proximity (nanometer range). Consistent with the observation that serum starvation activates Hippo (Fig. 4A), starved cells have 60% fewer YAP–TEAD complexes than cells cultured in FBS (Fig. 6, A–C). Importantly, starved cells incubated with CGS9343B have 2.2-fold more nuclear YAP–TEAD complexes than DMSO-treated cells (Fig. 6, A–C). We verified by Western blotting that the amount of TEAD is not significantly altered by serum starvation and/or calmodulin antagonism (Fig. 6D). Taken together, these data demonstrate that calmodulin impairs YAP–TEAD nuclear interactions when Hippo is activated by serum starvation. Calmodulin modulates the activity of numerous kinases in response to changes in intracellular free Ca2+ concentration (27Cheung W.Y. Calmodulin plays a pivotal role in cellular regulation.Science. 1980; 207: 19-27Crossref PubMed Scopus (1839) Google Scholar). To gain insight into the molecular mechanism by which calmodulin modulates Hippo signaling, we evaluated whether calmodulin directly influences LATS1 kinase. We used purified active LATS1 kinase (Active Motif). Cellular activation of LATS1 requires its phosphorylation at Ser909 and Thr1079 (28Praskova M. Xia F. Avruch J. MOBKL1A/MOBKL1B phosphorylation by MST1 and MST2 inhibits cell proliferation.Curr. Biol. 2008; 18: 311-321Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). To validate that the purified kinase was active, we evaluated its phosphorylation at Ser909 and Thr1079. Analysis of purified LATS1 by Western blotting with specific antibodies revealed that the LATS1 is phosphorylated at both Ser909 and Thr1079 (Fig. 7A). The activity of purified active LATS1 was measured in vitro by homogeneous time-resolved fluorescence (HTRF) using the KinEASE STK S1 kit. The assay quantifies phosphorylation of a peptide substrate by LATS1 using FRET between a donor antibody that recognizes phosphorylated sites on the peptide and an acceptor molecule that targets the peptide substrate. The effect of purified calmodulin on LATS1 activity was examined in the presence or the absence of Ca2+. Calmodulin significantly stimulates LATS1 kinase activity in a dose-dependent manner in the presence of Ca2+ (Fig. 7B). In contrast, calmodulin does not increase LATS1 kinase activity in the absence of Ca2+. Importantly, addition of pure myoglobin instead of calmodulin to the reaction mixture does not stimulate LATS1 kinase (Fig. 7B). The lack of effect of myoglobin, which has similar molecular weight to calmodulin, valid