Serumdeprivation inhibits the transcriptional co-activatorYAP and cell growth via phosphorylation of the 130-kDaisoform of Angiomotin by the LATS1/2 protein kinasesJacob J. Adlera, Derrick E. Johnsona, Brigitte L. Hellera, Lauren R. Bringmana, William P. Ranahana, Michael D. Conwellb,Yang Sunb, Andy Hudmona,c, and Clark D. Wellsa,1

aDepartment of Biochemistry and Molecular Biology, bDepartment of Ophthalmology, Glick Eye Institute, and cStark Neuroscience Research Institute, IndianaUniversity School of Medicine, Indianapolis, IN 46202

Edited* by Tony Pawson, Samuel Lunenfeld Research Institute, Toronto, ON, Canada, and approved September 17, 2013 (received for review May 3, 2013)

Large tumor suppressor (LATS)1/2 protein kinases transmit Hipposignaling in response to intercellular contacts and serum levels tolimit cell growth via the inhibition of Yes-associated protein (YAP).Here low serum and high LATS1 activity are found to enhance thelevels of the 130-kDa isoform of angiomotin (Amot130) throughphosphorylation by LATS1/2 at serine 175, which then forms abinding site for 14-3-3. Such phosphorylation, in turn, enables theubiquitin ligase atrophin-1 interacting protein (AIP)4 to bind, ubiq-uitinate, and stabilize Amot130. Consistently, the Amot130 (S175A)mutant, which lacks LATS phosphorylation, bound AIP4 poorly underall conditions and showed reduced stability. Amot130 and AIP4also promoted the ubiquitination and degradation of YAP in re-sponse to serum starvation, unlike Amot130 (S175A). Moreover,silencing Amot130 expression blocked LATS1 from inhibiting theexpression of connective tissue growth factor, a YAP-regulatedgene. Concordant with phosphorylated Amot130 specifically me-diating these effects, wild-type Amot130 selectively induced YAPphosphorylation and reduced transcription of connective tissuegrowth factor in an AIP4-dependent manner versus Amot130(S175A). Further, Amot130 but not Amot130 (S175A) stronglyinhibited the growth of MDA-MB-468 breast cancer cells. Thedominant-negative effects of Amot130 (S175A) on YAP signalingalso support that phosphorylated Amot130 transduces Hippo sig-naling. Likewise, Amot130 expression provoked premature growtharrest during mammary cell acini formation, whereas Amot130(S175A)-expressing cells formed enlarged and poorly differentiatedacini. Taken together, the phosphorylation of Amot130 by LATS isfound to be a key feature that enables it to inhibit YAP-dependentsignaling and cell growth.

breast cancer | Itch | growth control

The Hippo signaling pathway integrates changes in the cellularmicroenvironment such as cell–cell contacts (1) and levels of

mitogenic lipids (2, 3) to control cell growth and survival (4). Duringdevelopment, Hippo signaling regulates organ size (5), whereas inadults it has tumor-suppressive effects (6). Canonical Hippo sig-naling entails the activation of the mammalian STE20-like (MST)1/2 protein kinases, which phosphorylate and activate the largetumor suppressor (LATS)1/2 protein kinases. Active LATS1/2phosphorylate YAP (Yes-associated protein) (7) and TAZ (tran-scriptional coactivator with a PDZ-binding motif) to trigger theirbinding to 14-3-3 proteins and repression of their protranscriptionalactivities (8–11). Active YAP and TAZ are primarily nuclear, wherethey coactivate the TEAD family of growth-promoting transcrip-tion factors (12) and the proapoptotic transcription factor p73(13). Recently, actin dynamics induced by the loss of cell attachment(14) or matrix stiffness (15, 16) have also been shown to regulateYAP and TAZ through LATS1/2-dependent and -independentmechanisms. However, the mechanisms relating the differentmodes of regulation of YAP are unclear.Angiomotin (Amot) is a member of a structurally related family

of adaptor proteins that also includes AmotL1 and AmotL2 that

all bind and inhibit YAP and TAZ (17–19). Amot associates withcell junctions and binds apical polarity proteins, which underlie itsability to control cell shape and migration (20–23). The 130 kDaisoform of Amot (Amot130), unlike the 80-kDa isoform (Amot80)that promotes cell growth (24), binds and inhibits YAP throughcytosolic sequestration (17, 18) and by facilitating its degradation(25) in a manner that can be independent of YAP phosphorylationby LATS1/2 at residue Ser-127 (17, 18).This study finds that Amot130 both induces and transmits Hippo

signaling in response to serum deprivation in a manner thatrequires its direct phosphorylation by LATS1/2. This underliesa process where Amot130 then binds atrophin-1 interactingprotein (AIP)4 to promote YAP degradation and consequentlyto inhibit YAP-dependent transcription and cell growth.

ResultsSerum Deprivation and LATS1 Activity Control the Protein Levels ofAmot130.The serum factors sphingosine-1-phosphate (S-1-P) (2, 3)and lysophosphatidic acid (LPA) (2) activate YAP through Gprotein-coupled receptor-initiated signaling. Here the converseprocess, whereby serum starvation induces Hippo signaling, wasinvestigated in breast cancer and nontransformed model celllines. Initially, the effects of serum starvation were measured onthe Hippo pathway proteins Amot130, AmotL1, LATS1, YAP,and TAZ by immunoblot. The phosphorylation of LATS1 atSer-909, a surrogate measure of activity, increased significantlyby 24 h, whereas the levels of YAP and TAZ declined (Fig. 1A)as reported (3). Conversely, the levels of Amot130 increasedbetween 24 and 30 h, unlike AmotL1, which was unchanged.

Significance

This study defines a unique mechanism controlling the activa-tion of Hippo signaling and consequent inhibition of cellgrowth. Specifically, serum starvation is found to induce thelarge tumor suppressor (LATS)1/2 kinases to phosphorylate andthus stabilize the 130 kDa isoform of the membrane-associatedpolarity protein angiomotin (Amot130). As a consequence,Amot130 recruits the E3 protein-ubiquitin ligase atrophin-1interacting protein 4. This multiprotein complex then signals thedegradation of Yes-associated protein (YAP) and the inhibitionof cell growth. These findings significantly modify our currentview that YAP phosphorylation by LATS1/2 is sufficient for itsinhibition in mammals and thus for growth arrest.

Author contributions: J.J.A., D.E.J., W.P.R., A.H., and C.D.W. designed research; J.J.A., D.E.J.,L.R.B., W.P.R., and C.D.W. performed research; J.J.A., D.E.J., B.L.H., L.R.B., M.D.C., Y.S., andA.H. contributed new reagents/analytic tools; J.J.A., D.E.J., M.D.C., and C.D.W. analyzeddata; and J.J.A. and C.D.W. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail: wells4@iupui.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1308236110/-/DCSupplemental.

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Serum starvation was found to significantly increase the levelsof Amot130 in all lines tested, including MDA-MB-468, humanembryonic kidney (HEK) 293T, and BT-474 cells (Fig. S1 A–D),which all show relatively high basal levels of Amot130. Thedramatic reduction in Amot130 steady-state levels upon rein-troduction of medium with 10% (vol/vol) serum highlights thatthere is a tight regulation of Amot130 protein stability (Fig. 1Band Fig. S1A). The temporal concordance between LATS1 ac-tivation and increased levels of Amot130 at 24 h suggests thatLATS1 activity may contribute to the stability of Amot130.Consistently, Amot130 protein (Fig. 1C and Fig. S1 E and F) butnot AMOT mRNA levels (Fig. S1G) were significantly reducedin cells silenced for expression of LATS1 by either siRNA orstable expression of shRNA.

Amot130 Is Phosphorylated by LATS1 and LATS2 at Serine 175. Theconsensus LATS phosphorylation motifs (HVRSLS) in Amot130(Ser-175), AmotL1 (Ser-262), and AmotL2 (Ser-159) (26) (Fig.2A) were measured for LATS1/2-catalyzed phosphorylation.This initially involved an array of 15-residue peptides encom-passing each motif, control peptides with an alanine at the pre-dicted site of phosphorylation (Fig. 2B and Fig. S2A), and theSer-127 motif in YAP as a positive control. Peptides were syn-thesized as individual immobilized spots on a membrane. Apurified fragment of active LATS2 phosphorylated the Amot130motif similarly to the YAP motif, which were both significantlyhigher than the Amot130 control peptide (Fig. 2B). AmotL1motif peptides were also phosphorylated to significantly higherlevels than the control peptide. However, the AmotL2 motif andcontrol peptides were phosphorylated to a similarly low level(Fig. S2A). The serine outside the LATS consensus site in con-trol peptides may explain their basal levels of phosphorylation.

The phosphorylation of Flag-tagged wild-type Amot130 versusFlag-tagged Amot130 (S175A) mutant by purified LATS2 andimmunoprecipitated Flag-tagged LATS1 activated with MST2was measured as described (27). Wild-type Amot130 was phos-phorylated 10-fold more than Amot130 (S175A) by LATS2 andover 2-fold more by LATS1 (Fig. 2C and Fig. S2B).The 14-3-3 family of proteins binds specifically to phosphor-

ylated serine residues (28), including phospho-Ser-127 in YAP(1). Based upon similarity with Amot130 Ser-175 (Fig. S2C), theability of LATS1 to induce 14-3-3 binding to this site was de-fined. Endogenous association of Amot130 with 14-3-3 wasconfirmed by immunoprecipitation of pan 14-3-3 with Amotfrom HEK 293T cells grown to high density (Fig. S2D). Thecoimmunoprecipitation of YFP-tagged Amot130 with Flag-tag-ged 14-3-3γ was then found to be induced in cells expressingMST2 and LATS1, but not from cells expressing either kinase inisolation or control vector (Fig. 2D). This is consistent with thelow phosphorylation of exogenously expressed LATS1 in theabsence of coexpressed MST2 (Fig. S2E). Further, 14-3-3γ failedto bind Amot130 (S175A) (Fig. 2E). Thus, the binding of 14-3-3γ

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Fig. 1. Serum starvation and LATS1 increase Amot130 protein levels. (A) Thelevels of endogenous proteins at the indicated times after initiation of serumstarvation were measured by immunoblot (IB) from lysates of MDA-MB-468cells. Pixel intensities of endogenous proteins normalized to GAPDH and theratios of phosphorylated Ser-909 (pS909) LATS1 to total LATS1 are provided.(B) A graph of the mean ratios of endogenous Amot130 to GAPDH from fourexperiments (n = 4) from HEK 293T cells grown in DMEM with 10% serum orfollowing 24 h of no serum followed by add back of DMEM containing 0% or10% serum for 5 min. (C) A graph showing the mean ratio of endogenousAmot130 to GAPDH from three experiments (n = 3) detected by immunoblotfrom lysates prepared from HEK 293T cells stably expressing control or LATS1shRNA. Error bars represent ± SD. ***P < 0.0001; **P < 0.005.

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Fig. 2. LATS1 and LATS2 phosphorylate Amot130 at serine 175. (A) Sche-matic of the predicted LATS phosphorylation motifs in Amot130, AmotL1,and AmotL2. (B) Graph of the mean incorporation of [32P]phosphate fromthree experiments (n = 3) into the indicated immobilized 15-residue peptidesby purified active LATS2. Values represent photostimulated luminescencedensity (PSL per mm2). (C) Immunoblot detecting the ATP-γ-S incorporatedinto immunoprecipitated (IP) Flag-tagged Amot130 or Amot130 (S175A) bypurified LATS2 or immunoprecipitated Flag-tagged LATS1 using an antibodyagainst thiophosphate esters (Thiophos. ester) (Top) and the ratio of phos-phorylated Amot130 to total Amot130, Flag-tagged Amot130 with anti-bodies against Amot or Flag (Middle), or LATS1 (Bottom). (D) Immunoblot ofthe levels of YFP-tagged Amot130 in anti-Flag (14-3-3γ) immunoprecipita-tions and the indicated proteins in lysates from HEK 293T cells transfected asindicated. (E) Immunoblot of the levels of YFP-tagged Amot130 andAmot130 (S175A) in an anti-Flag (14-3-3γ) immunoprecipitation and the in-dicated proteins in lysates from HEK 293T cells transfected as indicated. Errorbars represent ± SD. **P < 0.005; n.d., no statistical difference.

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to Ser-175 in Amot130 is a unique interaction that indicates itsphosphorylation state in vivo.

Serum Starvation and LATS1 Activity Induce AIP4 to Bind andUbiquitinate Amot130. The Nedd4 ubiquitin ligase AIP4 bindsand ubiquitinates Amot130, resulting in a reduction in Amot130residence at actin fibers and a significant enhancement of thestability of Amot130 protein (25). Thus, the role of serum andLATS1 in promoting the Amot130–AIP4 complex was examined.Whereas Myc-tagged AIP4 coimmunoprecipitated with Amot130to a greater extent from lysates prepared from serum-starved cellsversus cells grown in 10% serum (Fig. 3A), this association wasundetectable in cells partially silenced for LATS1 regardlessof serum conditions. Conversely, the coprecipitation of AIP4with wild-type Amot130 but not with Amot130 (S175A) wasenhanced from lysates from cells expressing MST2 and LATS1(Fig. 3B). This is likely via direct binding, as LATS1 did not induceAIP4 to bind the Amot130 (P-Y1,2,3A) mutant that encodes al-anine at tyrosine residues in all motifs that bind AIP4 (Fig. S3A).Further, LATS1 expression, in cells grown to high confluence,promoted the AIP4-dependent ubiquitination of itself and of

Amot130 (Fig. 3C). Thus, serum starvation, through LATS1 acti-vation, promotes AIP4 to bind and ubiquitinate Amot130.Because ubiquitination of Amot130 reduces its association

with actin stress fibers and increases its stability (25), wild-typeAmot130 and Amot130 (S175A) were compared for similarbehaviors. YFP-tagged wild-type Amot130, similar to previousreports, localized mainly at intercellular contacts (21), likelythrough membrane association (Fig. S3 B–D) (29) and to a lesserextent at cortical and F-actin stress fibers (30) (Fig. S3E).Strikingly, Amot130 (S175A) highly concentrated at thickenedF-actin stress fibers (Fig. S3F), similar to the localization of theAmot130 (K481R) mutant, which has reduced ubiquitination byAIP4 (25). Thus, the nonphosphorylated forms of Amot130 andAmotL1 likely bind and rearrange actin (30, 31). Importantly,following the inhibition of translation using cycloheximide, therate of decay of Amot130 (S175A) protein was over twofoldhigher [similar to the Amot130 (K481R) mutant] than that ofwild-type Amot130 in both MDA-MB-468 and MCF7 cells (Fig.3D and Fig. S3 G and H). Thus, Amot130 (S175A) likely is lessstable, at least in part, due to its inability to bind and be ubiq-uitinated by AIP4.

The Amot130–AIP4 Complex Promotes the Ubiquitination of YAP inResponse to Serum Starvation. The ability of serum deprivation andLATS1 activity to regulate the ubiquitination and destabilizationof YAP (25) by promoting the Amot130–AIP4 complex wasinvestigated. The ubiquitination of Flag-tagged YAP2 was in-creased in HEK 293T cells under serum-starved conditions versuscontrol cells in a manner promoted by expression of AIP4 (Fig.4A) or wild-type Amot130, but not by Amot130 (S175A) (Fig. 4B).Consistently, wild-type Amot130 but not Amot130 (S175A) hadincreased ubiquitination following serum starvation. Together, theAmot130–AIP4 complex promotes the ubiquitination of YAP in amanner that is dependent on the phosphorylation of Amot130.Based on the differences in wild-type Amot130 versus Amot130

(S175A) to induce YAP ubiquitination, their effects on YAP sta-bility were defined. This analysis was carried out inMCF7 cells dueto their high AIP4 levels (25), which sensitize them to effects ofphosphorylation of Amot130. Expression of wild-type Amot130reduced the half-life of YAP to 8.9 h, following incubation withcycloheximide, as previously shown (25). However, cells expressingAmot130 (S175A) showed no detectable reduction of YAP sta-bility over this period (Fig. 4C). Because actin stress fibers arehighly implicated in the activation of YAP and TAZ (16), theredistribution of YAP to actin stress fibers by Amot130 (S175A)(Fig. 4 D and E and Fig. S4 A and B) is consistent with it havingrobust YAP binding (Fig. S4C) but not playing a role in sup-pressing YAP signaling. Overall, phosphorylation of Amot130 islikely central to its ability to redirect YAP away from actin stressfibers and to trigger the destruction of YAP.

Amot130 Mediates the Effects of Hippo Signaling on the Inhibition ofYAP-Dependent Transcription. The requirement of Amot130 forLATS1 to inhibit endogenous connective tissue growth factor(CTGF) transcript levels, a measure of YAP and TAZ activity(32, 33), was monitored by real-time quantitative PCR in MDA-MB-468 cells. This breast cancer cell line expresses high levels ofAmot and is a model for studying YAP signaling. Following in-fection of these cells with shRNA targeting Amot for 24 h, therewas an isoform-selective silencing of Amot130 but not Amot80.Cells expressing CFP-tagged LATS1 and a control shRNA showeda significant reduction in CTGF levels (Fig. 5A), whereas therewas no significant loss of CTGF transcription in LATS-expressingcells with silenced Amot130 levels. Interestingly, silencing ofAmot130 without LATS1 expression resulted in a significant in-crease in CTGF levels. Thus, Amot130 is required to transducethe inhibition of YAP by LATS1 in these cells.The roles of phosphorylation of Amot130 in YAP phosphory-

lation and YAP-dependent transcription were then investigated.Confluent MDA-MB-468 cells that exogenously expressed wild-typeAmot130 showed increased levels of phospho-Ser-127-YAP

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Fig. 3. Active LATS1 drives the association of Amot130 with AIP4 resultingin the ubiquitination and increased stability of Amot130. (A) An immunoblotof YFP-tagged Amot130 in immunoprecipitates of Myc-tagged AIP4 as wellas the total levels of indicated proteins in lysates from HEK 293T cells thatwere coinfected with the indicated combinations of shRNA and taggedproteins and then grown with or without 10% serum for 24 h. Pixel in-tensities of LATS1 over GAPDH (below the fourth panel) are indicated. (B)Immunoblot of YFP-tagged Amot130 or Amot130 (S175A) in lysates andimmunoprecipitations of Myc-tagged AIP4 (anti-Myc) prepared from HEK293T cells expressing the indicated combinations of proteins. (C) Immuno-blot of ubiquitinated Amot130 and AIP4 in immunoprecipitates of HA-Lys-0 ubiquitin (HA-K0 Ub) along with the indicated proteins in lysates from HEK293T cells expressing the indicated combinations of proteins. (D) A graph ofthe mean regression slopes from three independent experiments (n = 3) ofimmunoblots of the levels of YFP-tagged Amot130 or Amot130 (S175A) inlysates of MDA-MB-468 cells treated with vehicle (DMSO) or cycloheximide(CHX) for 0, 4, or 8 h. Error bars represent ± SD. ***P < 0.05.

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versus control cells as previously reported (Fig. 5B) (17). Con-versely, Amot130 (S175A) reduced phospho-Ser-127-YAP versuscontrol cells. To determine whether this translated into reducedlevels of YAP-regulated transcripts, CTGF mRNA levels weremeasured in cells following growth under conditions conduciveto low Hippo signaling (10% serum and low confluence) or highHippo signaling (10% serum and high confluence) (Fig. 5C andFig. S4D). The expression of wild-type Amot130 but not Amot130(S175A) significantly reduced the level of CTGF transcription incells with low Hippo signaling. Alternatively, cells with high levelsof intercellular contacts showed modestly reduced CTGF levelsupon wild-type Amot130 expression from presumably already lowbasal levels of YAP activity. However, Amot130 (S175A) ex-pression increased CTGF levels significantly above those of con-trol cells. This is consistent with it exerting a dominant-negativeeffect in cells with active Hippo signaling. Importantly, the silencingof AIP4 dramatically reduced the ability of wild-type Amot130expression to inhibit CTGF levels, and it resulted in a synergisticincrease of CTGF levels in cells expressing Amot130 (S175A)(Fig. 5D). Thus, AIP4 recruitment to Amot130 upon its phos-phorylation by LATS1/2 is proposed to be a key mechanismwhereby Hippo signaling inhibits YAP.

Amot130 (S175A) Lacks the Ability of Wild-Type Amot130 to InhibitCell Growth in Breast Cancer Cells. To measure the effects of ex-pression of wild-type Amot130 and Amot130 (S175A) on contactinhibition, the rate of growth of MDA-MB-468 cells on plastic inserum-free medium or medium containing 10% serum that stablyexpressed either protein or a control vector over 4 d was mea-sured by both cell counting and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTS)cell-proliferation assay (Fig. S5 A–E). Cells expressing wild-typeAmot130 grew at significantly slower rates in the presence orabsence of serum, whereas cells expressing Amot130 (S175A)grew similarly to cells expressing a control vector. Analysis of the

growth of these cells in Matrigel as three-dimensional coloniesrevealed that cells expressing wild-type Amot130 formed coloniesthat were approximately threefold smaller after 4 d versus thoseformed by cells expressing control or Amot130 (S175A) (Fig. 6A–C and Fig. S5F). Thus, in a cancer cell line with defectivegrowth control, the inability of Amot130 to be phosphorylatedprevents it from inducing the inhibition of cell growth.

Amot130 (S175A) Disrupts Growth Control During the Formation ofMammary Acini.The growth and differentiation of nontransformedMCF10A cells into hollow acini in Matrigel are highly sensi-tive to Hippo signaling (14, 34, 35). The effects of expression ofwild-type Amot130 and Amot130 (S175A) on this process weretherefore compared (Fig. 6 D–F). After 1 d of growth, the sizesof cell clusters were nearly identical between all conditions (Fig.S6A). However, MCF10A cells expressing wild-type Amot130formed significantly smaller clusters that stopped growing by day4 (Fig. S6 B–D). Conversely, cells expressing Amot130 (S175A)displayed a loss of growth inhibition, where colonies were sig-nificantly larger by days 4 and 8 versus control cells (Fig. 6 Dand F and Fig. S6 B–D). Furthermore, unlike control cells, whichformed hollow acini by day 14, cells expressing Amot130 (S175A)formed solid tumor-like structures (Fig. 6E). Taken together, thephosphorylation of Amot130 is strongly indicated to play a vitalrole in Hippo signaling involved in cell-growth arrest (Fig. 6G).

DiscussionAlthough atypical cadherins Fat and Dachsous are central toHippo activation in Drosophila, their nonessential role in murineliver development has led to the suggestion that Amot proteinsare part of a replacement pathway in vertebrates (36), wherethey are exclusively found. This concept is further extended bythis study, showing that Amot130 phosphorylation by LATS1/2is essential for Hippo signaling. This is found to be mediatedby inducing Amot130 to bind and thereby couple AIP4 to the

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Fig. 4. Amot130 and AIP4, but not Amot130 (S175A), cooperatively induce the ubiquitination and degradation of YAP. (A) Immunoblot with an anti-HAantibody used to detect ubiquitinated Flag-tagged YAP2 (anti-Flag) and Amot130 in immunoprecipitations with anti-Flag antibody from HEK 293T cellsexpressing HA-tagged Lys-0 ubiquitin (HA-K0 Ub) and the indicated combinations of proteins. Before lysis, cells were cultured in DMEM with 10% or 0%serum for 24 h. (B) Ubiquitinated proteins were immunoprecipitated with an anti-HA antibody from HEK 293T cells expressing the indicated combinations ofrecombinant proteins. Individual proteins were then detected by immunoblot as indicated. (C) The levels of YFP-tagged Amot130, YFP-tagged Amot130(S175A), CFP-tagged YAP2, and endogenous GAPDH were detected by immunoblot of lysates from MCF7 cells stably expressing these proteins followingtreatment for the indicated times with vehicle (DMSO) or CHX. (Lower) A graph of the ratios of CFP-tagged YAP2 to GAPDH and the resulting half-lives (t1/2)in cells expressing (▪) YFP-tagged Amot130 (S175A) or (♦) YFP-tagged Amot130. (D and E) Confocal images of fixed MCF7 cells stained with phalloidin-594 (foractin) and expressing CFP-tagged YAP2 in combination with (D) YFP-tagged Amot130 (S175A) or (E) YFP-tagged Amot130.

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ubiquitination and degradation of YAP. This also results in theubiquitination and stabilization of Amot130, which likely rein-forces such antigrowth signaling.Although serum components, such as LPA or S-1-P, have re-

cently been shown to repress Hippo signaling (2, 3, 37), this studydescribes how serum starvation directly activates this pathway.Here the levels of Amot130, but not AmotL1, are shown to di-rectly respond to serum starvation through phosphorylation byLATS, which in turn is a critical event for Hippo-induced growtharrest. Future experiments examining other inhibitors of YAP,such as PP1 (38), glucagon, and epinephrine (2), for their effectson Amot130 phosphorylation may further establish how thisevent is central to Hippo signaling.The multifactorial effects of Amot130 on promoting the deg-

radation of YAP (25) likely underlie a self-reinforcing process,where Amot130 both activates and transmits Hippo signaling.This process appears to initially require LATS1 to phosphorylate

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Fig. 5. Phosphorylation of Amot130 at Ser-175 underlies its role in mediatingHippo signaling to inhibit YAP. (A) Real-time quantitative PCR measurementsof the levels of CTGF mRNA in MDA-MB-468 cells coinfected for 24 h withlentivirus encoding combinations of Amot shRNA, CFP-tagged LATS1, or theindicated controls. (Lower) Immunoblots from paired cells validating Amot130-selective silencing and CFP-tagged LATS1 expression with pixel intensity valuesof Amot130 and Amot80 levels normalized to GAPDH. (B) Immunoblot ofphospho-Ser-127 (pS127) YAP, total YAP, and GAPDH from MDA-MB-468 cellsexpressing YFP-tagged Amot130, Amot130 (S175A), or control vector. (C) Real-time quantitative PCR measurements of CTGF mRNA levels in MDA-MB-468cells described in B and harvested at low (Left) or high confluence (Right).(D) CTGF mRNA levels from MDA-MB-468 cells coinfected with lentivirusexpressing YFP-tagged Amot130, Amot130 (S175A), or control vector incombination with control shRNA (n = 4) or shRNA targeting AIP4 (n = 3), andcultured at low confluence in 0% serum for 24 h. Error bars represent ± SD.***P < 0.0001; **P < 0.01; *P < 0.05; n.d., no statistical difference.

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Fig. 6. Phosphorylation of Amot130 at Ser-175 is essential for it to inhibit cellgrowth. (A–C) MDA-MB-468 cells stably expressing YFP-tagged Amot130,Amot130 (S175A), or control vector were seeded onto Matrigel and imagedafter 1 and 4 d. (A–C) Representative bright-field stereo images (A) and plotsof the mean pixel integrated intensity values per field from four experiments(n = 4) (B and C) at (B) day 1 and (C) day 4 are presented. (D–F) MCF10A cellsstably expressing proteins as inAwere seeded ontoMatrigel and grown for 14d. (D) Bright-field stereo images of colonies at day 8 and (E) confocal fluo-rescence images of representative acini stained with Hoechst for nuclei at day14 of growth. (F) Plots of the mean cross-sectional area per acini (μm2) fromthree experiments each with 60 acini (n = 180) at day 8 are graphed. (Lower)Immunoblot measuring YFP-tagged Amot130 or Amot130 (S175A) andGAPDH from cells extracted fromMatrigel at day 8. (G) Model of the proposedmechanism for activation of Amot130 by serum deprivation and LATS resultingin the inhibition of YAP activity. Error bars represent ± SD. ***P < 1.0 × 10−60;**P < 0.01; n.d., no statistical difference. (Unlabeled scale bars, 50 μm.)

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Amot130 to promote its association with AIP4 that, in turn, ubiq-uitinates and thereby enhances Amot130 levels. Amot130 bindingAIP4 also increases LATS1 signaling by preventing AIP4 from in-stigating LATS1 degradation (25, 39). In parallel, Amot130 directlyactivates LATS1/2 (40). Both events by increasing LATS1/2 ac-tivity would reinforce this positive feedback loop. The predictedrapid accumulation of active Amot130 and LATS1/2 would alsoensure pervasive targeting of YAP for ubiquitination and de-gradation by SCF-(β)-TRCP (9) and by AIP4. Consistent withAmot130 having such effects, its expression is sufficient to stronglyinhibit the growth of MCF10A cells during acini formation, whereasthe phosphorylation-incompetent mutant, Amot130 (S175A),induces a loss of growth control.Further work exploring the relationships between the phos-

phorylation and ubiquitination of Amot130 will likely also provideinsights into the signaling leading to growth inhibition induced byfactors that control cell shape. Because Amot also binds apicalpolarity proteins (21) to mediate cross-talk with Hippo signaling(20), the ability of polarity proteins to potentially induce Amot130phosphorylation is increasingly attractive as a mechanism wherebyit may integrate Hippo kinase activity with cell polarity. Further,the fact that Amot130 also binds and bundles actin preferentiallywhen it is unphosphorylated suggests that it may coordinate suchsignaling with actin dynamics that are critical for the entry of YAPinto the nucleus (15, 41).

Materials and MethodsCell Culture, Treatment, and Assays. HEK 293T, MDA-MB-468, and MCF7 cellswere cultured as previously described and used for immunoblot, immuno-precipitation, membrane fractionation, RNA isolation/real-time PCR, confocalfluorescence imaging, cell accumulation, and MTS cell-proliferation experi-ments. MCF10A cells were cultured in lrECM (Matrigel, 7.5 mg/mL or greaterprotein; BD Biosciences; 354230). Cell growth in three dimensions was carriedout after plating 200 thousand MDA-MB-468 cells or 300 thousand MCF10Acells onto Matrigel, and colonies or acini were imaged with a stereomicro-scope or confocal microscope as previously described (24). Cells were serum-starved in all cases with Opti-MEM Reduced Serum Medium (Gibco). Moreinformation on these methods with cells, including infections, antibodies,reagents, plasmids, transfections, and detailed protocols, is provided in SIMaterials and Methods.

In Vitro Kinase Assays. In vitro SPOT phosphorylation assays including peptidesequences and immunoprecipitation in vitro kinase assays are described indetail in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Dannel McCollum for helpful discussions.HA-tagged Lys-0 ubiquitin was obtained through Addgene from T. Dawson(The Johns Hopkins University). Flag-tagged YAP2 and LATS1 constructs wereobtained through Addgene from M. Sudol (Weis Center for Research). Thiswork was supported by National Institutes of Health/National Cancer Insti-tute Grants R01CA151765 (to C.D.W.) and R01NS078171 (to A.H.) and De-partment of Defense Grant W81XWH (to C.D.W.).

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