Serum Deprivation Inhibits the Transcriptional Co-Activator YAP And

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

Jacob 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, Indiana University 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 Hippo all bind and inhibit YAP and TAZ (17–19). Amot associates with signaling in response to intercellular contacts and serum levels to cell junctions and binds apical polarity proteins, which underlie its limit cell growth via the inhibition of Yes-associated protein (YAP). ability to control cell shape and migration (20–23). The 130 kDa Here low serum and high LATS1 activity are found to enhance the isoform of Amot (Amot130), unlike the 80-kDa isoform (Amot80) levels of the 130-kDa isoform of angiomotin (Amot130) through that promotes cell growth (24), binds and inhibits YAP through phosphorylation by LATS1/2 at serine 175, which then forms a cytosolic sequestration (17, 18) and by facilitating its degradation binding site for 14-3-3. Such phosphorylation, in turn, enables the (25) in a manner that can be independent of YAP phosphorylation ubiquitin ligase atrophin-1 interacting protein (AIP)4 to bind, ubiq- by LATS1/2 at residue Ser-127 (17, 18). uitinate, and stabilize Amot130. Consistently, the Amot130 (S175A) This study ﬁnds that Amot130 both induces and transmits Hippo mutant, which lacks LATS phosphorylation, bound AIP4 poorly under signaling in response to serum deprivation in a manner that all conditions and showed reduced stability. Amot130 and AIP4 requires its direct phosphorylation by LATS1/2. This underlies also promoted the ubiquitination and degradation of YAP in re- a process where Amot130 then binds atrophin-1 interacting protein (AIP)4 to promote YAP degradation and consequently

sponse to serum starvation, unlike Amot130 (S175A). Moreover, CELL BIOLOGY silencing Amot130 expression blocked LATS1 from inhibiting the to inhibit YAP-dependent transcription and cell growth. expression of connective tissue growth factor, a YAP-regulated Results gene. Concordant with phosphorylated Amot130 specifically mediating these effects, wild-type Amot130 selectively induced YAP Serum Deprivation and LATS1 Activity Control the Protein Levels of phosphorylation and reduced transcription of connective tissue Amot130. The serum factors sphingosine-1-phosphate (S-1-P) (2, 3) growth factor in an AIP4-dependent manner versus Amot130 and lysophosphatidic acid (LPA) (2) activate YAP through G (S175A). Further, Amot130 but not Amot130 (S175A) strongly protein-coupled receptor-initiated signaling. Here the converse inhibited the growth of MDA-MB-468 breast cancer cells. The process, whereby serum starvation induces Hippo signaling, was dominant-negative effects of Amot130 (S175A) on YAP signaling investigated in breast cancer and nontransformed model cell also support that phosphorylated Amot130 transduces Hippo sig- lines. Initially, the effects of serum starvation were measured on naling. Likewise, Amot130 expression provoked premature growth the Hippo pathway proteins Amot130, AmotL1, LATS1, YAP, and TAZ by immunoblot. The phosphorylation of LATS1 at arrest during mammary cell acini formation, whereas Amot130 fi (S175A)-expressing cells formed enlarged and poorly differentiated Ser-909, a surrogate measure of activity, increased signi cantly acini. Taken together, the phosphorylation of Amot130 by LATS is by 24 h, whereas the levels of YAP and TAZ declined (Fig. 1A) found to be a key feature that enables it to inhibit YAP-dependent as reported (3). Conversely, the levels of Amot130 increased between 24 and 30 h, unlike AmotL1, which was unchanged. signaling and cell growth. fi breast cancer | Itch | growth control Signi cance

fi he Hippo signaling pathway integrates changes in the cellular This study de nes a unique mechanism controlling the activa- microenvironment such as cell–cell contacts (1) and levels of tion of Hippo signaling and consequent inhibition of cell T fi mitogenic lipids (2, 3) to control cell growth and survival (4). During growth. Speci cally, serum starvation is found to induce the development, Hippo signaling regulates organ size (5), whereas in large tumor suppressor (LATS)1/2 kinases to phosphorylate and adults it has tumor-suppressive effects (6). Canonical Hippo sig- thus stabilize the 130 kDa isoform of the membrane-associated naling entails the activation of the mammalian STE20-like (MST) polarity protein angiomotin (Amot130). As a consequence, 1/2 protein kinases, which phosphorylate and activate the large Amot130 recruits the E3 protein-ubiquitin ligase atrophin-1 tumor suppressor (LATS)1/2 protein kinases. Active LATS1/2 interacting protein 4. This multiprotein complex then signals the phosphorylate YAP (Yes-associated protein) (7) and TAZ (tran- degradation of Yes-associated protein (YAP) and the inhibition of cell growth. These findings significantly modify our current scriptional coactivator with a PDZ-binding motif) to trigger their view that YAP phosphorylation by LATS1/2 is sufficient for its binding to 14-3-3 proteins and repression of their protranscriptional inhibition in mammals and thus for growth arrest. activities (8–11). Active YAP and TAZ are primarily nuclear, where they coactivate the TEAD family of growth-promoting transcrip- 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., tion factors (12) and the proapoptotic transcription factor p73 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., and (13). Recently, actin dynamics induced by the loss of cell attachment A.H. contributed new reagents/analytic tools; J.J.A., D.E.J., M.D.C., and C.D.W. analyzed (14) or matrix stiffness (15, 16) have also been shown to regulate data; and J.J.A. and C.D.W. wrote the paper. YAP and TAZ through LATS1/2-dependent and -independent The authors declare no conflict of interest. mechanisms. However, the mechanisms relating the different *This Direct Submission article had a prearranged editor. modes of regulation of YAP are unclear. 1To whom correspondence should be addressed. E-mail: [email protected]. Angiomotin (Amot) is a member of a structurally related family This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of adaptor proteins that also includes AmotL1 and AmotL2 that 1073/pnas.1308236110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1308236110 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 A Serum Starved (Hours) The phosphorylation of Flag-tagged wild-type Amot130 versus 0 1 3 6 9 1224262830 Flag-tagged Amot130 (S175A) mutant by purified LATS2 and Amot130 immunoprecipitated Flag-tagged LATS1 activated with MST2 IB: Amot was measured as described (27). Wild-type Amot130 was phos- Amot130 / GAPDH 1.0 0.9 0.8 0.90.9 1.0 1.3 1.3 1.5 1.5 phorylated 10-fold more than Amot130 (S175A) by LATS2 and IB: AmotL1 over 2-fold more by LATS1 (Fig. 2C and Fig. S2B). IB: pS909- The 14-3-3 family of proteins binds specifically to phosphor- LATS1 ylated serine residues (28), including phospho-Ser-127 in YAP pS909-LATS1 / 1.0 0.5 0.4 0.81.1 0.9 2.5 2.3 1.6 0.5 Total LATS1 (1). Based upon similarity with Amot130 Ser-175 (Fig. S2C), the IB: LATS1 ability of LATS1 to induce 14-3-3 binding to this site was defined. Endogenous association of Amot130 with 14-3-3 was IB: YAP fi YAP / GAPDH 1.0 1.0 1.0 1.21.2 1.1 0.6 0.7 0.5 0.0 con rmed by immunoprecipitation of pan 14-3-3 with Amot from HEK 293T cells grown to high density (Fig. S2D). The IB: TAZ coimmunoprecipitation of YFP-tagged Amot130 with Flag-tag- TAZ / GAPDH 1.0 0.8 1.0 1.31.4 1.2 0.5 0.7 0.5 0.1 ged 14-3-3γ was then found to be induced in cells expressing IB: GAPDH MST2 and LATS1, but not from cells expressing either kinase in isolation or control vector (Fig. 2D). This is consistent with the B C low phosphorylation of exogenously expressed LATS1 in the N=4 N=3 0.8 *** ** 1.2 absence of coexpressed MST2 (Fig. S2E). Further, 14-3-3γ failed ** γ 0.6 0.8 to bind Amot130 (S175A) (Fig. 2E). Thus, the binding of 14-3-3 0.4 0.4 0.2

Steady State Levels 0.0 0.0 Normalized Amot130 Normalized Amot130 Steady State Levels Consensus LATS Recognition Motif Serum Starved - 24 h 24 h shControl + - A AmotL1(S262) Add Back of -+ 10 % Serum - - 5 min shLATS1 HVRSLS(175) AmotL2(S159) PDZ Binding P- Y 1 P- Y 2 P- Y 3 Motif Fig. 1. Serum starvation and LATS1 increase Amot130 protein levels. (A) The Amot130 ACCH Domain levels of endogenous proteins at the indicated times after initiation of serum LPTY PPEY PPEY Amot80 starvation were measured by immunoblot (IB) from lysates of MDA-MB-468 YAP2 AIP4 cells. Pixel intensities of endogenous proteins normalized to GAPDH and the Flag-Amot130 ++-- + - ratios of phosphorylated Ser-909 (pS909) LATS1 to total LATS1 are provided. B C Flag-Amot130 -+- + -+ (B) A graph of the mean ratios of endogenous Amot130 to GAPDH from four N=3 n.d. (S175A) 800 experiments (n = 4) from HEK 293T cells grown in DMEM with 10% serum or 2 ** GST-LATS2 -- - - ++ following 24 h of no serum followed by add back of DMEM containing 0% or Flag-LATS1 & YFP-MST2 - - ++-- 10% serum for 5 min. (C) A graph showing the mean ratio of endogenous 400 IB: Thiophos. Amot130 to GAPDH from three experiments (n = 3) detected by immunoblot ester IP: Flag

PSL / mm PSL p-Amot130 / Total Amot130 1.0 0.4 1.0 0.1 from lysates prepared from HEK 293T cells stably expressing control or LATS1 0 shRNA. Error bars represent ± SD. ***P < 0.0001; **P < 0.005. IB: Amot IB: Flag

Amot130 Amot130 (S175A) IB: LATS1 Serum starvation was found to signiﬁcantly increase the levels (S175) YAP (S127) of Amot130 in all lines tested, including MDA-MB-468, human D E embryonic kidney (HEK) 293T, and BT-474 cells (Fig. S1 A–D), YFP-MST2 - +-+ YFP-Control + - - CFP-LATS1 - ++ - YFP-Amot130 - + - which all show relatively high basal levels of Amot130. The -- YFP-Amot130 +++ + YFP-Amot130 (S175A) + dramatic reduction in Amot130 steady-state levels upon rein- YFP-MST2 Flag-14-3-3γ + ++ + + ++ troduction of medium with 10% (vol/vol) serum highlights that CFP-LATS1 + ++ there is a tight regulation of Amot130 protein stability (Fig. 1B IB: Amot IP: Flag Flag-14-3-3γ + ++ IP: F and Fig. S1A). The temporal concordance between LATS1 ac- IB: Flag IB: Amot

(14-3-3γ) lag tivation and increased levels of Amot130 at 24 h suggests that IB: Flag LATS1 activity may contribute to the stability of Amot130. (14-3-3γ) IB: Amot

Consistently, Amot130 protein (Fig. 1C and Fig. S1 E and F)but L

ysa IB:Amot ﬁ L

not AMOT mRNA levels (Fig. S1G)weresigni cantly reduced y sa

IB: LATS1 t in cells silenced for expression of LATS1 by either siRNA or e

IB: LATS1 te stable expression of shRNA. IB: YFP (MST2) IB: YFP (MST2) Amot130 Is Phosphorylated by LATS1 and LATS2 at Serine 175. The consensus LATS phosphorylation motifs (HVRSLS) in Amot130 Fig. 2. LATS1 and LATS2 phosphorylate Amot130 at serine 175. (A) Sche- (Ser-175), AmotL1 (Ser-262), and AmotL2 (Ser-159) (26) (Fig. matic of the predicted LATS phosphorylation motifs in Amot130, AmotL1, 2A) were measured for LATS1/2-catalyzed phosphorylation. and AmotL2. (B) Graph of the mean incorporation of [32P]phosphate from This initially involved an array of 15-residue peptides encom- three experiments (n = 3) into the indicated immobilized 15-residue peptides passing each motif, control peptides with an alanine at the pre- by purified active LATS2. Values represent photostimulated luminescence 2 γ dicted site of phosphorylation (Fig. 2B and Fig. S2A), and the density (PSL per mm ). (C) Immunoblot detecting the ATP- -S incorporated into immunoprecipitated (IP) Flag-tagged Amot130 or Amot130 (S175A) by Ser-127 motif in YAP as a positive control. Peptides were syn- fi thesized as individual immobilized spots on a membrane. A puri ed LATS2 or immunoprecipitated Flag-tagged LATS1 using an antibody against thiophosphate esters (Thiophos. ester) (Top) and the ratio of phos- purified fragment of active LATS2 phosphorylated the Amot130 fi phorylated Amot130 to total Amot130, Flag-tagged Amot130 with anti- motif similarly to the YAP motif, which were both signi cantly bodies against Amot or Flag (Middle), or LATS1 (Bottom). (D) Immunoblot of higher than the Amot130 control peptide (Fig. 2B). AmotL1 γ fi the levels of YFP-tagged Amot130 in anti-Flag (14-3-3 ) immunoprecipita- motif peptides were also phosphorylated to signi cantly higher tions and the indicated proteins in lysates from HEK 293T cells transfected as levels than the control peptide. However, the AmotL2 motif and indicated. (E) Immunoblot of the levels of YFP-tagged Amot130 and control peptides were phosphorylated to a similarly low level Amot130 (S175A) in an anti-Flag (14-3-3γ) immunoprecipitation and the in- (Fig. S2A). The serine outside the LATS consensus site in con- dicated proteins in lysates from HEK 293T cells transfected as indicated. Error trol peptides may explain their basal levels of phosphorylation. bars represent ± SD. **P < 0.005; n.d., no statistical difference.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1308236110 Adler et al. Downloaded by guest on September 24, 2021 Amot130 (Fig. 3C). Thus, serum starvation, through LATS1 acti- A B YFP- MST2 +++++ + vation, promotes AIP4 to bind and ubiquitinate Amot130. Serum Starved - - ++ - + - + -+ Because ubiquitination of Amot130 reduces its association YFP-Amot130 ++++ Flag-LATS1 ﬁ YFP-Control +-+ -- -with actin stress bers and increases its stability (25), wild-type Myc-AIP4 ++++ shControl -+ + - YFP-Amot130 - - ++ -- Amot130 and Amot130 (S175A) were compared for similar YFP-Amot130 behaviors. YFP-tagged wild-type Amot130, similar to previous shLATS1 - +- + ---- ++ IP: Myc (S175A) reports, localized mainly at intercellular contacts (21), likely IB: Amot Myc-AIP4 +++++ + –

IP: Myc through membrane association (Fig. S3 B D) (29) and to a lesser IP: Myc IB: Amot ﬁ (AIP4) extent at cortical and F-actin stress bers (30) (Fig. S3E). IB: Myc Strikingly, Amot130 (S175A) highly concentrated at thickened IB: Amot (AIP4) Lysate F-actin stress ﬁbers (Fig. S3F), similar to the localization of the

IB: LATS1 IB: Amot Lysate Amot130 (K481R) mutant, which has reduced ubiquitination by LATS1 / GAPDH 1.0 0.5 0.7 0.4 IB: Flag AIP4 (25). Thus, the nonphosphorylated forms of Amot130 and (LATS1) IB: GAPDH AmotL1 likely bind and rearrange actin (30, 31). Importantly, IB: YFP (MST2) following the inhibition of translation using cycloheximide, the rate of decay of Amot130 (S175A) protein was over twofold C YFP- higher [similar to the Amot130 (K481R) mutant] than that of Amot130 - - + + - - + + Myc-AIP4 - - ++ - - ++ D wild-type Amot130 in both MDA-MB-468 and MCF7 cells (Fig. 0.12 3D and Fig. S3 G and H). Thus, Amot130 (S175A) likely is less Flag-LATS1 ---- ++++ N=3 HA-K0 Ub ++++ ++++ stable, at least in part, due to its inability to bind and be ubiq- *** uitinated by AIP4. IB: Amot 0.08 IP: HA IB: Myc The Amot130–AIP4 Complex Promotes the Ubiquitination of YAP in (AIP4) Response to Serum Starvation. The ability of serum deprivation and IB: HA 0.04 (Free K0 Ub) LATS1 activity to regulate the ubiquitination and destabilization of YAP (25) by promoting the Amot130–AIP4 complex was IB: Amot Apparent Decay Rate Lysate

(Fraction Degraded / hour) investigated. The ubiquitination of Flag-tagged YAP2 was in- 0.00 IB: Myc creased in HEK 293T cells under serum-starved conditions versus (AIP4) control cells in a manner promoted by expression of AIP4 (Fig. CELL BIOLOGY IB: Flag 4A) or wild-type Amot130, but not by Amot130 (S175A) (Fig. 4B). (LATS1) Amot130 Amot130 (S175A) Consistently, wild-type Amot130 but not Amot130 (S175A) had increased ubiquitination following serum starvation. Together, the Fig. 3. Active LATS1 drives the association of Amot130 with AIP4 resulting – in the ubiquitination and increased stability of Amot130. (A) An immunoblot Amot130 AIP4 complex promotes the ubiquitination of YAP in a of YFP-tagged Amot130 in immunoprecipitates of Myc-tagged AIP4 as well manner that is dependent on the phosphorylation of Amot130. as the total levels of indicated proteins in lysates from HEK 293T cells that Based on the differences in wild-type Amot130 versus Amot130 were coinfected with the indicated combinations of shRNA and tagged (S175A) to induce YAP ubiquitination, their effects on YAP sta- proteins and then grown with or without 10% serum for 24 h. Pixel in- bility were defined. This analysis was carried out in MCF7 cells due tensities of LATS1 over GAPDH (below the fourth panel) are indicated. (B) to their high AIP4 levels (25), which sensitize them to effects of Immunoblot of YFP-tagged Amot130 or Amot130 (S175A) in lysates and phosphorylation of Amot130. Expression of wild-type Amot130 immunoprecipitations of Myc-tagged AIP4 (anti-Myc) prepared from HEK reduced the half-life of YAP to 8.9 h, following incubation with 293T cells expressing the indicated combinations of proteins. (C) Immuno- cycloheximide, as previously shown (25). However, cells expressing blot of ubiquitinated Amot130 and AIP4 in immunoprecipitates of HA-Lys- Amot130 (S175A) showed no detectable reduction of YAP sta- 0 ubiquitin (HA-K0 Ub) along with the indicated proteins in lysates from HEK bility over this period (Fig. 4C). Because actin stress fibers are 293T cells expressing the indicated combinations of proteins. (D) A graph of = highly implicated in the activation of YAP and TAZ (16), the the mean regression slopes from three independent experiments (n 3) of redistribution of YAP to actin stress fibers by Amot130 (S175A) immunoblots of the levels of YFP-tagged Amot130 or Amot130 (S175A) in lysates of MDA-MB-468 cells treated with vehicle (DMSO) or cycloheximide (Fig. 4 D and E and Fig. S4 A and B) is consistent with it having (CHX) for 0, 4, or 8 h. Error bars represent ± SD. ***P < 0.05. robust YAP binding (Fig. S4C) but not playing a role in sup- pressing YAP signaling. Overall, phosphorylation of Amot130 is likely central to its ability to redirect YAP away from actin stress fi to Ser-175 in Amot130 is a unique interaction that indicates its bers and to trigger the destruction of YAP. phosphorylation state in vivo. Amot130 Mediates the Effects of Hippo Signaling on the Inhibition of Serum Starvation and LATS1 Activity Induce AIP4 to Bind and YAP-Dependent Transcription. The requirement of Amot130 for Ubiquitinate Amot130. The Nedd4 ubiquitin ligase AIP4 binds LATS1 to inhibit endogenous connective tissue growth factor and ubiquitinates Amot130, resulting in a reduction in Amot130 (CTGF) transcript levels, a measure of YAP and TAZ activity residence at actin fibers and a significant enhancement of the (32, 33), was monitored by real-time quantitative PCR in MDA- MB-468 cells. This breast cancer cell line expresses high levels of stability of Amot130 protein (25). Thus, the role of serum and – Amot and is a model for studying YAP signaling. Following in- LATS1 in promoting the Amot130 AIP4 complex was examined. fection of these cells with shRNA targeting Amot for 24 h, there Whereas Myc-tagged AIP4 coimmunoprecipitated with Amot130 was an isoform-selective silencing of Amot130 but not Amot80. to a greater extent from lysates prepared from serum-starved cells Cells expressing CFP-tagged LATS1 and a control shRNA showed versus cells grown in 10% serum (Fig. 3A), this association was asignificant reduction in CTGF levels (Fig. 5A), whereas there undetectable in cells partially silenced for LATS1 regardless was no significant loss of CTGF transcription in LATS-expressing of serum conditions. Conversely, the coprecipitation of AIP4 cells with silenced Amot130 levels. Interestingly, silencing of with wild-type Amot130 but not with Amot130 (S175A) was Amot130 without LATS1 expression resulted in a significant in- enhanced from lysates from cells expressing MST2 and LATS1 crease in CTGF levels. Thus, Amot130 is required to transduce (Fig. 3B). This is likely via direct binding, as LATS1 did not induce the inhibition of YAP by LATS1 in these cells. AIP4 to bind the Amot130 (P-Y1,2,3A) mutant that encodes al- The roles of phosphorylation of Amot130 in YAP phosphory- anine at tyrosine residues in all motifs that bind AIP4 (Fig. S3A). lation and YAP-dependent transcription were then investigated. Further, LATS1 expression, in cells grown to high confluence, Confluent MDA-MB-468 cells that exogenously expressed wild-type promoted the AIP4-dependent ubiquitination of itself and of Amot130 showed increased levels of phospho-Ser-127-YAP

Adler et al. PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 CFP-YAP2 A Serum Starved - + +- B C Serum Starved -+- + YFP-Amot130 WT YFP-Amot130 (S175A) YFP-Amot130 + +++ CHX (Hours) Myc-AIP4 - -++ YFP-Amot130 + +-- 003 629612 3 19 Flag-YAP2 + +++ YFP-Amot130 - -++ IB: Amot HA-K0 Ub + +++ (S175A) Flag-YAP2 (Ub-YFP- + +++ IB: GAPDH Amot130) HA-K0 Ub + +++

(Ub-Flag- IP: Flag IB:Amot IB: YAP IB: HA

YAP2) IP: HA IB: Flag IB: Amot (YAP2) IB: GAPDH IB: Flag IB: HA 1.2 (Free K0 Ub) t1/2 = N.A. (> 48 h) YFP-Amot130 (YAP2) (S175A) Lysate IB: Amot 0.8 YFP-Amot130 Lysate IB: Amot WT IB: Flag 0.4 t IB: Myc 1/2 = 8.9 h (YAP2) (AIP4) YAP/GAPDH 0.0 036912 CHX Treatment (Hours)

D CFP-YAP2 )A571S(031tomA-PFY Phalloidin E CFP-YAP2 031tomA-PFY nidiollahP

20 μm 20 μm 20 μm 20 μm 20 μm 20 μm

Fig. 4. Amot130 and AIP4, but not Amot130 (S175A), cooperatively induce the ubiquitination and degradation of YAP. (A) Immunoblot with an anti-HA antibody used to detect ubiquitinated Flag-tagged YAP2 (anti-Flag) and Amot130 in immunoprecipitations with anti-Flag antibody from HEK 293T cells expressing 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 of recombinant 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 following

treatment 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 (for actin) and expressing CFP-tagged YAP2 in combination with (D) YFP-tagged Amot130 (S175A) or (E) YFP-tagged Amot130.

versus control cells as previously reported (Fig. 5B) (17). Con- growth of these cells in Matrigel as three-dimensional colonies versely, Amot130 (S175A) reduced phospho-Ser-127-YAP versus revealed that cells expressing wild-type Amot130 formed colonies control cells. To determine whether this translated into reduced that were approximately threefold smaller after 4 d versus those levels of YAP-regulated transcripts, CTGF mRNA levels were formed by cells expressing control or Amot130 (S175A) (Fig. 6 measured in cells following growth under conditions conducive A–C and Fig. S5F). Thus, in a cancer cell line with defective to low Hippo signaling (10% serum and low confluence) or high growth control, the inability of Amot130 to be phosphorylated Hippo signaling (10% serum and high confluence) (Fig. 5C and prevents it from inducing the inhibition of cell growth. Fig. S4D). The expression of wild-type Amot130 but not Amot130 (S175A) significantly reduced the level of CTGF transcription in Amot130 (S175A) Disrupts Growth Control During the Formation of cells with low Hippo signaling. Alternatively, cells with high levels Mammary Acini. The growth and differentiation of nontransformed of intercellular contacts showed modestly reduced CTGF levels MCF10A cells into hollow acini in Matrigel are highly sensi- upon wild-type Amot130 expression from presumably already low tive to Hippo signaling (14, 34, 35). The effects of expression of basal levels of YAP activity. However, Amot130 (S175A) ex- wild-type Amot130 and Amot130 (S175A) on this process were pression increased CTGF levels significantly above those of con- therefore compared (Fig. 6 D–F). After 1 d of growth, the sizes trol cells. This is consistent with it exerting a dominant-negative of cell clusters were nearly identical between all conditions (Fig. effect in cells with active Hippo signaling. Importantly, the silencing S6A). However, MCF10A cells expressing wild-type Amot130 of AIP4 dramatically reduced the ability of wild-type Amot130 formed significantly smaller clusters that stopped growing by day expression to inhibit CTGF levels, and it resulted in a synergistic 4(Fig. S6 B–D). Conversely, cells expressing Amot130 (S175A) increase of CTGF levels in cells expressing Amot130 (S175A) displayed a loss of growth inhibition, where colonies were sig- (Fig. 5D). Thus, AIP4 recruitment to Amot130 upon its phos- nificantly larger by days 4 and 8 versus control cells (Fig. 6 D phorylation by LATS1/2 is proposed to be a key mechanism and F and Fig. S6 B–D). Furthermore, unlike control cells, which whereby Hippo signaling inhibits YAP. formed hollow acini by day 14, cells expressing Amot130 (S175A) formed solid tumor-like structures (Fig. 6E). Taken together, the Amot130 (S175A) Lacks the Ability of Wild-Type Amot130 to Inhibit phosphorylation of Amot130 is strongly indicated to play a vital Cell Growth in Breast Cancer Cells. To measure the effects of ex- role in Hippo signaling involved in cell-growth arrest (Fig. 6G). pression of wild-type Amot130 and Amot130 (S175A) on contact inhibition, the rate of growth of MDA-MB-468 cells on plastic in Discussion serum-free medium or medium containing 10% serum that stably Although atypical cadherins Fat and Dachsous are central to expressed either protein or a control vector over 4 d was mea- Hippo activation in Drosophila, their nonessential role in murine sured by both cell counting and 3-(4,5-dimethylthiazol-2-yl)-5-(3- liver development has led to the suggestion that Amot proteins carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTS) are part of a replacement pathway in vertebrates (36), where cell-proliferation assay (Fig. S5 A–E). Cells expressing wild-type they are exclusively found. This concept is further extended by Amot130 grew at significantly slower rates in the presence or this study, showing that Amot130 phosphorylation by LATS1/2 absence of serum, whereas cells expressing Amot130 (S175A) is essential for Hippo signaling. This is found to be mediated grew similarly to cells expressing a control vector. Analysis of the by inducing Amot130 to bind and thereby couple AIP4 to the

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1308236110 Adler et al. Downloaded by guest on September 24, 2021 ubiquitination and degradation of YAP. This also results in the A 10 % Serum & Low Confluence B ubiquitination and stabilization of Amot130, which likely rein- 2.0 n.d. N=3 forces such antigrowth signaling. * * n.d. 10 % Serum Although serum components, such as LPA or S-1-P, have re- 1.6 * High Confluence cently been shown to repress Hippo signaling (2, 3, 37), this study ) 1.2 * YFP-Control + - - describes how serum starvation directly activates this pathway. CT 0.8 YFP-Amot130 - + - Here the levels of Amot130, but not AmotL1, are shown to di- ΔΔ ( YFP-Amot130 0.4 --+ rectly respond to serum starvation through phosphorylation by

mRNA Levels (S175A) Relative CTGF 0.0 LATS, which in turn is a critical event for Hippo-induced growth IB: Amot arrest. Future experiments examining other inhibitors of YAP, IB: LATS1 such as PP1 (38), glucagon, and epinephrine (2), for their effects Amot130 IB: pS127 YAP on Amot130 phosphorylation may further establish how this Amot130 / 1.0 1.0 0.1 0.1 event is central to Hippo signaling. GAPDH pS127 YAP / YAP 0.4 0.6 0.3 Amot80 The multifactorial effects of Amot130 on promoting the deg- IB: Amot Amot80 / IB: YAP radation of YAP (25) likely underlie a self-reinforcing process, GAPDH 1.0 1.0 0.9 0.7 where Amot130 both activates and transmits Hippo signaling. IB: GAPDH This process appears to initially require LATS1 to phosphorylate CFP-Control + - + - IB: GAPDH CFP-LATS1 - + - + shControl shAmot Day 1 A Day 1 Day 4 B 1.2 C 10 % Serum 10 % Serum n.d. N=4 Low Confluence High Confluence n.d. 0.8 n.d. N=3 N=3 1.2 n.d. * * * μ 2.0 Control 100 m 100μ m 0.4

) * Intensity / Field 0.8 1.5 Mean Integrated 0.0

CT * Control Amot130 Amot130 1.0 (S175A) ΔΔ ( 0.4 100μ m 100μ m Day 4 0.5 Amot130 C mRNA Levels

Relative CTGF n.d.

N=4 CELL BIOLOGY 1.6 ** 0.0 0.0 ** IB: Amot 0.8 100μ m 100μ m (S175A) Amot130 Intensity / Field IB: GAPDH Mean Integrated 0.0 Control Amot130 Amot130 YFP-Control + - - + - - D Day 8 E Day 14 (S175A) YFP-Amot130 - + - - + - Day 8

F ) YFP-Amot130 2 N=180 -- + --+ m 6000 *** (S175A) μ *** Control 100 m Control 20μ m 4000 D Serum Starved & Low Confluence *** 2.0 N=4 *** N=3 ** ** 2000 *** 6 1.6 Area / Acini ( μ 0 100μ m Mean Cross-Sectional ) μ Amot130 Amot130 20 m 1.2 4 IB: Amot

CT *** IB: GAPDH ΔΔ 0.8 ( ** 2 YFP-Control + - -

mRNA Levels 0.4 Relative CTGF 100μ m μ YFP-Amot130 - + - (S175A) (S175A) 20

Amot130 m 0.0 0 Amot130 YFP-Amot130 --+ IB: AIP4 (S175A) G Hippo Signaling Initiated Phosphorylated Amot130 IB: Amot 1 3 recruits AIP4 and 14-3-3 MST1/2 P 2 LATS1/2 14-3-3 IB: GAPDH LATS1/2 Phosphorylate P Amot130 Amot130 YFP-Control + - - + - - P S175 4 stabilization 127 P and YAP YAP Amot130 YFP-Amot130 - + - - + - S S175 induces the YAP Amot130 ub Amot130 degradation YFP-Amot130 AIP4 (S175A) --+ --+ Amot130 K481 of YAP resulting shControl shAIP4 ub in growth inhibition

Fig. 5. Phosphorylation of Amot130 at Ser-175 underlies its role in mediating Fig. 6. Phosphorylation of Amot130 at Ser-175 is essential for it to inhibit cell Hippo signaling to inhibit YAP. (A) Real-time quantitative PCR measurements growth. (A–C) MDA-MB-468 cells stably expressing YFP-tagged Amot130, of the levels of CTGF mRNA in MDA-MB-468 cells coinfected for 24 h with Amot130 (S175A), or control vector were seeded onto Matrigel and imaged lentivirus encoding combinations of Amot shRNA, CFP-tagged LATS1, or the after 1 and 4 d. (A–C) Representative bright-field stereo images (A)andplots indicated controls. (Lower) Immunoblots from paired cells validating Amot130- of the mean pixel integrated intensity values per field from four experiments selective silencing and CFP-tagged LATS1 expression with pixel intensity values (n = 4) (B and C)at(B) day 1 and (C) day 4 are presented. (D–F)MCF10Acells of Amot130 and Amot80 levels normalized to GAPDH. (B) Immunoblot of stably expressing proteins as in A were seeded onto Matrigel and grown for 14 phospho-Ser-127 (pS127) YAP, total YAP, and GAPDH from MDA-MB-468 cells d. (D)Bright-field stereo images of colonies at day 8 and (E) confocal fluo- expressing YFP-tagged Amot130, Amot130 (S175A), or control vector. (C)Real- rescence images of representative acini stained with Hoechst for nuclei at day time quantitative PCR measurements of CTGF mRNA levels in MDA-MB-468 14 of growth. (F) Plots of the mean cross-sectional area per acini (μm2)from cells described in B and harvested at low (Left)orhighconfluence (Right). three experiments each with 60 acini (n = 180) at day 8 are graphed. (Lower) (D) CTGF mRNA levels from MDA-MB-468 cells coinfected with lentivirus Immunoblot measuring YFP-tagged Amot130 or Amot130 (S175A) and expressing YFP-tagged Amot130, Amot130 (S175A), or control vector in GAPDH from cells extracted from Matrigel at day 8. (G) Model of the proposed combination with control shRNA (n = 4) or shRNA targeting AIP4 (n = 3), and mechanism for activation of Amot130 by serum deprivation and LATS resulting − cultured at low confluence in 0% serum for 24 h. Error bars represent ± SD. in the inhibition of YAP activity. Error bars represent ± SD. ***P < 1.0 × 10 60; ***P < 0.0001; **P < 0.01; *P < 0.05; n.d., no statistical difference. **P < 0.01; n.d., no statistical difference. (Unlabeled scale bars, 50 μm.)

Adler et al. PNAS Early Edition | 5of6 Downloaded by guest on September 24, 2021 Amot130 to promote its association with AIP4 that, in turn, ubiq- Materials and Methods uitinates and thereby enhances Amot130 levels. Amot130 binding Cell Culture, Treatment, and Assays. HEK 293T, MDA-MB-468, and MCF7 cells AIP4 also increases LATS1 signaling by preventing AIP4 from in- were cultured as previously described and used for immunoblot, immuno- stigating LATS1 degradation (25, 39). In parallel, Amot130 directly precipitation, membrane fractionation, RNA isolation/real-time PCR, confocal activates LATS1/2 (40). Both events by increasing LATS1/2 ac- ﬂuorescence imaging, cell accumulation, and MTS cell-proliferation experi- tivity would reinforce this positive feedback loop. The predicted ments. MCF10A cells were cultured in lrECM (Matrigel, 7.5 mg/mL or greater rapid accumulation of active Amot130 and LATS1/2 would also protein; BD Biosciences; 354230). Cell growth in three dimensions was carried ensure pervasive targeting of YAP for ubiquitination and de- out after plating 200 thousand MDA-MB-468 cells or 300 thousand MCF10A gradation by SCF-(β)-TRCP (9) and by AIP4. Consistent with cells onto Matrigel, and colonies or acini were imaged with a stereomicro- Amot130 having such effects, its expression is sufﬁcient to strongly scope or confocal microscope as previously described (24). Cells were serum- inhibit the growth of MCF10A cells during acini formation, whereas starved in all cases with Opti-MEM Reduced Serum Medium (Gibco). More the phosphorylation-incompetent mutant, Amot130 (S175A), information on these methods with cells, including infections, antibodies, induces a loss of growth control. reagents, plasmids, transfections, and detailed protocols, is provided in SI Further work exploring the relationships between the phos- Materials and Methods. phorylation and ubiquitination of Amot130 will likely also provide insights into the signaling leading to growth inhibition induced by In Vitro Kinase Assays. In vitro SPOT phosphorylation assays including peptide factors that control cell shape. Because Amot also binds apical sequences and immunoprecipitation in vitro kinase assays are described in polarity proteins (21) to mediate cross-talk with Hippo signaling detail in SI Materials and Methods. (20), the ability of polarity proteins to potentially induce Amot130 phosphorylation is increasingly attractive as a mechanism whereby ACKNOWLEDGMENTS. We thank Dannel McCollum for helpful discussions. it may integrate Hippo kinase activity with cell polarity. Further, HA-tagged Lys-0 ubiquitin was obtained through Addgene from T. Dawson the fact that Amot130 also binds and bundles actin preferentially (The Johns Hopkins University). Flag-tagged YAP2 and LATS1 constructs were obtained through Addgene from M. Sudol (Weis Center for Research). This when it is unphosphorylated suggests that it may coordinate such work was supported by National Institutes of Health/National Cancer Insti- signaling with actin dynamics that are critical for the entry of YAP tute Grants R01CA151765 (to C.D.W.) and R01NS078171 (to A.H.) and De- into the nucleus (15, 41). partment of Defense Grant W81XWH (to C.D.W.).

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