Stable MOB1 interaction with Hippo/MST is not essential for development and tissue growth control

The Hippo tumor suppressor pathway is essential for development and tissue growth control, encompassing a core cassette consisting of the Hippo (MST1/2), Warts (LATS1/2), and Tricornered (NDR1/2) kinases together with MOB1 as an important signaling adaptor. However, it remains unclear which regulatory interactions between MOB1 and the different Hippo core kinases coordinate development, tissue growth, and tumor suppression. Here, we report the crystal structure of the MOB1/NDR2 complex and define key MOB1 residues mediating MOB1’s differential binding to Hippo core kinases, thereby establishing MOB1 variants with selective loss-of-interaction. By studying these variants in human cancer cells and Drosophila, we uncovered that MOB1/Warts binding is essential for tumor suppression, tissue growth control, and development, while stable MOB1/Hippo binding is dispensable and MOB1/Trc binding alone is insufficient. Collectively, we decrypt molecularly, cell biologically, and genetically the importance of the diverse interactions of Hippo core kinases with the pivotal MOB1 signal transducer. The Hippo tumor suppressor pathway is essential for development and tissue growth control. Here the authors employ a multi-disciplinary approach to characterize the interactions of the three Hippo kinases with the signaling adaptor MOB1 and show how they differently affect development, tissue growth and tumor suppression.

T he Hippo pathway is vital for organ growth control and tissue homeostasis [1][2][3] , and its dysregulation has been linked to various human cancers 4 . Therefore, it is imperative to understand how major core components of the Hippo pathway are regulated in different cellular contexts in health and disease. The Hippo tumor suppressor pathway signals through three main levels: (i) a central core kinase cassette, (ii) downstream regulators of transcriptional programs, and (iii) diverse upstream regulators [1][2][3][4] . The Drosophila core cassette comprises the Warts (Wts) and Hippo (Hpo) kinases supported by the adaptor protein Mats 5,6 . The Hpo-Wts-Mats cassette acts together to inhibit Yorkie (Yki), which when uncontrolled promotes tissue overgrowth 7 . The Hippo core cassette is conserved from flies to humans, with human MST2, LATS1, and MOB1A compensating for Hpo, Wts, and Mats loss-of-function, respectively [8][9][10] . Thus, Drosophila is well suited for in vivo studies of essential molecular mechanisms in Hippo core signaling 5,11 .
MOB proteins are highly conserved amongst eukaryotes, constituting signal transducers in essential processes via their regulatory interactions with NDR/LATS 16 . In Drosophila, Mats (aka dMOB1 16 ) can function together with Wts and Hpo in Hippo signaling 10,23 . However, Mats also interacts genetically with Trc 24 , the fly counterpart of human NDR1/2 15 . Mammals express at least six MOBs, with MOB1A and MOB1B sharing 95% sequence identity 16 . MOB1A/B (aka MOB1) can function redundantly as regulators of LATS1/2 signaling 21, [25][26][27] , but are also required for NDR1/2 activation 16 , as an alternative branch 17,18 of the Hippo core cassette. Biochemical evidence suggests that MOB1 can associate with the highly conserved N-terminal regulatory domain (NTR) of NDR/LATS kinases to promote their activities 15,16,25 . MST1/2 phosphorylation of MOB1 can influence MOB1 binding to the NTR 19,20,22,28,29 , although it is yet to be determined whether this phosphorylation is required for NDR/LATS kinase activity in vivo. Nevertheless, MOB1 (Mats) is very likely acting as a central molecular switch in Hippo signaling 3,30 . However, we still lack structural and molecular insights on how the regulatory bindings of MOB1 to MST1/ 2, LATS1/2, or NDR1/2 are mediated and how MOB1 differentiates between these interactions. Two crystal structures of MOB1 bound to LATS1 were reported 19,20 , but the crystal structure of the MOB1/NDR complex has yet to be documented. The importance of stable MOB1 binding to MST1/2 (Hpo) is currently also debatable based on recently published biochemical data and the analysis of a chimeric conformation sensor 19,20,[28][29][30][31] . In general, the biological significance of MOB1 interactions with Hippo core kinases is not defined for tumor suppression, development, and tissue growth control (Supplementary Fig. 1a).
Here, we report our structural, biochemical, cell biological, and organismal studies of the importance of MOB1 interactions with Hippo core kinases. The overall crystal structure of MOB1 bound to NDR2 is very similar to the previously reported structures of MOB1/LATS1 19,20 . However, by comparing these crystal structures we could identify Asp63 as a key MOB1 residue that specifically mediates binding to LATS1. Thus, we characterized the interactions of Hippo core kinases with full-length MOB1 variants carrying specific point mutations, resulting in the discovery of MOB1 variants that are selectively impaired in their binding to MST1/2 (Hpo) or LATS1/2 (Wts) in human and fly cells. Using these MOB1 variants with selective loss-of-interaction, we found that a stable interaction of MOB1 with LATS1/2, but not with MST1/2, is essential for tumor suppressive properties of MOB1 in human cancer cells. By employing fly genetics, we discovered that the MOB1/Wts interaction is essential for development and tissue growth control, while stable MOB1 binding to Hpo is dispensable. Taken together, our study decrypts the nature and functional importance of the diverse interactions of Hippo core kinases with the central MOB1 signaling adaptor.

Results
Crystal structure of MOB1 bound to the NTR of human NDR2. To delineate the interaction of MOB1 with NDR2 on the atomic level, we determined the crystal structure of the MOB1/NDR2 complex at 2.1 Å using purified MOB1 (residues  and the NTR of NDR2 (residues 25-88) (Fig. 1a-c and Table 1). The structure of MOB1 adopts a globular shape consisting of nine α-helices (α1-α9) and two β-strands (Fig. 1a), as reported for unbound human MOB1 19,32 , frog MOB1 33 , and yeast Mob1p 34 . The overall structure of the MOB1/NDR2 complex is similar to the reported MOB1/LATS1 complex 19,20 , since NDR2 binds to MOB1 in a V-shaped structure composed of two antiparallel α-helices (Fig. 1a-c). In agreement with biochemical studies 15,17,25 , highly conserved positively charged residues of NDR2 bond with negatively charged electrostatic surfaces of MOB1 (Fig. 1b, c).
Taken together, we demonstrate in Figs. 1 and 2 that MOB1 relies on different residues to bind to NDR2 and LATS1. MST1/2 phosphorylation of MOB1 can play a significant role in modulating in vitro the diverse binding affinities of MOB1 to the NTRs of NDR/LATS kinases. MOB1/NDR2 complex formation is dramatically increased by prior MST1/2 phosphorylation of MOB1, while the MOB1/LATS1 interaction appears to be fully dependent on MST1/2 phosphorylation of MOB1. In this regard, a single substitution of Val647 to Tyr of LATS1 (or Val610 of LATS2) is sufficient to support MOB1/LATS complex formation independent of MOB1 phosphorylation. More specifically, LATS1(V647Y) and LATS2(V610Y) bound to nonphospho and phospho-MOB1 with much increased affinities as observed for LATS1 and LATS2 wild-type, hence indicating that a single substitution in LATS1 or LATS2 can switch the binding mode of LATS kinases. Noteworthy, these conclusions are solely based on our ITC assays ( Fig. 2 and Supplementary Fig. 3), hence the in vivo relevance has yet to be determined. In this regard, it has been documented that unphosphorylated MOB1 can bind to a LATS1 fragment in vitro 19,20 . Therefore, more research is needed to decipher the reason(s) for this discrepancy. Possibly our ITC assay requires higher concentrations to detect lower affinity interactions, or ITC assays with higher affinity kinase fragments are necessary. Certainly, identical kinase fragments will need to be tested to allow a proper comparison of our findings ( Fig. 2 and Supplementary Fig. 3) with previous studies 19,20 , but most importantly, the in vivo implications of MST1/2 (Hpo) phosphorylation of MOB1 need to be delineated in future studies. Fig. 1 The crystal structure of the MOB1/NDR2 complex reveals phosphorylation-independent interactions of MOB1 with NDR2 through conserved interfaces. a Overall crystal structure of the complex between human MOB1A (residues 33-216) and human NDR2 (residues 25-88). MOB1 and NDR2 are colored in magenta and green, respectively. The Zinc associated with MOB1 is indicated in gray. Secondary structure elements are highlighted. b The negatively charged surface of MOB1 recognizes positively charged residues on NDR2. The electrostatic surface potential of MOB1 is shown (red: negatively charged residues; blue: positively charged residues). NDR2 is shown as a ribbon representation colored in green. c The MOB1/NDR2 binding surfaces are highly conserved. The residues of MOB1 are gradually colored according to conservation scores (dark red: most conserved residues; dark cyan: least conserved residues). NDR2 is shown as a ribbon representation colored in green. d Structure based sequence alignment of MOB1 from indicated species. MOB1 residues mediating interactions with NDR2 are marked with brown (main-chain hydrogen bonds), green (side-chain hydrogen bonds), and magenta circles (van der Waals interactions). α helices are painted with cyan background. Thr12 and Thr35 phosphorylation sites are indicated by red arrows. e, f The interaction interfaces of MOB1 with the α1 (residues 25-56) and α2 (residues 61-84) helices of NDR2. MOB1 and NDR2 and their key interacting residues are shown in pink and green, respectively. Dashed lines represent hydrogen bonds. Side-chain nitrogen, oxygen, and sulfur atoms of MOB1 and NDR2 residues are colored in blue, red and gold, respectively. The hydrogen bond network connecting the α1 and α2 helices of NDR2 is also illustrated in f.
i for the intensity (I) of observation i of reflection h. R factor = Σ||F obs |−|F calc ||/Σ|F obs |, where F obs and F calc are the observed and calculated structure factors, respectively. R free = R factor calculated using 5% of the reflection data chosen randomly and omitted from the start of refinement. Data for the highest resolution shell are shown in parentheses Defining MOB1 variants with selective loss-of-interactions. To empower the translation of our structural and biochemical findings into studies of human cells and flies, we studied the interactions of full-length human MOB1 variants with human/fly Hippo core kinases ( Fig. 3    Thus, together with our structural data (Figs. 1 and 2) these findings indicate that Asp63 of MOB1 plays a specific role in MOB1 binding to fly and human LATS kinases.
In the hope to generate an alternative MOB1 variant displaying selective loss-of-interaction with MST1/2 and Hpo, we considered modifications of the MOB1 residues Lys153 and Arg154 as promising candidates due to their central roles in the phosphothreonine binding interface supporting MOB1/MST1 and MOB1/ MST2 interactions 20,28 . However, MOB1(K153A/R154A) and MOB1(K153E/R154E) displayed defective interactions with fulllength wild-type MST2 and LATS2, while NDR1 binding was intact ( Supplementary Fig. 9). MOB1(K153A/R154A) and MOB1 (K153E/R154E) were also defective in Wts and Hpo binding, while they bound to Trc in insect cells ( Supplementary Fig. 9). Thus, as suggested previously 37 , as central P0 phosphate coordinating residues 28, 37 , Lys153 and Arg154 are likely to represent the core of a more general phospho-serine/threonine binding domain of MOB1. This conclusion is reinforced by the finding that the region surrounding Lys153 and Arg154 of MOB1 also supports Praja2 binding 38 . As a result, we concluded that Lys153 and Arg154 modifications of MOB1 are not suitable to develop MOB1 variants with selective loss-of-interaction with MST1/2 and Hippo.
To complete the biochemical characterization, we performed additional experiments. First, we investigated the importance of Asp63 and Lys104/Lys105 of MOB1 for in vitro binding to Hippo core kinases using gel filtration chromatography, revealing that recombinant full-length MOB1(D63V) and MOB1(K104E/ K104E) displayed the same binding patterns as observed for full-length proteins expressed in human and fly cells ( Fig. 3c and Supplementary Fig. 10). Second, we performed ITC assays to determine the dissociation constant of full-length MOB1(K104E/ K105E) with the NTRs of NDR1, NDR2, LATS1, and LATS2 ( Supplementary Fig. 11). Unphosphorylated MOB1(K104E/ K105E) bound to all four NTRs comparable to unphosphorylated wild-type MOB1 (compare Fig. 2 D63V) is not a consequence of altered Thr12/Thr35 phosphorylation, but rather caused by the substitution of a key residue that is essential for MOB1/LATS complex formation. Our data ( Fig. 3 and Supplementary Figs. 7-13) further argue that a stable interaction of MST1/2 (Hpo) with MOB1 is not required for MOB1 phosphorylation by MST1/2 (Hpo).
Taken together, we discovered that distinct MOB1 residues mediate the interactions with the different mammalian and fly Hippo core kinases. Specifically, Asp63 and Lys104/Lys105 of MOB1 represent key residues mediating the differential binding properties of MOB1 with LATS1/2 (Wts) and MST1/2 (Hpo), respectively (Fig. 3d).

Testing of MOB1 variants in anchorage-independent growth.
To define which MOB1 interactions with Hippo core kinases are necessary for tumor suppression, we engineered pools of MCF-7 human breast cancer cells stably expressing either empty vector (EV), HA-MOB1 wild-type (wt), HA-MOB1(D63V), HA-MOB1 (K104E/K105E), or HA-MOB1(D63V/K104E/K105E) (Fig. 4a). Then, we determined proliferation and colony formation in twodimensional (2D) culture conditions. Specifically, we measured proliferation using IncuCyte live cell analysis technology (Fig. 4b) and performed colony formation assays (Fig. 4c, d) to determine Fig. 2 MOB1 binds differently to the NTR domains of NDR2 and LATS1 through key residues. a Structure based sequence alignment of the conserved Nterminal regulatory domain of NDR/LATS kinases that is required for MOB1 binding. NDR2 residues mediating interactions with MOB1 are marked with red circles. Residues mediating intramolecular interactions of NDR2 are denoted by blue triangles. Residues conserved from yeast to humans are highlighted in green, while residues conserved in at least seven of the conserved NDR/LATS family members are marked in cyan. α helices are indicated with gray rods. b Asp63 of MOB1 is involved in LATS1 binding, but does not contribute to the interaction of MOB1 with NDR2. MOB1 is shown in yellow, while NDR2 and LATS1 are indicated in green and cyan, respectively. Top panel, Phe31 of NDR2 does not interact with Asp63 of MOB1. Bottom panel, His646 of LATS1 forms a hydrogen bond with Asp63 of MOB1. c-f Isothermal titration calorimetry (ITC) assays measuring the dissociation constant (K d ) of indicated nonphosphorylated full-length MOB1 or MST2-phosphorylated MOB1 (phospho-MOB1) with wild-type and mutant NDR2  or LATS1 (640-703) variants. MOB1/NDR2 complex formation was dramatically increased by prior phosphorylation of MOB1 c. ITC measurements could not detect any interaction between LATS1 wild-type and non-phosphorylated MOB1, while phospho-MOB1 bound to LATS1 d. The NDR2(Y32V) mutant did not associate with non-phosphorylated MOB1, but bound to phospho-MOB1, although with a 20-fold decreased binding affinity compared to wild-type NDR2 (e). The mutation of Val647 to Tyr of LATS1 enabled LATS1 to bind to non-phosphorylated MOB1, and LATS1(V647Y) displayed a 5-fold increased binding affinity for phospho-MOB1 compared to wild-type LATS1 (f) cell survival based on the ability of single cells to grow into colonies 40 . Expression of all MOB1 variants resulted in decreased proliferation in 2D compared to controls (Fig. 4b). Likewise, except for MOB1(D63V/K104E/K105E), our MOB1 variants reduced colony formation (Fig. 4c, d). Transient expression of our MOB1 variants in HCT116 colon cancer cells also diminished proliferation and colony formation in 2D ( Supplementary  Fig. 14). These data show that MOB1(D63V) and MOB1(K104E/ K105E) suppress proliferation and colony formation similarly to MOB1(wt), suggesting that the interactions of MOB1 with LATS1/2 and MST1/2 are dispensable. Considering that MOB1 (D63V) and MOB1(K104E/K105E) still bind to NDR1/2 ( Fig. 3c and Supplementary Fig. 6) and that MOB1(K104E/K105E) is phosphorylated on Thr12 and Thr35 in MCF-7 cells comparable to wild-type MOB1 ( Supplementary Fig. 15), we are therefore tempted to conclude that MOB1 binding to NDR1/2 can be  sufficient to at least in part suppress cancer-related features in human cancer cells grown in 2D. Next, we performed anchorage-independent growth assays (Fig. 4e, f), a more stringent method to determine malignant transformation in three-dimensional (3D) tissue culture 41 . In contrast to our 2D observations (Fig. 4b-d), MOB1(wt) or MOB1 (K104E/K105E), but not MOB1(D63V) or MOB1(D63V/K104E/ K105E), significantly suppressed anchorage-independent growth in 3D (Fig. 4e, f and Supplementary Fig. 14). This suggests that MOB1 interactions with MST1/2 are dispensable, while MOB1 binding to LATS1/2 is important and MOB1 binding to NDR1/2 alone is insufficient in this 3D setting. In general, our anchorageindependent growth data of human cancer cells (Fig. 4e, f) mirrored our fly genetics discoveries (see Fig. 5 and 6 and Supplementary Fig. 16 below), suggesting that tissue culture experiments performed under more physiological conditions can reflect in vivo tissue overgrowth experiments.
MOB1/Hpo in fly development and tissue growth control. To study our MOB1 variants in a complex multicellular organism, we generated and characterized transgenic flies that ubiquitously expressed our myc-tagged MOB1 versions in a mats mutant background (Figs. 5 and 6 and Supplementary Fig. 16). As human MOB1 expression can rescue mats mutants 10,16 , this allowed us to determine the functional significance of altering MOB1 binding in vivo. Using PhiC31-mediated recombination we integrated our MOB1 variants at the same chromosomal location (89E11 on chromosome 3) under control of the ubiquitous ubiquitin-63E promoter (ubi > MOB1, Supplementary Fig. 16a). Western blotting of whole flies confirmed similar expression of myc-tagged MOB1 variants (Supplementary Fig. 16b). We then tested which MOB1 transgene can rescue the larval lethality of mats deficient flies 10 . As expected, wild-type MOB1 expression rescued the lethality of a null mats trans-heteroallelic combination (mats roo /mats e235 , Supplementary Fig. 16c). Likewise, mats deficient animals expressing MOB1(K104E/K105E) were viable and fertile ( Supplementary Fig. 16c), suggesting that stable MOB1/Hpo binding is dispensable for normal fly development. In contrast, neither MOB1(D63V) nor MOB1 (D63V/K104E/K105E) rescued mats mutants ( Supplementary  Fig. 16c), showing that MOB1/Wts complex formation is essential for normal development, while MOB1 binding to Trc alone is insufficient to promote normal development.
To test the rescue effect of our MOB1 mutations on the mats tissue overgrowth phenotype, we generated mats mutant clones in the head using the eyFLP/FRT system 42 . Expression of wild-type MOB1 and MOB1(K104E/K105E) fully rescued the overgrown and misshapen head phenotype of eyFLP mats animals (compare Fig. 5b, d with Fig. 5a, f). In contrast, expression of MOB1(D63V) or MOB1(D63V/K104E/K105E) only partially suppressed the mats overgrowth phenotype (compare Fig. 5c, e with Fig. 5a, f). Thus, stable MOB1 binding to Hpo is dispensable for tissue growth control, while MOB1/Wts complex formation is necessary.
Finally, we tested the effect of our MOB1 transgenes on Yki transcriptional activity by examining the levels of Expanded (Ex), a well-characterized Yki transcriptional target 43 . We generated mutant clones for mats in wing imaginal disks (the larval precursors to the adult wing) using the FLP/FRT system under control of the heat shock promoter (Fig. 6). While mats clones displayed a robust increase in Ex expression (Fig. 6a-e) expression of either wild-type MOB1 or MOB1(K104E/K105E) restored Ex levels to control levels ( Fig. 6f-j, p-t). In contrast, Ex levels (and therefore Yki activity) were still strongly upregulated when MOB1(D63V) or MOB1(D63V/K104E/K105E) were expressed in mats clones (Fig. 6k-o, u-y). Thus, in full agreement with the animal viability ( Supplementary Fig. 16) and head overgrowth data (Fig. 5), the interaction of MOB1 with Wts is required to repress Yki activity, while the MOB1/Hpo interaction is dispensable and MOB1/Trc complex formation alone is insufficient for a complete rescue.
As previously observed 10 , mats clones were usually small (Fig. 6a). Mats mutant clones were rarely recovered in the wing pouch, likely because to their tendency to delaminate due to excessive overgrowth. Interestingly, although Ex levels were strongly upregulated in both MOB1(D63V) or MOB1(D63V/ K104E/K105E) expressing clones, MOB1(D63V) expressing clones were larger and survived readily in the pouch (Fig. 6k), in contrast to MOB1(D63V/K104E/K105E) expressing clones, which were more similar to mats clones not expressing MOB1 (compare Fig. 6a, u). In this regard, we also noted that adult heads expressing MOB1(D63V/K104E/K105E) were more severely affected than adult heads expressing MOB1(D63V) in mats null tissue (compare the more rippled appearance of Fig. 5c, e). These findings collectively suggest that when MOB1 activity is weakened by loss of the MOB1/Wts interaction, MOB1 function is further compromised by loss of the Hpo/MOB1 interaction, while disruption of the Hpo/MOB1 interaction alone does not affect MOB1 function.

Discussion
Despite extensive progress in elucidating Hippo growth control signaling, studies comparing the biological significance of the regulatory interactions of MOB1 with Hippo core kinases have remained elusive. This point is crucial, since loss-of-function of MOB1 in flies and mice causes the most severe phenotypes of Hippo core cassette components 10,23,26,27 , indicating that MOB1 represents a multipurpose hub in Hippo core signaling. By combining genetics, structure, molecular, and cell biology our present study addresses this pressing issue. We uncovered key mechanisms promoting selective binding of MOB1 to Hippo core kinases in mammalian and fly cells. Precisely, we discovered that Asp63 of MOB1 is indispensable for interacting with LATS1/2 and Wts, while Lys104/Lys105 of MOB1 are essential for stable complex formation with MST1/2 and Hpo. Thus, MOB1 can differentiate between interactions in the Hippo core cassette, thereby most likely enabling MOB1 to regulate the specificity and amplitude of Hippo core kinase signaling. While our biochemical and molecular data regarding Asp63 of MOB1 are further supported by a comparison of crystal structures of MOB1/NDR2 and MOB1/LATS2 complexes, we can currently only speculate on the structural level concerning Lys104/Lys105 of MOB1 and stable complex formation with MST1/2 (Hpo). Based on available structural data 28 one can draw the conclusion that Pro106 of MOB1 significantly contributes to the MST1 binding surface. Thus, we are tempted to speculate that modifications of Lys104/Lys105 of MOB1 impact the neighboring Pro106 and thereby impair stable MOB1/MST1 complex formation. Possibly, the positively charged Lys104/Lys105 residues of MOB1 further bond with the negatively charged phosphorylated Thr residues on MST1/2 (Hpo). In this regard, the available crystal structures 20, 28 of MOB1/MST1 and MOB1/MST2 complexes cover only a fraction of the possible interactions between MOB1 and different phospho-threonine residues on MST1/2. More specifically, these structures cover MOB1 bound to phosphorylated Thr353 and Thr367 of MST1 and phosphorylated Thr378 of MST2, while MOB1 binding to phosphorylated Thr329, Thr340, Thr380, and Thr387 of MST1 and phosphorylated Thr349, Thr356, and Thr364 of MST2 have been reported 20,28 . The MOB1/MST1 and MOB1/MST2 crystal structures 20, 28 consistently highlighted Lys153 and R154 of MOB1 as central P0 phosphate coordinating residues of the phospho-threonine on MST1/2. Thus, we also tested the consequences of Lys153/R154 of MOB1, hoping to define an alternative MOB1 mutant that displays selective loss-of-interaction with human MST1/2 and fly Hippo. However, the testing of MOB1(K153A/R154A) and MOB1(K153E/R154E) revealed that Lys153/Arg154 of MOB1 are important for MST2 as well as LATS2 binding, supporting the previous notion that Lys153/Arg154 of MOB1 are likely to represent the core of a more general phospho-serine/threonine binding domain 37 . This interpretation is further supported by the finding that Lys153/Arg154 of MOB1 are part of the Praja2 binding region 38 .
Our data indicate that MOB1 binding to the NTRs of NDR1/2 vs. LATS1/2 also differs quantitatively. By determining interaction affinities, we observed an interaction of non-phosphorylated MOB1 with NDR2, while binding to LATS1 was not detectable. Phospho-MOB1 bound to LATS1, but interacted with NDR2 at a much higher affinity. These findings suggest that MOB1 generally displays a significantly higher affinity for NDR2, hence MOB1mediated Hippo signaling may preferentially signal through the NDR kinase branch, although our biological data indicate that MOB1/NDR (Trc) complex formation alone is insufficient to support development and normal tissue growth control. In this regard, His646 and Val647 of LATS1 (see this study), and possibly selective inhibitory MOB2 binding to the NTR of NDR1/2 44 , are promising candidates for the fine-tuning of MOB1-mediated signaling. Consequently, future studies are warranted to address these possibilities. In particular, crystal structures of full-length NDR kinases bound to full-length MOB1 in its nonphosphorylated vs. phosphorylated state will help to further our understanding with regard to the recently proposed autoinhibition model for MOB1 binding 19,20 . Furthermore, the in vivo importance of MST1/2 (Hpo) mediated phosphorylation of MOB1 (Mats) needs to be deciphered. In this regard, our conclusions regarding MOB1 phosphorylation are currently based on in vitro experiments, which must be cautiously interpreted regarding in vivo implications.
By discovering MOB1 variants displaying selective loss-ofinteractions and decrypting the biological significance of regulatory interactions of MOB1 in Hippo core signaling, we believe that our study helps to settle the recent controversy 3,19,20,30,31 regarding the importance of MOB1 binding to MST1/2 (Hpo). Ni et al. previously concluded that stable MST2 binding to MOB1 functions as an important step in activating the MST1/2-LATS1/2 kinase cascade 20 . However, no biological functions were addressed 20 . In this regard, using a chimeric sensor to measure Wts conformation in fly tissues, Vrabioiu and Struhl 31 found that MOB1 can act as a Hpo-independent activator of Wts, hence contrasting the model proposed by Ni et al. 20 . Manning and Harvey 30 proposed a unifying model, wherein MOB1 acts before and after Hpo-mediated phosphorylation of Wts and MOB1. However, in support of Luo and colleagues 20 , the Sicheri and Gingras laboratories recently showed that ternary MST1/2-MOB1-LATS1/2 (NDR1/2) complex formation is important, at least when tested in vitro 28,29 . Conversely, our study rather supports the model drawn by Vrabioiu and Struhl 31 , namely that a ternary complex is not required for MOB1/LATS1 activation in vivo. Therefore, it is currently difficult to reconcile all published data into one general model. Nonetheless, we are proposing an updated four-step model ( Supplementary Fig. 17) attempting to consolidate these models 20, 30, 31 with our discoveries reported here and other important biochemical data 19,21,22,28,29,35,36 . First, activated Hpo (MST1/2) phosphorylates MOB1 to release MOB1 from an auto-inhibitory conformation 19,20,22 . Significantly, this first step does not seem to require formation of a stable Hpo/MOB1 complex (see this study), suggesting that MST1/2 phosphorylation of MOB1 only requires a brief transient kinase-substrate interaction. However, we currently do not understand how MOB1 (Mats) phosphorylation actually fits into the regulation of Hippo core kinase signaling in vivo. Second, MOB1 binds to Wts (LATS1/2) to "open up" Wts 31 . In this second step the formation of a stable MOB1/Wts complex is essential (see this study and refs. 21,22,35,36 ). Third, "open" LATS1/2 is phosphorylated by MST1/2 in the C-terminal hydrophobic motif (HM) 20,21 . This third step can occur without formation of a stable ternary MST1/2-MOB1-LATS1/2 complex, as proposed by the MST1/2-binding deficient K104E/ K105E mutant characterized in this study. Fourth, HMphosphorylated LATS1/2 autophosphorylates on the activation loop 20, 21 , a step that can occur independent of MOB1 binding to LATS1/2 21 . Intriguingly, the MST1/2-MOB1-NDR1/2 signaling model is very similar, but different 17,45 , since MOB1 binding to NDR1/2 can occur independently of MOB1 phosphorylation (see this study). Even more importantly, it is crucial to note that our proposed model (Supplementary Fig. 17) is mainly supported by in vitro experiments.
Our study together with the report by Vrabioiu and Struhl 31 would argue that the MOB1/Hippo interaction is dispensable for tissue growth control by Hippo signaling. But we strongly caution from drawing a broad and general conclusion from these studies, since we cannot rule out the possibility that the MOB1/Hippo interaction is only dispensable in selective aspects of tissue growth control. Thus, our model ( Supplementary Fig. 17) may exemplify a mechanism for a switch-like activation of NDR/LATS kinases in specific biological contexts (for example, to ensure abrupt termination of growth responses), consequently being limited to specific biological settings. As a result, much more future research is warranted to further dissect these subtle, but important, differences in the activation mechanisms of LATS1/2 vs. NDR1/2 kinases in specific physiological contexts.
Surprisingly, our study further revealed that stable MOB1/Hpo interaction is dispensable for development, tissue growth control and suppression of Yki activity. We also discovered that the MOB1/Trc interaction alone is insufficient to normally support these processes, although it can be sufficient to decrease proliferation of human cancer cells. The MOB1/Wts interaction is essential for development, tissue growth control, and Yki regulation in Drosophila, but it can be dispensable for some tumor suppressive properties of MOB1 in human cells. Therefore, our study significantly advances our understanding of the biological importance of the regulatory interactions of MOB1 with Hippo core kinases, in addition to providing structural and molecular insights into the differential binding of MOB1 to Hippo core kinases.
In the course of our in vivo studies we noted further interesting aspects. While disruption of the stable MOB1/Hpo interaction alone did not have a detectable effect on MOB1 function (see K104E/K105E mutant), loss of the MOB1/Hpo interaction could affect MOB1 function in the context of disrupted MOB1/Wts interaction (see D63V/K104E/K105E mutant). This is illustrated by the observations that, in the eye overgrowth assay, D63V/K104E/K105E mutant tissues were noticeably more overgrown than in K104E/K105E mutants. In the wing clone experiments, only D63V/K104E/K105E mutants displayed a high frequency of clone delamination and loss in the wing pouch, indicative of a strong overgrowth phenotype and similar to the full mats mutant phenotype. Thus, in the context of a weakened MOB1 function (i.e. through disrupted MOB1/Wts binding), loss of the MOB1/Hpo interaction can further reduce the in vivo function of MOB1.
Another interesting aspect based on our in vivo work is that, although D63V/K104E/K105E rescued flies showed a strong eye overgrowth phenotype and Ex upregulation, these phenotypes were still markedly weaker than the full mats loss-of-function phenotypes, suggesting that D63V/K104E/K105E can at least partially rescue the mats mutant phenotype. This could be due to several reasons. First, as the MOB1/Trc interaction is not disrupted in the D63V/K104E/K105E mutant, this mutant may partially rescue the mats mutant phenotype through MOB1mediated Trc regulation. Indeed, Trc has been proposed to function partially redundantly with Wts, at least in some contexts 46 . Second, although our data indicate that the D63V/K104E/ K105E mutant is severely impaired in its ability to bind to Wts, it is nevertheless possible that some low-affinity Wts binding activity might remain, which possibly is sufficient to partly rescue the mats mutant phenotype. Third, we cannot exclude the possibility that other factors, besides the Hippo core kinases, may facilitate MOB1-mediated Hippo signaling. In this regard, other binding partners of MOB1 are worth considering, although the known MOB1 binders Praja2 38 and Dock8 47 are not conserved in flies. Therefore, future experiments are warranted to address this issue, taking into account recent interactome screens 29,48,49 .
Taken together, our study establishes a major foundation of MOB1-mediated Hippo core signaling. The Hippo pathway is essential for tissue growth control and homeostasis 1-3 , and its dysregulation has been linked to various human cancers 4 . Therefore, our study through providing notable insights into how MOB1 differentiates between Hippo core kinase defines a framework for how these different interactions may function in different cellular contexts in health and disease. We discovered that selected regulatory interactions of MOB1 are essential for development, tissue growth control, and Yki regulation, hence establishing MOB1 as the central hub of Hippo core signaling, besides providing structural and molecular insights into the Hippo core cassette, and establishing key research tools for future studies of the regulatory interactions of MOB1 in diverse diseaserelevant settings.
Structure refinement and molecular graphics. The crystal belonged to the space group P2 1 2 1 2 1 , and contained two MOB1A (33-216)-NDR2 (25-88) complexes in each asymmetric unit. MOB1 (33-216) molecules in the complex were located by the molecular replacement method with the CCP4 program Phaser 51, 52 using the published 32 structure of MOB1A (33-216) alone (PDB code: 1PI1) as the searching model. The model of the NDR2 (25-88) fragment was built manually with Coot 53 . After refinement by the CCP4 program REFMAC5 54 , the R work and R free factors were 15.3 and 24.4%, respectively. The quality of the final structure was verified by the CCP4 program PROCHECK 51 , revealing good stereochemistry according to the Ramachandran plot (99.0, 0.6, and 0.4% for favored, allowed, and disallowed regions, respectively). The final models include residues 33-212 and 33-216 of MOB1A for chains A and B, respectively, and residues 25-85 and 25-87 of NDR2 for chains C and D, respectively. All figures displaying protein structures were generated with PyMOL (http://www.pymol.org). Sequence conservation of MOB1 mapped onto the surface of its crystal structure was generated by the ConSurf server 55 .
Isothermal titration calorimetry assays. ITC experiments were performed at 25°C using an ITC200 system (MicroCal) and the following buffer system: 25 mM HEPES pH 7.5, 200 mM NaCl, and 1 mM EDTA. Proteins were centrifuged and degassed before experiments. Typically,~240 μM MOB1A was injected 20 times in 2 μl aliquots into a 200 μl sample containing~24 μM of NDR1, NDR2, LATS1, or LATS2 proteins in the sample cell. By varying the stoichiometry (n), the enthalpy for the reaction (ΔH), and the association constant (K a ), data were fitted with the non-linear least-square method using a single-site binding model with Origin for ITC version 7.0 (MicroCal).
Kinase assays. Human full-length MOB1A cDNA variants were inserted into the pMal-c2 vector (New England BioLabs) using BamHI and XhoI/SalI sites to generate pMal-MOB1A plasmids, which can express MOB1A N-terminally tagged by the maltose binding protein. Recombinant Mal-MOB1A proteins were expressed in E. coli BL21(DE3) at 30°C and purified using amylose resin (E8021, New England BioLabs). Briefly, single-bacteria colonies containing pMal-MOB1A were grown overnight at 37°C, followed by 1:10 dilution before IPTG was added at 100 µM and bacteria were incubated for another 3 h at 30°C. Subsequently, cultures were centrifuged at 1400×g for 10 min, followed by addition of Mal lysis buffer (20 mM Tris pH 7.5, 1 mM EDTA, 200 mM NaCl) and sonication at 70% amplitude (model:VCx 130, Sonics Vibra Cell). Lysates were centrifuged and the supernatant was clarified by filtration (40 µm cell strainer) before addition of amylose-resin and overnight incubation at 4°C. The following day, the protein-amylose resin mix was washed excessively with Mal lysis buffer, followed by elution of Mal-MOB1A proteins in 20 mM Tris pH 7.5 buffer containing 10 mM maltose. Recombinant Mal-MOB1A proteins were dialyzed in 20 mM Tris pH 7.5 containing 10% (v/v) glycerol, followed by quantification using SDS-PAGE and Coomassie staining. Recombinant full-length wild-type GST-MST1 (M9697) and GST-MST2 (S6573) were from Sigma. To produce immunopurified full-length wild-type HA-tagged Hpo kinase, Drosophila S2R + cells were transfected with pAW_HA-Hpo and processed for immunoprecipitation (IP) using anti-HA antibody and standard IP conditions as defined above. Immunopurified Hpo proteins were washed twice with MST1/2 (Hpo) kinase buffer (5 mM Tris pH 7.5, 2.5 mM beta-glycerophosphate, 1 mM EGTA, 1 mM Na 3 VO 4 , 4 mM MgCl 2 , 0.1 mM DTT), before kinase reactions were performed as follows: per reaction 200 ng of Mal-MOB1A proteins were incubated at 30°C for 30 min in 20 μl of kinase reaction buffer (5 mM Tris pH 7.5, 100 μM ATP, 2.5 mM beta-glycerophosphate, 1 mM EGTA, 1 mM Na 3 VO 4 , 4 mM MgCl 2 , 0.1 mM DTT) in the absence or presence of GST-MST1/2 (50 ng per reaction) or immunopurified Hpo. The reactions were stopped by the addition of Laemmli buffer, before proteins were separated by SDS-PAGE, followed by immunoblotting as outlined above.
Generation of stable cell line pools. To generate stable lentiviral cell pools using pLEX_HA-MOB1 plasmids, 8 × 10 E6 of the HEK293T packaging cells were transfected with 2.4 μg of pMD.G, 0.8 μg of p8.91, and 4.8 μg of pLEX plasmids using Lipofectamine 2000 (11668, Invitrogen) as recommended by the manufacturer. Tissue culture supernatants were harvested 24 h later, passed through a 0.45-μm filter and added to the target cell lines in the presence of 1 μg ml −1 polybrene. Infected cells were selected by growth in the presence of 2.5 μg ml −1 puromycin. Stable pools (uncloned mass culture) of cells were maintained in DMEM supplemented with 10% FBS and 0.6 μg ml −1 puromycin. Retroviral pools (uncloned mass culture) were generated using pCMV-R-retro based plasmids and maintained in the presence of G418 as described elsewhere 58,61 .
Proliferation and colony formation assays. HCT116 transiently transfected with pcDNA3-based plasmids were seeded 24 h post-transfection, or stable MCF-7 cell line pools were analyzed. Media were replenished every 72 h during the duration of experiments. For cell proliferation analysis, cells were seeded at defined densities (5 × 10 E4 cells per well) in triplicates in 12-well plates, followed by non-invasive IncuCyte live cell imaging (Essen BioScience) to measure the kinetics of cell growth/proliferation based on area (confluence) metrics. Phase-contrast images were continuously collected on IncuCyte ZOOM (Essen BioScience) for at least one week. A specific processing definition (Phase-contrast processing module) was applied to count objects (cells) for the duration of the assay. The Phase object area was expressed as relative cell confluency for each well at each time point and subsequently exported into GraphPad Prism Software for final analysis. To evaluate colony formation, 1,000 cells were seeded per well (6-well format). After 8-12 days, colonies were fixed with methanol/acidic acid (3:1) for 5 min at room temperature, stained with 0.5% (w/v) crystal violet for 15 min at room temperature, washed with water and finally scanned using the G:BOX HR gel documentation system (Syngene). Colonies composed of at least 50 cells were score as positive.
Soft agar assays for anchorage-independent growth. After trypsinization, cells were passed 4-5 times through a 21 G syringe, before 1 × 10 E4 cells were resuspended in complete medium (DMEM containing 10% FBS and appropriate antibiotics) with 0.6% agarose (16520050, Thermo Fisher Scientific), and subsequently cultured in wells (6-well format) underlaid by a layer of 1% agarose in complete medium and overlaid with complete medium without agarose. Top layer media were replenished every 72 h. After three weeks, colonies were stained with 2.5 mg ml −1 thiazolyl blue tetrazolium blue (MTT, Sigma), scanned, and quantified. Cell clusters of at least 50 cells were scored as colonies.
Drosophila melanogaster genetics. All flies were maintained at 25°C unless otherwise stated. The mats roo and mats e235 fly strains, carrying mats null alleles, have been reported 10 . The mats gene is located on chromosome 3 at 94A12 (FlyBase ID: FBgn0038965). The PhiC31 integrase-mediated site-specific recombination method was used to express human MOB1 variants from one identical chromosomal location (PBac{y[ + ]-attP-9A}VK00027 integrated at 89E11 on chromosome 3) (see also Supplementary Fig. 16a). Briefly, N-terminally myctagged MOB1 cDNA variants were cloned into the pKC26w-pUbiq vector that allows expression of the cloned fragments under the ubiquitin-63E promoter 62 . pKC26w-pUbiq_myc-MOB1 plasmids were injected into y[1] w[1118]; PBac{y[ + ]-attP-9A}VK00027 flies (FlyBase ID: FBst0009744) and stable transformants were identified by the presence of the mini-white + marker. Injections of embryos with pKC26w_pUbiq _myc-hMOB1A plasmids and the initial generation of stable transformants were performed by BestGene Inc. (Chino Hills, USA). The ubi > MOB1 transgenes were recombined with the mats roo allele on an FRT82B-carrying chromosome by meiotic recombination followed by selection in medium containing 100 µl of 25 mg ml −1 neomycin. Upon appearance of eggs, vials were heatshocked at 37°C for 1 h 1-2 times every day until pupae appeared. After balancing, the mats roo allele and FRT82B sites were confirmed by PCR genotyping (see below). To generate adult eyes mosaic for homozygous mutant and heterozygous wild-type tissue, these recombinant lines were crossed to eyFLP;; FRT82B GFP/TM6B (eyFLP expresses FLP only in the eye and head capsule 42 ). To generate mutant clones in wing imaginal disks, we crossed our lines to hsFLP; FRT82B GFP. Larvae were heatshocked at 37°C for 1 h 3 days after egg laying, followed by dissection at wandering L3 stage (6 or 7 days after egg laying).
Genotypes of transgenic flies used in this study. a-e. yw hsFLP/+ ;; FRT82B GFP/FRT82B mats roo f-j. yw hsFLP/+ ;; FRT82B GFP/FRT82B mats roo , ubi > MOB1-wt k-o. yw hsFLP/+ ;; FRT82B GFP/FRT82B mats roo , ubi > MOB1-DV p-t. yw hsFLP/+ ;; FRT82B GFP/FRT82B mats roo , ubi > MOB1-KEKE u-y. yw hsFLP/+ ;; FRT82B GFP/FRT82B mats roo , ubi > MOB1-DVKEKE Immunofluorescencse microscopy of Drosophila imaginal disks. All procedures were carried out at room temperature, protected from light, unless otherwise stated. Wandering L3 larvae were dissected in ice-cold phosphate-buffered saline (PBS). Briefly, larvae were torn in half, the anterior half inverted, and undesired tissues were removed (central nervous system, fat body, and salivary glands), leaving only the imaginal disks attached to the inverted carcass. Carcasses were then fixed in 4% paraformaldehyde in PBS at room temperature for 30 min, washed several times in PBS containing 0.1% Triton-X100 (PBST), followed by permeabilization for 25 min in PBS containing 0.3% Triton-X100 and 1 h pre-blocking with 10% normal goat serum (NGS) in PBST. Subsequently, samples were incubated with primary antibody at 4°C overnight. Then samples were washed with PBST, pre-blocked once more with 10% NGS in PBST, followed by incubation with secondary antibody for at least 2 h. Finally, samples were washed with PBST, stained for 20 min with 1 µg ml −1 DAPI in PBST, before washing once more with PBST and mounting of the disks in Vectashield mounting medium (Vector Labs). Samples were stored at 4°C until imaging. Confocal images were acquired on a Zeiss LSM510 laser scanning microscope using a water immersion ×40 objective. To visualize apical surfaces (where Expanded protein is localized), Z-stacks of between 8 and 20 × 1 µm sections were collected, and then processed as maximum intensity projections. Images were processed using ImageJ, and figures prepared using Adobe software.
Western blotting and PCR genotyping of transgenic flies. To analyze adult flies using immunoblotting, three flies per genotype were frozen at −80°C overnight, followed by homogenization on ice in 20 µl of Laemmli sample buffer per fly (1610737, Bio-Rad,) using a Squisher manual homogenizer (Zymo Research). After boiling samples at 95°C for 10 min, samples were centrifuged and supernatants transferred to fresh tubes. Finally, DTT was added to a final concentration of 50 mM and equal volumes of samples were analyzed by SDS-PAGE, followed by western blotting. For PCR genotyping, flies were collected, frozen in liquid nitrogen, and subsequently crushed using a micropestle, followed by preparation of genomic DNA using a Qiagen kit (69506). Genomic DNA was stored at -20°C, followed by PCR genotyping using the following primers: 5′-GCCGCTCAAGA-TAGCCAGAT-3′ and 5′-GCACACTTCCTGGAACCGCTCGCATC-3′ to detect the Roo transposon; and 5′-AGAGGCGCTTCGTCTACGGAGCGACA-3′ and 5′-CGGCAAGCAGGCATCGCCATGGGTC-3′ to detect the FRT site.
Statistical analysis. Graphics and statistical analyses were carried out using the GraphPad Prism software. Data are presented as mean ± s.e.m., unless stated otherwise. The significance of differences between the means or the population distributions was determined using two-tailed unpaired Student t-test. Differences were considered statistically significant when p-values were below 0.05 (*), 0.01 (**), or 0.001 (***). p-values are listed in the corresponding figure legends where appropriate.
Data availability. The coordinates and structure factor files of the complex of human MOB1 (residues 33-216) bound to NDR2 (residues 25-88) have been deposited in the Protein Data Bank (PDB) with accession number 5XQZ. All relevant data are available from the corresponding authors upon reasonable request.