Results Characterization of MOB1A mutants with selective binding to

selective binding to MST/NDR/LATS kinases in mammalian cells

To develop research tools that allow us to understand which protein-protein interaction(s) (PPIs) between MOB1A and MST/LATS/NDR are required for YAP-Hippo signalling, cell proliferation and suppression of cellular transformation, we designed and characterized a series of mutants with affected binding of MOB1A to MST/LATS/NDR based on our structural studies (Section 3.1.3). Specifically, we focused on characterising MOB1A variants carrying E51K, D63V, K104E/K105E, E51K/K104E/K105E, or D63V/ K104E/K105E point mutations. The nomenclature of these mutations used in this thesis has been simplified, respectively, as follows:

EK (E51K), DV (D63V), KEKE (K104E/K105E), EKKEKE

(E51K/K104E/K105E), DVKEKE (D63V/K104E/K105E).

Stavridi et al. (2003) reported that MOB1A has a flat surface rich in acidic residues on one side, which is believed to provide the structural basis for the association with the NDR1/2 and LATS1/2 kinases through electrostatic forces. Stavridi et al. (2003) found that the E51 of MOB1A is conserved from yeast to humans and speculated that this residue is very likely of functional importance. Furthermore, Hergovich et al. (2009) demonstrated that the substitution of Glu51 with Lys (E51K) interferes with the interaction with NDR1/2, highlighting the importance of the E51 residue of MOB1A for the interaction with the NDR1/2 kinases. In addition, based on our structural and biochemical studies (see section 3.1.2 and 3.1.3 above), we concluded that MOB1A(DV) displays an abolished interaction with LATS1 (Figure 3.3), while the interactions with NDR2 and MST2 remain fully intact (Figure 3.3). Furthermore, we also discovered that MOB1A (KEKE) displayed an abolished interaction with the MST2, but not with NDR2 and LATS1 (Figure 3.3). Following the discovery of these mutations which appear to be important for the interaction of hMOB1A with MST/LATS/NDR, these mutations were then characterized in mammalian cells using co-immunoprecipitation assays. To this end, hemagglutinin A (HA)-tagged MST1/2, LATS1/2, NDR1/2 and myc-tagged MOB1A variants were co-expressed in HEK 293 cells, followed by immunoprecipitation with

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anti-HA 12CA5 antibody, before analyses of protein complexes by Western blotting.

Firstly, the characterization of our MOB1A variants with respect to MST1/2 binding was carried out. The results are presented in Figure 3.4 below.

Figure 3.4. Characterization of the interaction of MOB1A variants with MST1/2. (a-d) Lysates of HEK293 cells expressing full-length HA-MST1 or HA-MST2 wild-type

(wt) together with indicated full-length MOB1A versions were subjected to immunoprecipitation (IP) using anti-HA. Complexes were studied by immunoblotting using anti-myc (top) and anti-HA (top middle). Input lysates were analysed with anti-myc (bottom middle) and anti-α-tubulin (bottom). Relative molecular weights are shown. The EK and KEKE mutations caused loss of binding to MST1/2, while the DV mutant bound normally. Complexes were studied by immunoblotting using anti-myc (top) and anti-HA (top middle). Input lysates were probed with anti-myc (bottom middle) and anti-α-tubulin (bottom). Relative molecular weights are shown.

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We investigated complex formation of MOB1A(wt) and MOB1A mutants with the MST1/2 kinases in HEK293 (Figure 3.4). To this end, N- terminally tagged versions of HA-MST1 and HA-MST2 were co-expressed with N-terminally tagged versions of myc-MOB1A(wt) and myc-MOB1A mutants, followed by co-immunoprecipitation using anti-HA. These experiments revealed that MOB1A(EK), MOB1(EKKEKE), and MOB1A(DVKEKE) cannot bind to MST1 and MST2 (Figures 3.4a and 3.4c). In sharp contrast, MOB1A(DV) associated with MST1 and MST2 like wild-type MOB1A (Figures 3.4a and 3.4c). Significantly, the MOB1A(KEKE) mutant could not form a complex with neither HA-MST1 nor HA-MST2 (Figures 3.4b and 3.4d). Collectively, these findings suggest that the MOB1A(EK) and MOB1A(KEKE) variants are deficient in MST1/2 binding, while MOB1A(DV) binds normally to MST1/2. Thus, we established in Figure 3.4 MOB1A mutants that allow us to test the significance of the MST1/2-MOB1A interaction in Hippo signalling. Furthermore, these findings (Figure 3.4) also further support our biochemical data presented in section 3.1.3 above.

Second, the PPIs of our panel of MOB1A mutants with LATS1/2 were examined. The results are shown in Figure 3.5 below.

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Figure 3.5. Characterization of the interaction of MOB1A variants with the LATS1/2 kinases.

(a-d) Lysates of HEK293 cells expressing full-length HA-tagged LATS1 or LATS2 wild-

type (wt) together with the indicated full-length MOB1A versions were subjected to immunoprecipitation (IP) using anti-HA 12CA5 antibody. Complexes were analysed by immunoblotting using anti-myc (top) and anti-HA antibody (top middle). Input lysates were analysed with anti-myc (bottom middle) and anti-a-tubulin antibody (bottom). Relative molecular weights are shown. The EK and DV mutations abolished MOB1 binding to LATS1/2, while the KEKE mutant bound normally. Complexes were analysed by immunoblotting using anti-myc (top) and anti-HA (top middle). Input lysates were analysed with anti-myc (bottom middle) and anti-a-tubulin (bottom). Relative molecular weights are shown.

We investigated complex formation of MOB1A(wt) and MOB1A mutants with the LATS1 and LATS2 kinases in HEK293 cells (Figure 3.5). To this end, N-terminally tagged versions of HA-LATS1 and HA-LATS2 were co- expressed with N-terminally tagged versions of myc-MOB1A wt and

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mutants, followed by co-immunoprecipitation using anti-HA. These findings uncovered that only MOB1A(wt) and MOB1A(KEKE) can bind to LATS1 and LATS2, while the remainder mutants did not to interact with LATS1 and LATS2 (Figure 3.5). Collectively, these findings show that the MOB1A(DV) and MOB1A(EK) variants are deficient in LATS1/2-binding, while MOB1A(KEKE) can normally associate with LATS1 and LATS2. Therefore, we established here a foundation that allows us to study the significance of LATS1/2-MOB1A complex formation in Hippo core signalling. Furthermore, our data presented here (Figure 3.5) also support the biochemical findings presented in section 3.1.3.

Third, the characterization of MOB1A mutants with respect to NDR1/2 binding was carried out. The corresponding results are presented in Figure 3.6 below.

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(a-d) Lysates of HEK293 cells expressing full-length HA-NDR1 or HA-NDR2 wild-type

(wt) together with the indicated full-length MOB1A versions were subjected to immunoprecipitation (IP) using anti-HA 12CA5 antibody. Complexes were studied by immunoblotting using anti-myc (top) and anti-HA antibody (top middle). Input lysates were analyzed with anti-myc (bottom middle) and anti-a-tubulin antibody (bottom). Relative molecular weights are shown. Only versions carrying the EK mutation displayed a reduction of binding to NDR1/2. Complexes were examined by immunoblotting using anti- myc (top) and anti-HA (top middle). Input lysates were investigated with anti-myc (bottom middle) and anti-a-tubulin (bottom). Relative molecular weights are shown.

N-terminally tagged versions of HA-NDR1 and HA-NDR2 were co- expressed with N-terminally tagged versions of myc-MOB1A(wt) and myc- MOB1A mutants, followed by co-immunoprecipitation using anti-HA. These findings uncovered that the MOB1A(EK) and MOB1A(EKKEKE) mutants cannot bind to NDR1 and NDR2, while the MOB1A(DV) and MOB1A(DVKEKE) interacted normally with NDR1 and NDR2 when compared to wild-type MOB1A (Figures 3.6a and 3.6c). In addition, we studied the interaction of MOB1A(KEKE) with HA-NDR1 and HA-NDR2, and found that the MOB1A(KEKE) can bind normally to NDR1 and NDR2 (Figures 3.6b and 3.6d). Collectively, our data shown in Figure 3.6 suggests that only MOB1A(EK) and MOB1A(EKKEKE) are deficient in NDR1/2 binding, while all other mutants tested displayed normal interactions with NDR1/2. Thus, we establish here (Figure 3.6) a research tools allowing us to study the significance of the interaction of MOB1A with NDR1/2 in the context of Hippo core signalling.

Taken together, in full agreement with published literature (Stavridi, Harris et al. 2003, Bichsel, Tamaskovic et al. 2004, Bothos, Tuttle et al. 2005, Hergovich, Bichsel et al. 2005, Hergovich, Schmitz et al. 2006, Vichalkovski, Gresko et al. 2008, Hergovich, Kohler et al. 2009, Kohler, Schmitz et al. 2010, Chen, Zhang et al. 2015, Hoa, Kulaberoglu et al. 2016) and the structural/biochemical studies presented in section 3.1.2 and 3.1.3, we found that MOB1A(wt) can stably bind to MST1/2, NDR1/2, and LATS1/2 kinases (Figures 3.4, 3.5, and 3.6). More importantly, we characterized a series of selected MOB1A with regard to MST/NDR/LATS

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binding. These experiments revealed that the MOB1A(EK) does not to interact with any of the NDR/LATS/MST kinases. In contrast, MOB1A(DV) selectively impaired the binding of MOB1A with LATS1/2, while the MOB1A(DV) normally bound to MST1/2 and NDR1/2 kinases. The MOB1A(KEKE) also interacted normally with LATS1/2 and NDR1/2 kinases, while the KEKE mutations selectively interfered with the interaction of MOB1A with MST1/2 kinases. In addition, the combination of the EK and DV with KEKE were tested for their ability to interact with MST1/2, LATS1/2, and NDR1/2 kinases, respectively. This revealed that the MOB1A(EKKEKE) does not interact stably with any of the tested kinases. The MOB1A(DVKEKE) mutant interacted only with NDR1/2 kinases, but did not bind to any of the other Hippo kinases tested. The binding properties of all mutants tested are summarized in Figure 3.7, including an illustration of the location of the key residues in the published structure of MOB1A.

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Figure 3.7. Generation of MOB1A variants to dissect the importance of MOB1A interactions with mammalian Hippo core cassette kinases.

(a) Model illustrating two key research questions regarding MOB1A as a hub of

mammalian Hippo core signalling: (1) Does MOB1A interact differently with MST1/2, LATS1/2 and NDR1/2 in human? (2) Which interactions of MOB1A with MST1/2, LATS1/2, or NDR1/2 are biologically important in the context of YAP-Hippo signalling?

(b) Schematic summary of the biochemical and molecular characterization of the indicated

MOB1A variants presented in Figures 3.1 to 3.6. (c) Model of human MOB1A(33-216) depicting the possibly opposing binding sites on MOB1A of NDR/LATS and MST1/2 kinases. Secondary structure elements of MOB1A are highlighted. The locations of Glu51, Asp63, and Lys104/Lys105 in MOB1 are indicated. (d) Schematic model of how the differential binding of MOB1A to MST1/2, LATS1/2 and NDR1/2 can be experimentally exploited using the DV and KEKE mutations in order to study the biological importance of these different interactions of MOB1A with Hippo core cassette kinases.

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