Results Testing selective MOB1A mutants as MST1/2 substrates

Considering that MST1 and MST2 can function as upstream kinase of MOB1A (Praskova, Xia et al. 2008), we tested whether our MOB1A mutants are phosphorylated by MST1 and MST2 similarly to wild-type MOB1A. To do so, we pursued two lines of in vitro kinase assays. First, radioactive in vitro kinase assays were performed to measure the total phosphorylation of MOB1A. Second, we utilized specific anti-phospho- antibodies raised against phospho-Thr12 (T12-P) and phosphor-Thr35 (T35- P) to measure MST1/2-mediated MOB1A phosphorylation in our in vitro kinase assays.

Prior to the characterization of our MOB1A variants as MST1/2 substrates, recombinant MAL-tagged MOB1A variants were bacterially expressed and then purified as outlined in section 2.2.2.6 above. Equal amounts of our MAL-MOB1A proteins were analysed by SDS-PAGE followed by Coomassie staining to validate the quality and purity of our protein preparations (Figure 3.12).

Figure 3.12. Bacterially expressed and purified MAL-MOB1A variants.

All MAL-MOB1A recombinant proteins were bacterially expressed and purified as described in section 2.2.2.7. The purified proteins were run on 8 % gel and the gels were stained with Coomassie staining as defined in section 2.2.2.7. The location of the recombinant MAL-MOB1A versions are pointed out by an arrow.

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After confirming equal quality and purity of our recombinant MAL- MOB1A proteins, equal amounts of recombinant proteins were used as substrates in in vitro kinase assays as outlined below.

First, in vitro kinase assays using radiolabelled ATP were performed as outlined in section 2.2.2.6. The results are presented in Figure 3.13 below.

Figure 3.13. Phosphorylation of MOB1A mutants by MST1/2.

GST-tagged full-length MST1 (a) and MST2 (b) kinases were incubated with purified bacterially expressed MAL-MOB1A proteins as described in section 2.2.2.6. As control, GST-MST1 and MST2 were incubated without MAL-MOB1A proteins (Top panel, lane

1). After the kinase reaction, proteins were separated by SDS-PAGE and detected by

autoradiography (top panel) or coommassie stain (bottom panel). All MAL-MOB1A variants, except for MOB1A(DVKEKE), displayed the same phosphorylation status. The autophosphorylation of MST1/2 are also visible.

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Recombinant GST-tagged MST1 and GST-MST2 kinases were incubated with recombinant MAL-MOB1A variants as substrates using [P32]γATP as a source of ATP, followed by SDS-PAGE and autoradiography. As shown in Figure 3.13, all MOB1A mutants tested, except for MOB1A(DVKEKE), were normally phosphorylated by MST1/2 when compared to wild type MOB1A. These results suggest that all MOB1A mutants, with the exception of MOB1A(DVKEKE), serve as MST1/2 substrates like wild-type MOB1A. Considering that MOB1A(EK) and MOB1A(KEKE) were phosphorylated like wild-type MOB1A by MST1/2 (Figure 3.13), although they cannot form a stable complex with MST1/2 (see section 3.1.4 above), these findings further suggested that MOB1A and MST1/2 do not need to stably interact for efficient phosphorylation of hMOB1A by MST1/2. Second, we utilized specific anti- phospho-antibodies raised against phospho-Thr12 (T12-P) and phosphor- Thr35 (T35-P) to measure MST1/2-mediated MOB1A phosphorylation in our in vitro kinase assays. However, before testing the phosphorylation status of our MOB1A variants on Thr12 and Thr35, we aimed to validate the specificities of the available anti-T12-P and anti-T35-P antibodies were tested. The results of these specificity tests are presented in Figure 3.14.

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Figure 3.14. Characterization of the anti-Thr12-P and anti-Thr35-P antibodies in the context of MST1/2-mediated phosphorylation of MOB1A.

(a,b) Recombinant full-length GST-MST1 or GST-MST2 wild-type (wt) were incubated

with full-length recombinant Mal-MOB1A(wt) or the T12A and T35A phospho-acceptor mutants. Following kinase reactions, the samples were analysed by Western blotting using indicated antibodies. Relative molecular weights are shown. Noteworthy, the anti-T12-P antibody only detected wild-type MOB1A when the kinase assays contained GST-MST1/2, while MOB1A (T12A) was not detected (a). The anti-T35-P antibody also specifically detected the phosphorylation of MOB1A on Thr35 (b).

Praskova et al. showed that MOB1A is phosphorylated at Thr12 and Thr35 by MST1/2. To develop research tools that allow the study of phosphorylation of MOB1A by MST1 and MST2 on Thr12 and Thr35, we characterised antibodies that specifically recognise MOB1A phosphorylated on Thr12 (anti-Thr12-P) and Thr35 (anti-Thr35-P). To test the specificity of these anti-phospho antibodies, we generated phosphor-acceptor mutants carrying Thr12 to Ala (T12A) or Thr35 to Ala (T35A) substitutions, and subsequently compared phosphorylation of recombinant MAL- MOB1A(T12A) and MAL-MOB1A(T35A) with the wild-type MAL-

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MOB1A (Figure 3.14). In vitro kinase assays. in vitro kinase assays were performed by incubating recombinant GST-tagged MST1/2 kinases with recombinant MAL-MOB1A variants in the presence of unlabelled ATP, followed by SDS-PAGE and Western blotting using the anti-T12-P and anti-T35-P antibodies. These experiments revealed that MOB1A(wt) display clearly detectable Thr12 and Thr35 phosphorylation (Figure 3.14, lanes 2). Demonstrating the specificity of the anti-phospho antibodies, MOB1A(T12A) and MOB1A(T35A) were not recognized by the anti-T12-P and anti-T35-P antibodies, respectively (Figure 3.14, compare lanes 2 and 3). This confirmed that the anti-T12-P and anti-T35-P antibodies can specifically detect the phosphorylation of MOB1A at Thr12 and Thr35 by MST1 and MST2. Kinase assays that only contained GST-MST1, GST- MST2, or MAL-hMOB1A recombinant proteins were used as additional negative controls (Figure 3.14), further confirming the specificity of the anti-phospho antibodies.

Next, the phosphorylation status of our MOB1A variants by MST1 and MST2 was tested. The results are shown below in Figure 3.15.

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Figure 3.15. Characterization of the MST1/2-mediated phosphorylation of MOB1A variants.

(a,b) Recombinant full-length GST-MST1 or GST-MST2 wild-type (wt) were incubated

with indicated full-length recombinant Mal-MOB1A variants. The reactions which only contained MST1 or MST2 were used as control. Following kinase reactions, the samples were analysed by immunoblotting using the specified antibodies. Relative molecular weights are indicated.

More specifically, given the specificity of these phosphor-specific antibodies (Figure 3.14), we sought to determine whether our MOB1A versions are affected with respect to phosphorylation by MST1 and MST2 on Thr12 and Thr35. In vitro kinase assays were performed by incubating recombinant GST-MST1 or GST-MST2 kinases with recombinant MAL-

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MOB1A variants in the presence of unlabelled ATP, before samples were analysed by SDS-PAGE and Western blotting (Figure 3.15). These experiments showed that our MOB1A versions are phosphorylated on Thr12 and Thr35 by MST1 and MST2 like wild-type MOB1A (Figure 3.15). Collectively, our findings presented in this section suggest that MOB1A and MST1/2 do not need to stably interact for phosphorylation (Figure 3.7). Even more importantly, these findings propose that changes in MST1/2- mediated phosphorylation of MOB1A are very unlikely the cause of the observed alterations of binding patterns of our MOB1A variants.

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3.3.4. Results - Testing selective MOB1A mutants as Hpo