Supplementary Figure S6 from MARK2/MARK3 Kinases Are Catalytic Codependencies of YAP/TAZ in Human Cancer

A-E, Western blot analysis of MARK2 specific substrates phosphorylation. Labeling as described in Fig 3D. Data are representative of two independent experiments. F, Lolli-pop illustration of MARK2-dependent phosphorylation sites on MST1/2 and MAP4K1-4,6,7 identified using mass spectrometry-based phosphoproteomics. SARAH= Sav/Rassf/Hpo domain, CNH= Citron homology domain. G, IP–western blot analysis evaluating the phosphorylation p-LATS2 (T1041) in the presence or absence of MARK2 or MARK3 following NF2 overexpression in HEK-293T cells. Data are representative of two independent experiments. H, IP–western blot analysis evaluating the phosphorylation p-LATS2 (T1041) after NF2 mutant overexpression in HEK-293T cells. Data are representative of two independent experiments. I, IP–western blot analysis evaluating p-LATS1 (T1079) in the presence of NF2 together with MARK2 overexpression in HEK-293T cells. Data are representative of two independent experiments. J, IP–western blot analysis evaluating the interaction of NF2 and MAP4K4,6,7 in the presence or absence of MARK2 overexpression in HEK-293T cells. Data are representative of two independent experiments. K, IP–western blot analysis evaluating the phosphorylation p-LATS1 (T1079) following indicated gene overexpression in HEK-293T cells. L, IP–western blot analysis evaluating the phosphorylation p-LATS1 (T1079) following indicated gene overexpression in HEK-293T cells. Data are representative of two independent experiments. M, IP–western blot analysis evaluating p-LATS1 (T1079) in the presence of MAP4K4 or MAP4K6 together with MARK2,3, kinase-dead MARK2K82H or empty vector control overexpression in HEK-293T cells. Data are representative of two independent experiments. N, O, IP–western blot analysis evaluating p-LATS1 (T1079) in the presence of MAP4K4 or MST1 mutants. Data are representative of two independent experiments. P, Coomassie stain of recombinant proteins used in in vitro kinase assays (Fig.4D, 3E), purified from bacteria (GST-YAP, GST-TAZ) and insect cells. Q, R, In vitro kinase assay using recombinant full-length GST-YAP (P) or synthetically synthesized S128-phosphorylated peptide (Q) together with recombinant LATS2 followed by Mass spectrometry analysis of double phosphorylated YAP peptides. F was created with BioRender.com.