Fig. 5: Elucidation of the MAP4K2/3 LOF missense mutations.
From: Functional annotation of the Hippo pathway somatic mutations in human cancers
a, b Illustration of the protein domains and the identified LOF missense mutations for MAP4K2/3. c Illustration of the in vitro kinase assay used for validating the MAP4K2/3 LOF missense mutations. d, e The MAP4K2/3 LOF missense mutations target their kinase activities in vitro. The SFB-tagged LOF mutants of MAP4K2 (d) and MAP4K3 (e) were expressed in HEK293T cells, purified using S protein beads, washed with high-salt buffer containing 250 mM NaCl, and subjected to in vitro kinase assay. A representative blot of two independent experiments is shown. f, g The MAP4K2/3 LOF missense mutations abolish their kinase activities in vivo. The MST/MAP4K-8KO HEK293A cells were transfected with Myc-LATS1 and the indicated SFB-tagged LOF mutants of MAP4K2 (f) and MAP4K3 (g) and subjected to Western blot. A representative blot of two independent experiments is shown. h Protein sequence alignment reveals a potential autophosphorylation site for MAP4K-family kinases. The predicted autophosphorylation site for MAP4Ks was highlighted. i MAP4K3 activity is required for its S170 phosphorylation. The MST/MAP4K-8KO cells were transfected with the indicated SFB-MAP4K3 constructs and subjected to pulldown assay. A representative blot of two independent experiments is shown. j Mutation of the predicted autophosphorylation site inhibits MAP4Ks. The MST/MAP4K-8KO HEK293A cells were transfected with the indicated constructs and subjected to immunofluorescent staining. Nucleus was visualized by Dapi. Scale bar, 20 μm. Arrows showed the cells expressing the indicated constructs. k, l The MAP4K2/3 autophosphorylation is dramatically inhibited by their LOF missense mutations. The MST/MAP4K-8KO HEK293A cells were transfected with the indicated SFB-tagged LOF mutants of MAP4K2 (k) and MAP4K3 (l) and subjected to pulldown assay. A representative blot of two independent experiments is shown. m Characterization of the MAP4K2 LOF missense mutations-induced effects on its autophosphorylation ability. The MST/MAP4K-8KO HEK293A cells were transfected with HA-MAP4K2 and the indicated SFB-MAP4K2 LOF mutants and subjected to pulldown assay. A representative blot of two independent experiments is shown. n, o Characterization of the MAP4K3 LOF missense mutations-induced effects on its autophosphorylation ability. The MST/MAP4K-8KO HEK293A cells were transfected with Myc-MAP4K3 and the indicated SFB-MAP4K3 LOF mutants and subjected to pulldown assay. A representative blot of two independent experiments is shown. p, q Characterization of the MAP4K2/3 LOF missense mutations-induced effects on their ATP-binding abilities. The MST/MAP4K-8KO HEK293A cells were transfected with the indicated SFB-tagged LOF mutants of MAP4K2 (p) and MAP4K3 (q), purified using S protein beads, washed with high-salt buffer containing 250 mM NaCl, and subjected to γ-ATP-based in vitro kinase assay. A representative blot of two independent experiments is shown. Source data are provided as a Source Data file.
