Fig. 1.

LATS1 and LATS2 directly phosphorylate Ser894 in the TSS motif of INCENP in vitro. (A) Schematics of human INCENP and alignment of primary sequences around the TSS motif of INCENP from various organisms. The conserved TSS motifs of human and mouse INCENP contain a putative LATS phosphorylation site (S894 in human numbering). Red and green letters indicate putative phosphorylated amino acids of LATS and Aurora-B, respectively. Hs, human; Mm, mouse; Gg, chicken; Xl, frog; Ce, nematode; Dm, fruit fly; Sp, fission yeast; Sc, budding yeast. (B) In vitro kinase assays were performed with immunocomplexes of either 6Myc-LATS1 or LATS2 (kinases), 3FLAG-MOB1A (kinase activator), and purified GST-fused INCENP-C (amino acids 884–899)-WT or -S894A protein (substrate) in the presence of [γ-³²P] ATP. Phosphorylation of LATS1, LATS2 (p-LATS1/2), and the C-terminus of INCENP (p-INCENP) was detected by autoradiography (³²P). Simply Blue staining was used as a loading control. WT and KD indicate wild-type and kinase dead, respectively. (C) Dot-blot analysis with two kinds of anti-pS894 polyclonal antibodies (#V5543 and #V5544). Phosphorylated and non-phosphorylated S894 peptides were blotted on PVDF membranes at the indicated concentrations, followed by western blotting. (D) In vitro LATS1/2-kinase assays were performed as in (B), except for the presence of [γ-³²P] ATP. Phosphorylation of INCENP was assessed by western blotting with anti-pS894, anti-GST, and anti-Myc antibodies. Vec, vector; L1, LATS1; L2, LATS2. See Fig. S1 for uncropped gel images of SDS-PAGE (autoradiography and Simply Blue staining) in Fig. 1B and western blot in Fig. 1D. Black boxes indicate the cropped regions. Molecular sizes (kDa) are based on prestained protein markers.