Extended Data Fig. 2: Multisite phosphorylation of Lgl.
(a) Binding affinity of the Llgl1 P-site peptide to the aPKCι kinase domain (2P, phosphorylated at pT412 and pT564) measured by fluorescence anisotropy. (b) Crystal structure of the Llgl2 substrate peptide (sticks) bound to the aPKCι kinase domain (yellow surface) with the C-terminal Ser site positioned as the phospho-acceptor, residues are numbered from Ser at residue 0. Superposed is the final electron density map calculated using sigma-A weighted mFo-DFc coefficients. The ordered part of the Llgl2 P-site is shaded orange with the −2R and S phospho-acceptor site indicated by an underline. (c) Specificity of phospho-Llgl antibodies used in this study. Western blot analysis using phospho-specific antibodies against pS653 site or pS645/pS649 sites for different GFP-Llgl2 phospho-acceptor site serine mutations in the P-site of loop (10-11). Phosphorylation of WT and the indicated mutants of Llgl2 is shown upon co-expression with myc-tagged aPKCι. The western blot shows the specificity of the two different phospho-specific antibodies against either the pS653 site or pS645/pS649 sites (Llgl2). Representative western blot of n = 2 experiments.
