(A,B) Cardiomyocytes (12 × 10⁶ cells) were infected with adenoviruses for expression of wild-type MAP4K4 and treated without or with calyculin A (CalA; 200 nM, 10 min). MAP4K4 was immunoprecipitated and separated by SDS–PAGE. Gels were stained with Coomassie Brilliant Blue R (A). Bands 1 and 2 were excised and taken for phosphoproteomics analysis. Band 3 was taken for identification using proteomics. Similar results were obtained with at least three separate cardiomyocyte preparations. (B) MAP4K4 protein sequence showing the positions of phosphorylation sites in untreated cardiomyocytes (green), in cells with CalA treatment (yellow) or in untreated and treated cells (cyan). The kinase domain (green), citron homology domain (CNH, blue), the helix C-terminal to the kinase domain (E295–K311; red) and the predicted coiled-coil region (black, bold type in italics) are shown. The Δ23 region (deletion of which eliminates kinase activity) is underlined. (C,D) HEK293 cells were transfected with constructs for FLAG-tagged wild-type (WT) MAP4K4, MAP4K4 with the indicated deletions [924–1233, Δ303–552, Δ552–924, Δ304–326 (i.e. Δ23)] or point mutations (T319A/S324A/S326A, T309A/T319A/S324A/S326A). MAP4K4 was immunoprecipitated using antibodies to the FLAG epitope and immunoblotted for FLAG. (E) Ribbon representation of the MAP4K4 kinase region (PDB:4ZK5 peptide A), including the kinase domain (silver, residues 1–294) and additional C-terminal αhelix (red, residues 295–311).