Fig. 6
Ser1116, Leu1120, and Leu1121 serve as a di-leucine signaling motif upon PKC activation. a Alignment of amino acid sequences of the C-terminal cytoplasmic region of mammalian ATP11C. Serine and threonine residues shown in bold were conserved among human (h), mouse (m), bovine (b), and dog (d). Arrowheads exhibit critical residues for endocytosis, and the residue marked with an asterisk is involved in endosomal localization of ATP11C after PKC activation. b HeLa cells were transiently co-transfected with expression vectors for FLAG-tagged CDC50A and HA-tagged WT of ATP11C, or mutants of HA-tagged ATP11C, in which each bold residue in a was replaced with alanine (see also Supplementary Fig. 3a) or aspartate. c HeLa cells were transiently co-transfected with expression vectors for FLAG-tagged CDC50A and HA-tagged WT of ATP11C, or mutants of HA-tagged ATP11C. In the mutants, alanine replaced all serine and threonine residues in the C-terminus of ATP11C shown in a (9-Ala), all except Ser1116 (8-Ala(S1116)), all except Ser1126 (8-Ala[S1126]), or all except Ser1116 and Ser1126 (7-Ala(S1116/1126)). b, c The cells were treated with vehicle alone (Mock) or 400 nM of PMA (PMA) for 15 min and fixed. The fixed cells were then incubated with anti-HA antibody, followed by incubation with Cy3-conjugated anti-rat secondary antibody. Scale bars, 20 μm
