Phosphopeptide analysis demonstrates 2 phosphopeptides.

(A) Tryptic-chymotryptic peptides were analyzed from [³²P]-orthophosphate-labeled CD33 isolated from PMA-stimulated CD33-transfected COS cells (i) or U937 cells (ii). Samples were initially purified by SDS-PAGE, blotted onto PVDF, and then digested sequentially with trypsin and chymotrypsin. Isolated phosphopeptides were examined by 2-dimensional thin-layer chromatography as described in “Materials and methods.” Two peptides of similar mobilities were identified in each sample. (B) Amino acid sequence of the cytoplasmic domain of CD33, starting with Lys283, with serine residues indicated by asterisks (*) and gaps indicating expected points of cleavage following combined trypsin-chymotrypsin digestion.