Expression and phosphorylation of truncated CD33 constructs on CHOK1 cells.

(A) FACS analysis of CD33 expression on the surface of CHOK1 cells, as detected by My9 anti-CD33 antibody. All constructs were expressed at about the same level, except construct ΔE, which was expressed at an approximately 20 times reduced level. (B) Autoradiograph of CD33 with tails of various length (as defined in text) after PMA-dependent phosphorylation with [³²P] in vivo. The applied amount of ΔE was 20 times that of the other constructs to compensate for reduced expression. Constructs ΔC through ΔF show phosphorylation in a PMA-dependent way, suggesting PKC activity on CD33 in vivo. The PMA-dependent phosphorylation of ΔC suggests Ser307 protein kinase as one PKC phosphorylated motif. The increase in phosphorylation after 15 minutes of PMA treatment compared to DMSO treatment alone is shown. (C) Amino acid sequence of the cytoplasmic tail of human CD33. Letters ΔB through ΔF indicate the length of the various constructs; the most C-terminal amino acid of each construct is shown. Putative phosphorylation sites Ser307 and Ser342 are indicated.