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Fig. 7. (A) Surface expression of ALCAM on K562-ALCAMSer, K562-ALCAMThr and wild-type K562-ALCAM. K562 cells were transfected with expression constructs coding for mutant ALCAM proteins and were sorted to obtain homogeneous cell populations expressing similar levels of mutant ALCAM. Expression was analyzed by flow cytometry. The shaded histograms represent isotype control staining and the white histograms represent staining with ALCAM-antibody AZN-L50. (B) Replacement of serine- or threonine residues in the cytoplasmic domain of ALCAM does not affect spontaneous or CytD-induced adhesion. Adhesion of K562-ALCAMSer, K562-ALCAMThr and K562-ALCAM was analyzed after treatment of cells with or without CytD (2.5 µg/ml) as in Fig. 1B. Wild-type and mutant ALCAM-expressing cells show similar patterns of adhesion. Data are representative of three experiments. (C) No evidence for phosphorylation of ALCAM after treatment with [³²P]-phosphate. K562 and K562-ALCAM cells were incubated overnight with [³⁵S]methionine/cysteine (a) or for 3 hours with [³²P]-disodiumphosphate (b). Cells were pre-incubated with 50 nM PMA for 16 hours prior to [³²P]-phosphate labeling (PKC downregulation), or 15 minutes after [³²P]-phosphate labeling (PKC activation), respectively. ALCAM was immunoprecipitated from labeled cell lysates with 1 µg of AZN-L51. ALCAM was detected in [³⁵S]methionine/cysteine labeled K562-ALCAM lysate, but no [³²P]phosphate-labeled ALCAM could be detected, not even after PMA stimulation. Long-term PMA treatment resulted in decreased overall protein phosphorylation. Similar observations were made with KG1 cells (not shown).

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