MUC1 is all integral membrane protein expressed on the apical surface of epithelial cells where it acts as a signaling receptor. Its cytoplasmic tail (CT) contains seven, highly conserved tyrosine residues, some of which are constitutively phosphorylated and serve as recognition sites for SH2 domain proteins involved in intracellular signal transduction. However, no studies have determined which MUC1 tyrosines are phosphorylated or which signaling pathways are activated in response to stimulation of its ectodomain. In this report, we used our previously characterized CD8/MUC1 chimeric protein that is tyrosine phosphorylated on the MUC1 CT in response to extracellular treatment with CD8 antibody and performed site-directed mutagenesis of all seven tyrosines, both individually and in multiple combinations, to identify the particular sites of stimulated phosphorylation. We observed four phosphorylation sites, three present in sequence motifs with known signaling potential (Y-20, Y-46, and Y-60) and one previously uncharacterized (Y-29). These results are discussed in the context of the role of MUC1 in signal transduction. (C) 2003 Elsevier Inc. All rights reserved.