Figure 3. CEACAM4 is tyrosine phosphorylated and binds directly to specific SH2 domains. (A) HEK-293 cells were transfected with plasmids encoding GFP-tagged CEACAM3, CEACAM4, or CEACAM3/4 WT. As indicated, cells were cotransfected with v-Src. Phosphorylated CEACAMs were detected with an anti-pTyr mAb (top). Equivalent expression of the receptors (2nd panel), v-Src (3rd panel), and protein content (bottom panel) was verified by Western blotting of WCLs with mAb against GFP, v-Src, or tubulin, respectively. (B) HEK-293 cells were transfected with CEACAM4-GFP, with or without v-Src as in A. Lysates containing p-CEACAM4 or CEACAM4 were prepared and used to probe protein domain microarrays encompassing the indicated SH2 domains spotted in the form of GST-fusion proteins. p-CEACAM4 or CEACAM4-GFP, bound to individual spots, were detected by anti-GFP antibodies and quantified. Extent of p-CEACAM4 binding versus CEACAM4 binding was calculated, and the values were normalized to the amount of immobilized GST-SH2 domains at individual spots, as detected by an anti-GST mAb. Bars represent the mean ± sd of 4 determinations. (C) Lysates as in B were used in GST pull-down assays with the indicated SH2 domains fused to GST or with GST alone. Precipitates were probed with an anti-GFP mAb for the presence of CEACAM4 (upper). Equal amounts of the GST-fusion proteins used in the pull down were verified by Coomassie staining of the membrane (lower). (D) Peptide spot membranes harboring synthetic 15-mer peptides surrounding the indicated tyrosine residues of the CEACAM4 cytoplasmic domain in the unphosphorylated (Y) or the tyrosine-phosphorylated (pY) form were probed with GST-PI3K-N-SH2, GST-PI3K-C-SH2, GST-c-Yes-SH2, GST-Nck2-SH2, or GST only. Bound GST-fusion proteins were detected with anti-GST mAb. (E) Spot membranes harboring the indicated 15-mer peptides derived from the CEACAM3 cytoplasmic domain were probed with GST or GST-c-Yes-SH2 as in D.