The CEACAM1-L Ser503 residue is crucial for inhibition of colon cancer cell tumorigenicity

CEACAM1 (also known as biliary glycoprotein, C-CAM or CD66a) is a cell adhe sion molecule of the immunoglobulin family behaving as a tumor inhibitory p rotein in colon, prostate, liver, endometrial and breast cancers. Inhibitio n of tumor development is dependent upon the presence of the long 71-73 ami no acid cytoplasmic domain of the CEACAM1 protein (CEACAM1-L). We have rece ntly defined a number of cis-acting motifs within the long cytoplasmic doma in participating in tumor cell growth inhibition. These are Tyr488, corresp onding to an Immunoreceptor Tyrosine-based Inhibition Motif, as web as the three terminal lysine residues of the protein. In this study, we provide ev idence that treatment with phorbol esters leads to increased phosphorylatio n of ill vivo P-32-labeled CEACAM1-L in mouse CT51 carcinoma cells, in the mouse 1MEA 7R.1 liver carcinoma cells and in 293 human embryonic kidney cel ls transfected with the Ceacam1-L cDNA. Basal level Ser phosphorylation was abrogated by treatment with the staurosporine inhibitor, but not by the pr otein kinase C-specific inhibitor calphostin C or other inhibitors such as H7 or sphingosine. Specific inhibitors of protein kinase A or calmodulin ki nase had only minimal effects on the levels of basal or PMA-induced Ser pho sphorylation, Furthermore, PMA treatment of the CT51 cells induced cell spr eading and cellular relocalization of the CEACAM1-L protein. Since Ser503 h as been described as a PMA-induced phosphorylation site in other cell syste ms, we investigated whether Ser503 was involved in these responses in mouse intestinal cells, No differences were noticed in the basal or the PMA-indu ced phosphorylation levels, kinase inhibitor sensitivity or the PMA-induced relocalization of the protein between the wild-type and the Ser503Ala muta nt CEACAM1-L. However, we provide evidence that Ser503 participates in CEAC AM1-L-mediated tumor inhibition as its mutation to an Ala led to im vivo tu mor development, contrary to the tumor inhibitory phenotype observed with t he wild-type CEACAM1-L protein.