Phosphorylation of Extracellular Domains of T-Lymphocyte Surface Proteins

The extracellular accumulation of ATP after activation of T-lymphocytes, as well as the presence of ecto-protein kinases in these cells, led us to propose that T cell surface receptors could be regulated through the reversible phosphorylation of their extracellular domains (ectodomains). Here, in a model system, we used T cell transfectants which express T cell antigen receptor chains lacking intracellular and transmembrane protein domains and 32Pi metabolic labeling of cells to definitively demonstrate phosphorylation of ectodomains of T cell surface proteins. We show that alphabetaTCR ectodomains were phosphorylated intracellularly and constitutively on serine and threonine residues and were then expressed on the T cell surface in phosphorylated form. TCR ectodomains also could be phosphorylated at the cell surface when extracellular [gamma-32P]ATP or [gamma-32P]GTP were used as phosphate donors with the same cells. Consensus phosphorylation sites for serine and threonine protein kinases were found to be strongly evolutionary conserved in both alpha and beta TCR chains constant regions. These results are consistent with the hypothesis, where T cell surface proteins which are phosphorylated intracellularly on their ectodomains, could subsequently be expressed at the cell surface and then be reversibly modified by ectoprotein phosphatase(s) and by ectokinase(s). Such modifications may change T cells cognate interactions by, e.g. affecting TCR-multimolecular complex formation and antigen binding affinity. It is suggested that alphabetaTCR ectodomain phosphorylation could serve as a potential mechanism for regulation of alphabetaTCR-mediated T-lymphocytes response.