Ecto‐phosphorylation of CD98 regulates lymphocytes‐epithelia interactions

Abstract

Ecto‐phosphorylation has been shown to play an important role in several cellular functions. Here we investigate the ecto‐phosphorylation of membrane glycoprotein CD98 by ecto‐protein kinases (ePK) and its functional consequences. Our results show that recombinant CD98 is phosphorylated by purified casein kinase 2 (CK2) and by ePK from Jurkat cells. Km and Vmax for the phosphorylation of CD98 by CK2 are 0.84 μM and 8.7e+6 μmol/min, respectively. In intestinal epithelial Caco2‐BBE cells, CD98 is also ecto‐phosphorylated by ePK from Jurkat cells. Interestingly, activation of CD98 in CHO cells expressing CD98 enhances the ecto‐phosphorylation of CD98 and increases the attachment of Jurkat cells to CHO cells, suggesting that the ecto‐phosphorylation of CD98 is important for cell‐cell interactions. To examine the role of CD98 ectodomain phosphorylation in cell‐cell interactions, we generated CD98‐CD69 and CD69‐CD98 constructs, as well as CD98 lacking PDZ‐binding domain or mutated at predicted serine phosphorylation sites and transfected these constructs into CHO cells. Cell adhesion assays show that mutation at S305‐S307‐S309 or deletion of extracellular C‐terminal or PDZ‐binding domain of CD98 significantly decreases the binding of Jurkat cells to CHO cells. In conclusion, we demonstrate, for the first time, that CD98 is ecto‐phosphorylated by ePK and its phosphorylation affects cell‐cell interactions.