Phosphorylation of CD20 in cells from a hairy cell leukemia cell line. Evidence for involvement of calcium/calmodulin-dependent protein kinase II

Hairy cell leukemia is an uncommon B cell lymphoproliferative disease of unknown etiology. We previously observed that CD20, a membrane protein involved in B cell activation, is hyperphosphorylated on hairy cells and that these cells have unusually high levels of intracellular free Ca2+. Therefore, we used a hairy cell line, HCLL-7876, to study the potential involvement of Ca(2+)-activated protein kinases in CD20 phosphorylation. Addition of the Ca2+ ionophore, ionomycin, increased CD20 phosphorylation both in activated B cells and in cells from the hairy cell line; addition of EGTA to either cell type decreased basal levels of CD20 phosphorylation. Ionomycin treatment of these cells resulted in increased kinase activity of cytosolic extracts toward syntide-2, a synthetic peptide substrate for calcium/calmodulin-dependent kinase II (CaM-KII), with kinetics similar to those of CD20 phosphorylation in the cell line. CD20 isolated from the cell line was a substrate for purified CaM-KII in vitro. Phosphopeptide maps of CD20 from untreated hairy cells or ionomycin-treated HCLL-7876 cells were similar to maps of CD20 that had been phosphorylated in vitro by CaM-KII. These results suggest that the unusually high levels of intracytoplasmic Ca2+ in hairy cells may enhance the phosphorylation of key surface proteins.