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 prot ein involved in B cell activation, is hyperphosphorylated on hairy cel ls and that these cells have unusually high levels of intracellular fr ee Ca2+. Therefore, we used a hairy cell line, HCLL-7876, to study the potential involvement of Ca2+-activated protein kinases in CD20 phosp horylation. 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. lonomycin treatment of these cells resulted in increased kinase activity of cytosolic extracts toward syntide-2, a synthetic peptide substrate for calcium/calmodulin-dependent kinase I I (CaM-KII), with kinetics similar to those of CD20 phosphorylation in the cell line. CD20 isolated from the cell line was a substrate for p urified CaM-KII in vitro. Phosphopeptide maps of CD20 from untreated h airy cells or ionomycin-treated HCLL-7876 cells were similar to maps o f CD20 that had been phosphorylated in vitro by CaM-KII. These results suggest that the unusually high levels of intracytoplasmic Ca2+ in ha iry cells may enhance the phosphorylation of key surface proteins.