Genot E M, Meier K E, Licciardi K A, Ahn N G, Uittenbogaart C H, Wietzerbin J, Clark E A, Valentine M A
Unite INSERM 196, Institut Curie, Paris, France.
J Immunol. 1993 Jul 1;151(1):71-82.
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.
毛细胞白血病是一种病因不明的罕见B细胞淋巴增殖性疾病。我们之前观察到，参与B细胞活化的膜蛋白CD20在毛细胞上发生过度磷酸化，且这些细胞的细胞内游离Ca2+水平异常高。因此，我们使用毛细胞系HCLL-7876来研究Ca(2+)激活的蛋白激酶在CD20磷酸化中的潜在作用。添加Ca2+离子载体离子霉素可增加活化B细胞和毛细胞系细胞中CD20的磷酸化；向任何一种细胞类型中添加EGTA均可降低CD20磷酸化的基础水平。用离子霉素处理这些细胞会导致胞质提取物对syntide-2（一种钙/钙调蛋白依赖性激酶II（CaM-KII）的合成肽底物）的激酶活性增加，其动力学与细胞系中CD20磷酸化的动力学相似。从细胞系中分离出的CD20在体外是纯化的CaM-KII的底物。未经处理的毛细胞或经离子霉素处理的HCLL-7876细胞的CD20磷酸肽图谱与体外经CaM-KII磷酸化的CD20图谱相似。这些结果表明，毛细胞中异常高的胞质内Ca2+水平可能会增强关键表面蛋白的磷酸化。
