Valentine M A, Meier K E, Rossie S, Clark E A
Department of Microbiology, University of Washington, Seattle 98195.
J Biol Chem. 1989 Jul 5;264(19):11282-7.
CD20, a B cell integral membrane protein, regulates B cell activation and is differently phosphorylated in resting and activated cells. We have previously shown that CD20 phosphorylation is increased in activated cells and in phorbol ester-treated resting cells. Phosphorylation is also altered by agents which signal B cell proliferation, such as anti-IgM and a B cell growth factor. The present study was designed to address whether protein kinase C (PKC) or other kinases used CD20 as a substrate. When purified PKC was incubated with isolated CD20, both the 35- and 37-kDa CD20 proteins were phosphorylated in vitro. Intact resting B cells were next incubated with the protein kinase inhibitors H-7, H-8, and W-7. No change in basal CD20 phosphorylation was observed in the presence of W-7 and H-8, indicating that the protein cyclic nucleotide-dependent and calmodulin-dependent kinases were not actively phosphorylating CD20. Surprisingly, the PKC inhibitor H-7 increased CD20 phosphorylation at concentrations above 25-50 microM. To assess whether PKC either activated a phosphatase or inactivated a kinase affecting CD20 phosphorylation, tryptic phosphopeptide mapping of CD20 was performed. These studies revealed that addition of phorbol 12-myristate 13-acetate increased phosphorylation of some peptides differing from those which had increased phosphorylation following addition of H-7. Furthermore, signalling through surface immunoglobulin increased phosphorylation of CD20 peptides distinct from those hyperphosphorylated following addition of phorbol 12-myristate 13-acetate. These results demonstrate that 1) CD20 has multiple phosphorylation sites, as predicted from sequence data, and 2) whereas PKC can use CD20 as substrate, at least one other unidentified kinase phosphorylates CD20 in resting cells. Our data also predict that activation of B cells via the antigen receptor (surface IgM) may activate other protein kinases in addition to PKC.
CD20是一种B细胞整合膜蛋白，可调节B细胞活化，且在静息细胞和活化细胞中的磷酸化情况不同。我们之前已经表明，活化细胞和佛波酯处理的静息细胞中CD20的磷酸化水平会升高。磷酸化也会被一些引发B细胞增殖的因子改变，比如抗IgM和一种B细胞生长因子。本研究旨在探讨蛋白激酶C（PKC）或其他激酶是否将CD20用作底物。当将纯化的PKC与分离出的CD20一起孵育时，35 kDa和37 kDa的CD20蛋白在体外均被磷酸化。接下来，将完整的静息B细胞与蛋白激酶抑制剂H-7、H-8和W-7一起孵育。在W-7和H-8存在的情况下，未观察到基础CD20磷酸化有变化，这表明蛋白环核苷酸依赖性激酶和钙调蛋白依赖性激酶并未积极地使CD20磷酸化。令人惊讶的是，PKC抑制剂H-7在浓度高于25 - 50 μM时会增加CD20的磷酸化。为了评估PKC是否激活了一种磷酸酶或使一种影响CD20磷酸化的激酶失活，对CD20进行了胰蛋白酶磷酸肽图谱分析。这些研究表明，添加佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯会增加一些肽段的磷酸化，这些肽段与添加H-7后磷酸化增加的肽段不同。此外，通过表面免疫球蛋白发出的信号会增加CD20肽段的磷酸化，这些肽段与添加佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯后过度磷酸化的肽段不同。这些结果表明：1）如序列数据所预测的，CD20有多个磷酸化位点；2）虽然PKC可以将CD20用作底物，但至少还有一种未鉴定的激酶在静息细胞中使CD20磷酸化。我们的数据还预测，通过抗原受体（表面IgM）激活B细胞可能除了激活PKC外还会激活其他蛋白激酶。
