The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation
The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation
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DOI：
10.1038/nature04255
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发表时间：
2005-11-24
期刊：
影响因子：
64.8
通讯作者：
Alt, FW
中科院分区：
文献类型：
--
作者：
Basu, U;Chaudhuri, J;Alt, FW
抗体由b系细胞产生，由免疫球蛋白重链（IgH）和轻链（IgL）组成，具有氨基末端可变区和羧基末端恒定区。在对抗原的反应中，B细胞经历两种类型的基因组改变来增加抗体多样性。通过体细胞超突变将点突变引入IgH和IgL可变区，可以增加对抗原的亲和力。此外，通过IgH类开关重组（CSR）改变表达的IgH恒定区外显子可以改变抗体效应函数（1-3）。体细胞超突变和CSR都需要b细胞特异性激活诱导的胞苷脱氨酶蛋白（AID）（4-6）， AID通过其单链dna特异性胞苷脱氨酶活性启动这些反应（7-11）。在生化分析中，复制蛋白A （RPA）是一种ssdna结合蛋白（12），与活化B细胞磷酸化的AID结合，并增强含有体细胞超突变或CSR靶序列的转录双链（ds）DNA上的AID活性。这种需要磷酸化的AID- rpa关联可能提供了一种允许AID进入活化B细胞中的dsDNA靶点的机制（13,14）。在这里，我们发现来自B细胞的AID在一个共识蛋白激酶a （PKA）位点被磷酸化，PKA是生理上的AID激酶。因此，来自非淋巴细胞的AID可以被重组PKA功能性磷酸化，从而允许与RPA相互作用，促进转录的dsDNA底物的脱氨作用。此外，AID的主要PKA磷酸化位点突变保留了ssDNA脱胺活性，但显著降低了rpa依赖性的dsDNA脱胺活性，严重损害了AID在体内影响CSR的能力。我们得出结论，PKA在B细胞AID活性的翻译后调控中起关键作用。
Antibodies, which are produced by B-lineage cells, consist of immunoglobulin heavy (IgH) and light (IgL) chains that have amino-terminal variable regions and carboxy-terminal constant regions. In response to antigens, B cells undergo two types of genomic alterations to increase antibody diversity. Affinity for antigen can be increased by introduction of point mutations into IgH and IgL variable regions by somatic hypermutation. In addition, antibody effector functions can be altered by changing the expressed IgH constant region exons through IgH class switch recombination (CSR)(1-3). Somatic hypermutation and CSR both require the B-cell-specific activation-induced cytidine deaminase protein (AID)(4-6), which initiates these reactions through its single-stranded (ss)DNA-specific cytidine deaminase activity(7-11). In biochemical assays, replication protein A (RPA), a ssDNA-binding protein(12), associates with phosphorylated AID from activated B cells and enhances AID activity on transcribed double-stranded (ds)DNA containing somatic hypermutation or CSR target sequences. This AID-RPA association, which requires phosphorylation, may provide a mechanism for allowing AID to access dsDNA targets in activated B cells(13,14). Here we show that AID from B cells is phosphorylated on a consensus protein kinase A (PKA) site and that PKA is the physiological AID kinase. Thus, AID from non-lymphoid cells can be functionally phosphorylated by recombinant PKA to allow interaction with RPA and promote deamination of transcribed dsDNA substrates. Moreover, mutation of the major PKA phosphorylation site of AID preserves ssDNA deamination activity, but markedly reduces RPA-dependent dsDNA deamination activity and severely impairs the ability of AID to effect CSR in vivo. We conclude that PKA has a critical role in post-translational regulation of AID activity in B cells.