Basu Uttiya, Chaudhuri Jayanta, Alpert Craig, Dutt Shilpee, Ranganath Sheila, Li Gang, Schrum Jason Patrick, Manis John P, Alt Frederick W
The Howard Hughes Medical Institute, The Children's Hospital, The CBR Institute for Biomedical Research, and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Nature. 2005 Nov 24;438(7067):508-11. doi: 10.1038/nature04255. Epub 2005 Oct 26.
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). Somatic hypermutation and CSR both require the B-cell-specific activation-induced cytidine deaminase protein (AID), which initiates these reactions through its single-stranded (ss)DNA-specific cytidine deaminase activity. In biochemical assays, replication protein A (RPA), a ssDNA-binding protein, 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. 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.
由B淋巴细胞产生的抗体由免疫球蛋白重链（IgH）和轻链（IgL）组成，这些链具有氨基末端可变区和羧基末端恒定区。作为对抗原的反应，B细胞经历两种类型的基因组改变以增加抗体多样性。通过体细胞高频突变在IgH和IgL可变区引入点突变可增加对抗原的亲和力。此外，通过IgH类别转换重组（CSR）改变表达的IgH恒定区外显子可改变抗体效应功能。体细胞高频突变和CSR都需要B细胞特异性激活诱导的胞苷脱氨酶蛋白（AID），它通过其单链（ss）DNA特异性胞苷脱氨酶活性启动这些反应。在生化分析中，复制蛋白A（RPA），一种ssDNA结合蛋白，与活化B细胞中磷酸化的AID结合，并增强AID对含有体细胞高频突变或CSR靶序列的转录双链（ds）DNA的活性。这种需要磷酸化的AID-RPA结合可能提供一种机制，使AID能够在活化的B细胞中接近dsDNA靶标。在这里，我们表明B细胞来源的AID在一个共有蛋白激酶A（PKA）位点上被磷酸化，并且PKA是生理性的AID激酶。因此，非淋巴细胞来源的AID可被重组PKA进行功能性磷酸化，以允许与RPA相互作用并促进转录dsDNA底物的脱氨作用。此外，AID主要PKA磷酸化位点的突变保留了ssDNA脱氨活性，但显著降低了RPA依赖性dsDNA脱氨活性，并严重损害了AID在体内影响CSR的能力。我们得出结论，PKA在B细胞中AID活性的翻译后调控中起关键作用。
