Effects of p47phox C terminus phosphorylations on binding interactions with p40phox and p67phox -: Structural and functional comparison of p40phox and p67phox SH3 domains
Effects of p47phox C terminus phosphorylations on binding interactions with p40phox and p67phox -: Structural and functional comparison of p40phox and p67phox SH3 domains
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DOI：
10.1074/jbc.m412897200
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发表时间：
2005-04-08
影响因子：
4.8
通讯作者：
Fieschi, F
中科院分区：
文献类型：
--
作者：
Massenet, C;Chenavas, S;Fieschi, F
中性粒细胞NADPH氧化酶在感染反应中产生超氧阴离子。该反应是由胞质因子p47（phox）和p67（phox）以及一个小G蛋白Rac与膜性黄细胞色素b（558）结合而激活的。另一种胞质因子p40（phox）与该复合体有关，据报道起调节作用。据报道，NADPH氧化酶激活级联的启动与p47(phox) C末端的359/370和379丝氨酸的磷酸化是连续的。这些丝氨酸围绕着一个聚脯氨酸基序，该基序可以与p40（phox）的Src同源3（SH3）模块（SH3(p40)）或p67（phox）的c端SH3（C-SH3(p67)）相互作用。后者在静息状态下对p47（phox）具有更高的亲和力。磷酸化后SH3结合偏好的改变已经在早期被假设。在这里，我们报道了SH3（p40）单独或与p47（phox）的富含12个残基脯氨酸区域的复合物在1.46埃分辨率下的晶体结构。利用固有色氨酸荧光测量，我们比较了严格的脯氨酸基序和整个C端肽与SH3（p40）和C-SH3（p67）的亲和力。这些数据表明SH3（p40）可以与一个公认的聚脯氨酸基序相互作用，也可以与p47(phox) C末端的一个非规范基序相互作用。这两种SH3的静电表面非常不同，因此C-SH3p67的结合偏好可归因于聚脯氨酸基序识别，特别是Arg-368（p47）的结合模式。非规范基序对与两个SH3的相互作用贡献相同。测定了359/370和379残基丝氨酸磷酸化对两个SH3结构域亲和力的影响。我们的结论与之前的建议相反，Ser-359/370的磷酸化不会改变SH3与SH3的结合亲和力，而Ser-379的磷酸化对这两种相互作用都有不稳定的影响。因此，除了磷酸化诱导的两个SH3之间的转换之外，NADPH氧化酶激活级联必须发生其他机制。
The neutrophil NADPH oxidase produces superoxide anions in response to infection. This reaction is activated by association of cytosolic factors, p47(phox) and p67(phox), and a small G protein Rac with the membranous flavocytochrome b(558). Another cytosolic factor, p40(phox), is associated to the complex and is reported to play regulatory roles. Initiation of the NADPH oxidase activation cascade has been reported as consecutive to phosphorylation on serines 359/370 and 379 of the p47(phox) C terminus. These serines surround a polyproline motif that can interact with the Src homology 3 (SH3) module of p40(phox) (SH3(p40)) or the C-terminal SH3 of p67(phox) (C-SH3(p67)). The latter one presents a higher affinity in the resting state for p47(phox). A change in SH3 binding preference following phosphorylation has been postulated earlier. Here we report the crystal structures of SH3(p40) alone or in complex with a 12-residue proline-rich region of p47(phox) at 1.46 angstrom resolution. Using intrinsic tryptophan fluorescence measurements, we compared the affinity of the strict polyproline motif and the whole C terminus peptide with both SH3(p40) and C-SH3(p67). These data reveal that SH3(p40) can interact with a consensus polyproline motif but also with a noncanonical motif of the p47(phox) C terminus. The electrostatic surfaces of both SH3 are very different, and therefore the binding preference for C-SH3p67 can be attributed to the polyproline motif recognition and particularly to the Arg-368(p47) binding mode. The noncanonical motif contributes equally to interaction with both SH3. The influence of serine phosphorylation on residues 359/370 and 379 on the affinity for both SH3 domains has been checked. We conclude that contrarily to previous suggestions, phosphorylation of Ser-359/370 does not modify the SH3 binding affinity for both SH3, whereas phosphorylation of Ser-379 has a destabilizing effect on both interactions. Other mechanisms than a phosphorylation induced switch between the two SH3 must therefore take place for NADPH oxidase activation cascade to start.