Activation of p47PHOX, a cytosolic subunit of the leukocyte NADPH oxidase -: Phosphorylation of Ser-359 or Ser-370 precedes phosphorylation at other sites and is required for activity
Activation of p47PHOX, a cytosolic subunit of the leukocyte NADPH oxidase -: Phosphorylation of Ser-359 or Ser-370 precedes phosphorylation at other sites and is required for activity
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DOI：
10.1074/jbc.273.52.35147
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发表时间：
1998-12-25
影响因子：
4.8
通讯作者：
Babior, BM
中科院分区：
文献类型：
--
作者：
Johnson, JL;Park, JW;Babior, BM
白细胞NADPH氧化酶催化氧还原为超氧化物（O-2（自由基阴离子），以吞噬细胞和B淋巴细胞中的NADPH为代价。该酶在静息细胞中处于休眠状态，但当细胞暴露于适当的刺激时变得活跃。在氧化酶激活过程中，高碱性胞质氧化酶成分p47（PHOX）变成 在几个丝氨酸上磷酸化并迁移到质膜。我们在此报告，与相同细胞相比，表达 p47(PHOX) S359A/S370A 或 p47PHOX S359K/S370K 双突变的 p47(PHOX) 缺陷 B 淋巴母细胞显示酶活性和 p47PHOX 磷酸化水平显着降低 表达野生型 p47PHOX。另外，当细胞受到刺激时，这些突变的 p47PHOX 蛋白无法转位到质膜。相反，在第359和370位含有天冬氨酸或谷氨酸的突变体中观察到正常的磷酸化和易位，但氧化酶活性仍然大大降低。这些结果意味着位置 359 和/或 370 处的负电荷是 足以使 p47(PHOX) 发生磷酸化和易位，但需要磷酸化羟基氨基酸独有的功能来支持 O-2（自由基阴离子）的产生。这些发现加上早期研究的结果（Inanami, O.、Johnson, J, L.、McAdara, J. K.、El Benna, J.、Faust, L. P.、Newburger, P, E. 和 Babior， B, M. (1998) J. Biol。化学。 273, 9539-9543)，表明氧化酶激活需要 1) p47(PHOX) 上至少两个丝氨酸的顺序磷酸化：Ser-359 或 Ser-370，然后是 Ser-303 或 Ser-304； 2) 在第一次磷酸化发生后的某个时刻，p47(PHOX) 易位至膜上。
The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (O-2(radical anion) at the expense of NADPH in phagocytes and B lymphocytes, The enzyme is dormant in resting cells but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(PHOX) becomes phosphorylated on several serines and migrates to the plasma membrane. We report here that p47(PHOX)-deficient B lymphoblasts expressing the p47(PHOX) S359A/S370A or p47PHOX S359K/S370K double mutation show dramatically reduced levels of enzyme activity and phosphorylation of p47PHOX as compared with the same cells expressing wild type p47PHOX. I, addition, these mutant p47PHOX proteins fails to translocate to the plasma membrane when the cells are stimulated. In contrast, normal phosphorylation and translocation are seen in mutants containing aspartate or glutamate at positions 359 and 370, but oxidase activity is still greatly reduced. These results imply that a negative charge at position 359 and/or 370 is sufficient to allow the phosphorylation and translocation of p47(PHOX) to take place but that features unique to a phosphorylated hydroxyamino acid are required to support O-2(radical anion) production. These findings, plus those from an earlier study (Inanami, O., Johnson, J, L., McAdara, J. K., El Benna, J., Faust, L. P., Newburger, P, E., and Babior, B, M. (1998) J. Biol. Chem. 273, 9539-9543), suggest that oxidase activation requires 1) the sequential phosphorylation of at least two serines on p47(PHOX): Ser-359 or Ser-370, followed by Ser-303 or Ser-304; and 2) the translocation of p47(PHOX) to the membrane at some point after the first phosphorylation takes place.