Harraz Maged M, Park Andrea, Abbott Duane, Zhou Weihong, Zhang Yulong, Engelhardt John F
Department of Anatomy & Cell Biology, The University of Iowa, Iowa City, Iowa 52242, USA.
Antioxid Redox Signal. 2007 Nov;9(11):1803-13. doi: 10.1089/ars.2007.1579.
Rac-dependent NADPH oxidases generate reactive oxygen species used in cell signaling and microbial killing or both. Whereas the mechanisms leading to NADPH oxidase activation are fairly well studied, the mechanisms that control downregulation of this enzyme complex remain unclear. We hypothesized that reactive oxygen species produced by NADPH oxidase may autoregulate the complex by inhibiting Rac activity. To this end, we searched for binding partners of Rac1 and identified a tyrosine-phosphorylated fragment of MKK6 that bound to Rac1 under redox-stress conditions. Constitutively active MKK6 interacted directly with Rac1 in vitro, and this interaction was enhanced when MKK6 was phosphorylated on tyrosine 219. Both Rac1 and Rac2 immunoprecipitated an MKK6 fragment under conditions that elevate cellular peroxide levels in 293 and RAW cells, respectively. Constitutively active and wild-type MKK6 enhanced Rac-GTPase activity in vitro, and their overexpression inhibited PMA-induced NADPH oxidase activation in RAW cells. In contrast, a Y219F mutant of MKK6 only partially enhanced Rac1 GTPase activity, and its overexpression did not alter PMA-induced NADPH oxidase activation in RAW cells. Last, MKK6 deficiency led to an increase in Rac1-GTP levels in brain tissue. Our findings suggest that MKK6 downregulates NADPH oxidase activity by enhancing Rac-GTPase activity.
Rac 依赖性 NADPH 氧化酶产生活性氧，用于细胞信号传导和杀灭微生物，或两者兼而有之。虽然导致 NADPH 氧化酶激活的机制已得到相当充分的研究，但控制该酶复合物下调的机制仍不清楚。我们推测，NADPH 氧化酶产生的活性氧可能通过抑制 Rac 活性对该复合物进行自动调节。为此，我们寻找了 Rac1 的结合伙伴，并鉴定出一个在氧化还原应激条件下与 Rac1 结合的 MKK6 酪氨酸磷酸化片段。组成型活性 MKK6 在体外直接与 Rac1 相互作用，当 MKK6 的酪氨酸 219 位点被磷酸化时，这种相互作用增强。在分别提高 293 细胞和 RAW 细胞中细胞过氧化物水平的条件下，Rac1 和 Rac2 均免疫沉淀出一个 MKK6 片段。组成型活性和野生型 MKK6 在体外增强了 Rac 鸟苷三磷酸酶活性，它们的过表达抑制了 RAW 细胞中佛波酯（PMA）诱导的 NADPH 氧化酶激活。相比之下，MKK6 的 Y219F 突变体仅部分增强了 Rac1 鸟苷三磷酸酶活性，其过表达并未改变 RAW 细胞中 PMA 诱导的 NADPH氧化酶激活。最后，MKK6 缺陷导致脑组织中 Rac1 - GTP 水平升高。我们的研究结果表明，MKK6 通过增强 Rac 鸟苷三磷酸酶活性下调 NADPH 氧化酶活性。
