Identification of Thr29 as a Critical Phosphorylation Site That Activates the Human Proton Channel Hvcn1 in Leukocytes
Identification of Thr29 as a Critical Phosphorylation Site That Activates the Human Proton Channel Hvcn1 in Leukocytes
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DOI：
10.1074/jbc.c109.082727
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发表时间：
2010-02-19
影响因子：
4.8
通讯作者：
DeCoursey, Thomas E.
中科院分区：
文献类型：
--
作者：
Musset, Boris;Capasso, Melania;DeCoursey, Thomas E.
电压门控质子通道和NADPH氧化酶在呼吸爆发期间在吞噬细胞中协同作用，此时产生活性氧以杀死微生物入侵者。激活NADPH氧化酶的试剂还显著增强质子通道门控，促进其在电荷补偿和pH（i）调节中的作用。"增强门控模式"似乎反映蛋白激酶C（PKC）磷酸化。在这里，我们研究了人类电压门控质子通道中PKC-δ磷酸化位点的两个候选者，H（V）1（Hvcn1），Thr（29）和Ser（97），都在细胞内N端。通过使用PKC-δ的体外激酶测定，在单突变体S97A或T29A中，以及在双突变体T29A/S97A中，通道磷酸化降低。通过在LK35.2细胞（一种B细胞杂交瘤）中表达野生型（WT）或突变型H（V）1通道来评估增强的门控。佛波醇肉豆蔻酸醋酸酯的刺激增强了WT通道门控，并且这种作用可通过PKC抑制剂GF109203 X治疗逆转。单突变体T29A或双突变体T29A/S97A对佛波醇肉豆蔻酸酯或GF109203X没有反应。相反，S97A突变体的反应类似于用WT H（V）1转染的细胞。我们的结论是，在这些条件下，直接磷酸化的质子通道分子在Thr（29）是主要负责质子通道门控的增强。这种磷酸化对吞噬细胞呼吸爆发期间质子传导的激活至关重要。
Voltage-gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Agents that activate NADPH oxidase also enhance proton channel gating profoundly, facilitating its roles in charge compensation and pH(i) regulation. The "enhanced gating mode" appears to reflect protein kinase C (PKC) phosphorylation. Here we examine two candidates for PKC-delta phosphorylation sites in the human voltage-gated proton channel, H(V)1 (Hvcn1), Thr(29) and Ser(97), both in the intracellular N terminus. Channel phosphorylation was reduced in single mutants S97A or T29A, and further in the double mutant T29A/S97A, by an in vitro kinase assay with PKC-delta. Enhanced gating was evaluated by expressing wild-type (WT) or mutant H(V)1 channels in LK35.2 cells, a B cell hybridoma. Stimulation by phorbol myristate acetate enhanced WT channel gating, and this effect was reversed by treatment with the PKC inhibitor GF109203X. The single mutant T29A or double mutant T29A/S97A failed to respond to phorbol myristate acetate or GF109203X. In contrast, the S97A mutant responded like cells transfected with WT H(V)1. We conclude that under these conditions, direct phosphorylation of the proton channel molecule at Thr(29) is primarily responsible for the enhancement of proton channel gating. This phosphorylation is crucial to activation of the proton conductance during the respiratory burst in phagocytes.