Londino James D, Gulick Dexter L, Lear Travis B, Suber Tomeka L, Weathington Nathaniel M, Masa Luke S, Chen Bill B, Mallampalli Rama K
Acute Lung Injury Center of Excellence, Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, U.S.A.
Medical Specialty Service Line, Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, PA, U.S.A.
Biochem J. 2017 Oct 10;474(20):3543-3557. doi: 10.1042/BCJ20170548.
The IFN gamma receptor 1 (IFNGR1) binds IFN-γ and activates gene transcription pathways crucial for controlling bacterial and viral infections. Although decreases in IFNGR1 surface levels have been demonstrated to inhibit IFN-γ signaling, little is known regarding the molecular mechanisms controlling receptor stability. Here, we show in epithelial and monocytic cell lines that IFNGR1 displays K48 polyubiquitination, is proteasomally degraded, and harbors three ubiquitin acceptor sites at K277, K279, and K285. Inhibition of glycogen synthase kinase 3 beta (GSK3β) destabilized IFNGR1 while overexpression of increased receptor stability. We identified critical serine and threonine residues juxtaposed to ubiquitin acceptor sites that impacted IFNGR1 stability. In CRISPR-Cas9 IFNGR1 generated knockout cell lines, cellular expression of plasmids encoding ubiquitin acceptor site mutations demonstrated significantly impaired STAT1 phosphorylation and decreased STAT1-dependent gene induction. Thus, IFNGR1 undergoes rapid site-specific polyubiquitination, a process modulated by GSK3β. Ubiquitination appears to be necessary for efficient IFNGR1-dependent gamma gene induction and represents a relatively uncharacterized regulatory mechanism for this receptor.
干扰素γ受体1（IFNGR1）结合干扰素-γ并激活对控制细菌和病毒感染至关重要的基因转录途径。尽管已证明IFNGR1表面水平的降低会抑制干扰素-γ信号传导，但对于控制受体稳定性的分子机制知之甚少。在此，我们在上皮细胞和单核细胞系中表明，IFNGR1显示K48多聚泛素化，经蛋白酶体降解，并在K277、K279和K285处有三个泛素受体位点。糖原合酶激酶3β（GSK3β）的抑制使IFNGR1不稳定，而其过表达则增加了受体稳定性。我们确定了与泛素受体位点相邻的关键丝氨酸和苏氨酸残基，它们影响IFNGR1的稳定性。在CRISPR-Cas9生成的IFNGR1基因敲除细胞系中，编码泛素受体位点突变的质粒的细胞表达显示STAT1磷酸化显著受损，STAT1依赖性基因诱导减少。因此，IFNGR1经历快速的位点特异性多聚泛素化，这一过程由GSK3β调节。泛素化似乎是有效的IFNGR1依赖性γ基因诱导所必需的，并且代表了该受体一种相对未被充分表征的调节机制。
