The Arabidopsis immune receptor FLS2 perceives bacterial flagellin epitope flg22 to activate defenses through the central cytoplasmic kinase BIK1. The heterotrimeric G proteins composed of the non-canonical Gα protein XLG2, the Gβ protein AGB1, and the Gγ proteins AGG1 and AGG2 are required for FLS2-mediated immune responses through an unknown mechanism. Here we show that in the pre-activation state, XLG2 directly interacts with FLS2 and BIK1, and it functions together with AGB1 and AGG1/2 to attenuate proteasome-mediated degradation of BIK1, allowing optimum immune activation. Following the activation by flg22, XLG2 dissociates from AGB1 and is phosphorylated by BIK1 in the N terminus. The phosphorylated XLG2 enhances the production of reactive oxygen species (ROS) likely by modulating the NADPH oxidase RbohD. The study demonstrates that the G proteins are directly coupled to the FLS2 receptor complex and regulate immune signaling through both pre-activation and post-activation mechanisms.
- Thorsten Nürnberger, University of Tubingen, Germany
© 2016, Liang et al.
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Trans-differentiation of hepatic stellate cells (HSCs) to activated state potentiates liver fibrosis through release of extracellular matrix (ECM) components, distorting the liver architecture. Since limited antifibrotics are available, pharmacological intervention targeting activated HSCs may be considered for therapy. A-kinase anchoring protein 12 (AKAP12) is a scaffolding protein that directs protein kinases A/C (PKA/PKC) and cyclins to specific locations spatiotemporally controlling their biological effects. It has been shown that AKAP12’s scaffolding functions are altered by phosphorylation. In previously published work, observed an association between AKAP12 phosphorylation and HSC activation. In this work, we demonstrate that AKAP12’s scaffolding activity toward the endoplasmic reticulum (ER)-resident collagen chaperone, heat-shock protein 47 (HSP47) is strongly inhibited by AKAP12’s site-specific phosphorylation in activated HSCs. CRISPR-directed gene editing of AKAP12’s phospho-sites restores its scaffolding toward HSP47, inhibiting HSP47’s collagen maturation functions, and HSC activation. AKAP12 phospho-editing dramatically inhibits fibrosis, ER stress response, HSC inflammatory signaling, and liver injury in mice. Our overall findings suggest a pro-fibrogenic role of AKAP12 phosphorylation that may be targeted for therapeutic intervention in liver fibrosis.
PCSK9 negatively regulates low-density lipoprotein receptor (LDLR) abundance on the cell surface, leading to decreased hepatic clearance of LDL particles and increased levels of plasma cholesterol. We previously identified SURF4 as a cargo receptor that facilitates PCSK9 secretion in HEK293T cells (Emmer et al., 2018). Here, we generated hepatic SURF4-deficient mice (Surf4fl/fl Alb-Cre+) to investigate the physiologic role of SURF4 in vivo. Surf4fl/fl Alb-Cre+ mice exhibited normal viability, gross development, and fertility. Plasma PCSK9 levels were reduced by ~60% in Surf4fl/fl Alb-Cre+ mice, with a corresponding ~50% increase in steady state LDLR protein abundance in the liver, consistent with SURF4 functioning as a cargo receptor for PCSK9. Surprisingly, these mice exhibited a marked reduction in plasma cholesterol and triglyceride levels out of proportion to the partial increase in hepatic LDLR abundance. Detailed characterization of lipoprotein metabolism in these mice instead revealed a severe defect in hepatic lipoprotein secretion, consistent with prior reports of SURF4 also promoting the secretion of apolipoprotein B. Despite a small increase in liver mass and lipid content, histologic evaluation revealed no evidence of steatohepatitis or fibrosis in Surf4fl/fl Alb-Cre+ mice. Acute depletion of hepatic SURF4 by CRISPR/Cas9 or liver-targeted siRNA in adult mice confirms these findings. Together, these data support the physiologic significance of SURF4 in the hepatic secretion of PCSK9 and APOB-containing lipoproteins and its potential as a therapeutic target in atherosclerotic cardiovascular diseases.