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Scaffold proteins tether and orient components of a signaling cascade to facilitate signaling. Although much is known about how scaffolds colocalize signaling proteins, it is unclear whether scaffolds promote signal amplification. Here, we used arrestin-3, a scaffold of the ASK1-MKK4/7-JNK3 cascade, as a model to understand signal amplification by a scaffold protein. We found that arrestin-3 exhibited >15-fold higher affinity for inactive JNK3 than for active JNK3, and this change involved a shift in the binding site following JNK3 activation. We used systems biochemistry modeling and Bayesian inference to evaluate how the activation of upstream kinases contributed to JNK3 phosphorylation. Our combined experimental and computational approach suggested that the catalytic phosphorylation rate of JNK3 at Thr-221 by MKK7 is two orders of magnitude faster than the corresponding phosphorylation of Tyr-223 by MKK4 with or without arrestin-3. Finally, we showed that the release of activated JNK3 was critical for signal amplification. Collectively, our data suggest a "conveyor belt" mechanism for signal amplification by scaffold proteins. This mechanism informs on a long-standing mystery for how few upstream kinase molecules activate numerous downstream kinases to amplify signaling.
The NAD+-dependent deacetylase SIRT2 is unique amongst sirtuins as it is effective in the cytosol, as well as the mitochondria. Defining the role of cytosolic acetylation state in specific tissues is difficult since even physiological effects at the whole body level are unknown. We hypothesized that genetic SIRT2 knockout (KO) would lead to impaired insulin action, and that this impairment would be worsened in HF fed mice. Insulin sensitivity was tested using the hyperinsulinemic-euglycemic clamp in SIRT2 KO mice and WT littermates. SIRT2 KO mice exhibited reduced skeletal muscle insulin-induced glucose uptake compared to lean WT mice, and this impairment was exacerbated in HF SIRT2 KO mice. Liver insulin sensitivity was unaffected in lean SIRT2 KO mice. However, the insulin resistance that accompanies HF-feeding was worsened in SIRT2 KO mice. It was notable that the effects of SIRT2 KO were largely disassociated from cytosolic acetylation state, but were closely linked to acetylation state in the mitochondria. SIRT2 KO led to an increase in body weight that was due to increased food intake in HF fed mice. In summary, SIRT2 deletion in vivo reduces muscle insulin sensitivity and contributes to liver insulin resistance by a mechanism that is unrelated to cytosolic acetylation state. Mitochondrial acetylation state and changes in feeding behavior that result in increased body weight correspond to the deleterious effects of SIRT2 KO on insulin action.
Methylmercury is a toxic environmental contaminant that elicits significant toxicity in humans. The central nervous system is the primary target of toxicity, and is particularly vulnerable during development. Rho-associated protein kinase 1 (ROCK-1) is a major downstream effector of the small GTPase RhoA and a direct substrate of caspase-3. The activation of ROCK-1 is necessary for membrane blebbing during apoptosis. In this work, we examined whether MeHg could affect the RhoA/ROCK-1 signaling pathway in primary cultures of mouse astrocytes. Exposure of cells with 10 μM MeHg decreased cellular viability after 24 h of incubation. This reduction in viability was preceded by a significant increase in intracellular and mitochondrial reactive oxygen species levels, as well as a reduced NAD/NADH ratio. MeHg also induced an increase in mitochondrial-dependent caspase-9 and caspase-3, while the levels of RhoA protein expression were reduced or unchanged. We further found that MeHg induced ROCK-1 cleavage/activation and promoted LIMK1 and MYPT1 phosphorylation, both of which are the best characterized ROCK-1 downstream targets. Inhibiting ROCK-1 and caspases activation attenuated the MeHg-induced cell death. Collectively, these findings are the first to show that astrocytes exposed to MeHg showed increased cleavage/activation of ROCK-1, which was independent of the small GTPase RhoA.
Copyright © 2018. Published by Elsevier Ltd.
Wnt/β-catenin signaling is necessary for normal lung development, and abnormal Wnt signaling contributes to the pathogenesis of both bronchopulmonary dysplasia (BPD) and idiopathic pulmonary fibrosis (IPF), fibrotic lung diseases that occur during infancy and aging, respectively. Using a library of human normal and diseased human lung samples, we identified a distinct signature of nuclear accumulation of β-catenin phosphorylated at tyrosine 489 and epithelial cell cytosolic localization of β-catenin phosphorylated at tyrosine 654 in early normal lung development and fibrotic lung diseases BPD and IPF. Furthermore, this signature was recapitulated in murine models of BPD and IPF. Image analysis of immunofluorescence colocalization demonstrated a consistent pattern of elevated nuclear phosphorylated β-catenin in the lung epithelium and surrounding mesenchyme in BPD and IPF, closely resembling the pattern observed in 18-week fetal lung. Nuclear β-catenin phosphorylated at tyrosine 489 associated with an increased expression of Wnt target gene AXIN2, suggesting that the observed β-catenin signature is of functional significance during normal development and injury repair. The association of specific modifications of β-catenin during normal lung development and again in response to lung injury supports the widely held concept that repair of lung injury involves the recapitulation of developmental programs. Furthermore, these observations suggest that β-catenin phosphorylation has potential as a therapeutic target for the treatment and prevention of both BPD and IPF.
Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
In , cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring (CR). A single essential formin, Cdc12, localizes to the cell middle upon mitotic onset and nucleates the F-actin of the CR. Cdc12 medial recruitment is mediated in part by its direct binding to the F-BAR scaffold Cdc15. Given that Cdc12 is hyperphosphorylated in M phase, we explored whether Cdc12 phosphoregulation impacts its association with Cdc15 during mitosis. We found that Cdk1, a major mitotic kinase, phosphorylates Cdc12 on six N-terminal residues near the Cdc15-binding site, and phosphorylation on these sites inhibits its interaction with the Cdc15 F-BAR domain. Consistent with this finding, a mutant with all six Cdk1 sites changed to phosphomimetic residues () displays phenotypes similar to , in which the Cdc15-binding motif is disrupted; both show reduced Cdc12 at the CR and delayed CR formation. Together, these results indicate that Cdk1 phosphorylation of formin Cdc12 antagonizes its interaction with Cdc15 and thereby opposes Cdc12's CR localization. These results are consistent with a general role for Cdk1 in inhibiting cytokinesis until chromosome segregation is complete.
© 2018 Willet et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
An increase in hepatic glucose production (HGP) is a key feature of type 2 diabetes. Excessive signaling through hepatic Gs-linked glucagon receptors critically contributes to pathologically elevated HGP. Here, we tested the hypothesis that this metabolic impairment can be counteracted by enhancing hepatic Gi signaling. Specifically, we used a chemogenetic approach to selectively activate Gi-type G proteins in mouse hepatocytes in vivo. Unexpectedly, activation of hepatic Gi signaling triggered a pronounced increase in HGP and severely impaired glucose homeostasis. Moreover, increased Gi signaling stimulated glucose release in human hepatocytes. A lack of functional Gi-type G proteins in hepatocytes reduced blood glucose levels and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Additionally, we delineated a signaling cascade that links hepatic Gi signaling to ROS production, JNK activation, and a subsequent increase in HGP. Taken together, our data support the concept that drugs able to block hepatic Gi-coupled GPCRs may prove beneficial as antidiabetic drugs.
Purpose - The purpose of this study was to identify the molecular defect in the disease-causing human arrestin-1 C147F mutant.
Methods - The binding of wild-type (WT) human arrestin-1 and several mutants with substitutions in position 147 (including C147F, which causes dominant retinitis pigmentosa in humans) to phosphorylated and unphosphorylated light-activated rhodopsin was determined. Thermal stability of WT and mutant human arrestin-1, as well as unfolded protein response in 661W cells, were also evaluated.
Results - WT human arrestin-1 was selective for phosphorylated light-activated rhodopsin. Substitutions of Cys-147 with smaller side chain residues, Ala or Val, did not substantially affect binding selectivity, whereas residues with bulky side chains in the position 147 (Ile, Leu, and disease-causing Phe) greatly increased the binding to unphosphorylated rhodopsin. Functional survival of mutant proteins with bulky substitutions at physiological and elevated temperature was also compromised. C147F mutant induced unfolded protein response in cultured cells.
Conclusions - Bulky Phe substitution of Cys-147 in human arrestin-1 likely causes rod degeneration due to reduced stability of the protein, which induces unfolded protein response in expressing cells.
β-Arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptors and β-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether β-arrestins are able to bind second messenger kinase-phosphorylated, but inactive receptors as well. Because heterologous phosphorylation is a common phenomenon among GPCRs, this mode of β-arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, G-coupled GPCR, or epidermal growth factor receptor stimulation promotes β-arrestin2 recruitment to unliganded AT angiotensin receptor (ATR). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and β-arrestins, formed by phosphorylated serine-threonine clusters in the receptor's C terminus and two conserved phosphate-binding lysines in the β-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters β-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor-β-arrestin interaction, but also governs the structural rearrangements within β-arrestins. Furthermore, we found that β-arrestin2 binds to PKC-phosphorylated ATR in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of β-arrestins and reveal their novel role in receptor cross-talk.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
A viable vascular endothelial layer prevents vasomotor dysfunction, thrombosis, inflammation, and intimal hyperplasia. Injury to the endothelium occurs during harvest and "back table" preparation of human saphenous vein prior to implantation as an arterial bypass conduit. A subfailure overstretch model of rat aorta was used to show that subfailure stretch injury of vascular tissue leads to impaired endothelial-dependent relaxation. Stretch-induced impaired relaxation was mitigated by treatment with purinergic P2X7 receptor (P2X7R) inhibitors, brilliant blue FCF (FCF) and A740003, or apyrase, an enzyme that catalyzes the hydrolysis of ATP. Alternatively, treatment of rat aorta with exogenous ATP or 2'(3')-O-(4-Benzoyl benzoyl)-ATP (BzATP) also impaired endothelial-dependent relaxation. Treatment of human saphenous vein endothelial cells (HSVEC) with exogenous ATP led to reduced nitric oxide production which was associated with increased phosphorylation of the stress activated protein kinase, p38 MAPK. ATP- stimulated p38 MAPK phosphorylation of HSVEC was inhibited by FCF and SB203580. Moreover, ATP inhibition of nitric oxide production in HSVEC was prevented by FCF, SB203580, L-arginine supplementation and arginase inhibition. Finally, L-arginine supplementation and arginase inhibition restored endothelial dependent relaxation after stretch injury of rat aorta. These results suggest that vascular stretch injury leads to ATP release, activation of P2X7R and p38 MAPK resulting in endothelial dysfunction due to arginase activation. Endothelial function can be restored in both ATP treated HSVEC and intact stretch injured rat aorta by P2X7 receptor inhibition with FCF or L-arginine supplementation, implicating straightforward therapeutic options for treatment of surgical vascular injury.
Despite its central role in protein degradation little is known about the molecular mechanisms that sense, maintain, and regulate steady state concentration of ubiquitin in the cell. Here, we describe a novel mechanism for regulation of ubiquitin homeostasis that is mediated by phosphorylation of ubiquitin at the Ser57 position. We find that loss of Ppz phosphatase activity leads to defects in ubiquitin homeostasis that are at least partially attributable to elevated levels of Ser57 phosphorylated ubiquitin. Phosphomimetic mutation at the Ser57 position of ubiquitin conferred increased rates of endocytic trafficking and ubiquitin turnover. These phenotypes are associated with bypass of recognition by endosome-localized deubiquitylases - including Doa4 which is critical for regulation of ubiquitin recycling. Thus, ubiquitin homeostasis is significantly impacted by the rate of ubiquitin flux through the endocytic pathway and by signaling pathways that converge on ubiquitin itself to determine whether it is recycled or degraded in the vacuole.