Background Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure (HF). The chronic adrenergic stimulation that accompanies HF is a signaling abnormality that leads to the up-regulation of G protein-coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part due to uncoupling of the β-adrenergic receptor (βAR) system. In this study we explored the possibility that enhanced GRK2 expression and activity, as seen during HF, can negatively affect cardiac metabolism as part of its pathogenic profile. Methods and Results Positron Emission Tomography (PET) studies revealed that transgenic mice with cardiac-specific overexpression of GRK2 negatively impacted cardiac metabolism by inhibiting glucose uptake and desensitization of insulin signaling, which increases after ischemic injury and precedes HF development. Mechanistically, GRK2 interacts with and directly phosphorylates insulin receptor substrate-1 (IRS1) in cardiomyocytes causing insulin-dependent negative signaling feedback including inhibition of membrane translocation of the glucose transporter, GLUT4. This identifies IRS1 as a novel non-receptor target for GRK2 and represents a new pathological mechanism for this kinase in the failing heart. Importantly, inhibition of GRK2 activity prevents post-ischemic defects in myocardial insulin signaling and improves cardiac metabolism via normalized glucose uptake, which appears to participate in GRK2-targeted prevention of HF. Conclusions Our data provide novel insight into how GRK2 is pathological in the injured heart. Moreover, it appears to be a critical mechanistic link within neurohormonal crosstalk governing cardiac contractile signaling/function through βARs and metabolism through the insulin receptor.
Rationale GRK2 is abundantly expressed in the heart and its expression and activity is increased in injured or stressed myocardium. This up-regulation has been shown to be pathological. GRK2 can promote cell death in ischemic myocytes and its inhibition by a peptide comprised of the last 194 amino acids of GRK2 (known as βARKct) is cardioprotective. Objective The aim of this study was to elucidate the signaling mechanism that accounts for the pro-death signaling seen in the presence of elevated GRK2 and the cardioprotection afforded by the βARKct. Methods and Results Using in vivo mouse models of ischemic injury and also cultured myocytes we found that GRK2 localizes to mitochondria providing novel insight into GRK2-dependent pathophysiological signaling mechanisms. Mitochondrial localization of GRK2 in cardiomyocytes was enhanced after ischemic and oxidative stress, events that induced pro-death signaling. Localization of GRK2 to mitochondria was dependent upon phosphorylation at residue Ser670 within its extreme carboxyl-terminus by extracellular signal-regulated kinases (ERKs), resulting in enhanced GRK2 binding to heat shock protein 90 (Hsp90), which chaperoned GRK2 to mitochondria. Mechanistic studies invivo and invitro showed that ERK regulation of the C-tail of GRK2 was an absolute requirement for stress-induced, mitochondrial-dependent pro-death signaling, and blocking this led to cardioprotection. Elevated mitochondrial GRK2 also caused increased Ca2+-induced opening of the mitochondrial permeability transition pore, a key step in cellular injury. Conclusions We identify GRK2 as a pro-death kinase in the heart acting in a novel manner through mitochondrial localization via ERK regulation.
The phosphorylation of eIF4E1 at serine 209 by MNK1 or MNK2 has been shown to initiate oncogenic mRNA translation, a process that favours cancer development and maintenance. Here, we interrogate the MNK-eIF4E axis in diffuse large B-cell lymphoma (DLBCL) and show a distinct distribution of MNK1 and MNK2 in germinal centre B-cell (GCB) and activated B-cell (ABC) DLBCL. Despite displaying a differential distribution in GCB and ABC, both MNKs functionally complement each other to sustain cell survival. MNK inhibition ablates eIF4E1 phosphorylation and concurrently enhances eIF4E3 expression. Loss of MNK protein itself downregulates total eIF4E1 protein level by reducing eIF4E1 mRNA polysomal loading without affecting total mRNA level or stability. Enhanced eIF4E3 expression marginally suppresses eIF4E1-driven translation but exhibits a unique translatome that unveils a novel role for eIF4E3 in translation initiation. We propose that MNKs can modulate oncogenic translation by regulating eIF4E1-eIF4E3 levels and activity in DLBCL.
The reversible conjugation of ubiquitin and ubiquitin-like (UbL) proteins to protein substrates plays a critical role in the regulation of many cellular pathways. The removal of ubiquitin from target proteins is performed by ubiquitin proteases also known as deubiquitylases (DUBs). Owing to their substrate specificity and the central role ubiquitylation plays in cell signaling pathways, DUB are attractive targets for therapeutic development. The development of DUB inhibitors requires assays that are amenable to high-throughput screening and provide rapid assessment of inhibitor selectivity. Determination of inhibitor selectivity at an early stage of drug discovery will reduce drug failure in the clinic as well as reduce overall drug development costs. We have developed two novel assays, UbLEnterokinase light chain and UbL-Granzyme B, for quantifying ubiquitin and UbL protease activity. In our quest to discover and characterize novel chemical entities, we have combined these assays with a previously developed assay in a multiplex format. This multiplex format allows for the detection of three distinct protease activities simultaneously, in a single well. We have demonstrated that the multiplex format is able to distinguish between selective and nonselective protease inhibitors. Specifically, we have used this assay format to characterize P022077, a selective ubiquitin-specific protease 7 inhibitor discovered at Progenra.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.