Stevan R. Hubbard

Drosophila melanogaster (fruit fly) insulin receptor (D‐IR) is highly homologous to the human counterpart. Like the human pathway, D‐IR responds to numerous insulin‐like peptides to activate cellular signals that regulate growth, development, and lipid metabolism in fruit flies. Allelic mutations in the D‐IR kinase domain elevate life expectancy in fruit flies. We developed a robust heterologous expression system to express and purify wild‐type and longevity‐associated mutant D‐IR kinase domains to investigate enzyme kinetics and substrate specificities. D‐IR exhibits remarkable similarities to the human insulin receptor kinase domain but diverges in substrate preferences. We show that longevity‐associated mutations reduce D‐IR catalytic activity. Deletion of the unique kinase insert domain portion or mutations proximal to activating tyrosines do not influence kinase activity, suggesting their potential role in substrate recruitment and downstream signaling. Through biochemical investigations, this study enhances our comprehension of D‐IR's role in Drosophila physiology, complementing genetic studies and expanding our knowledge on the catalytic functions of this conserved signaling pathway.

Summary: Currently authorized vaccines for SARS-CoV-2 have been highly successful in preventing infection and lessening disease severity. The vaccines maintain effectiveness against SARS-CoV-2 Variants of Concern but the heavily mutated, highly transmissible Omicron variant poses an obstacle both to vaccine protection and monoclonal antibody therapies. Analysis of the neutralization of Omicron spike protein-pseudotyped lentiviruses showed a 26-fold relative resistance (compared to D614G) to neutralization by convalescent sera and 26-34-fold resistance to Pfizer BNT162b2 and Moderna vaccine-elicited antibodies following two immunizations. A booster immunization increased neutralizing titers against Omicron by 6-8-fold. Previous SARS-CoV-2 infection followed by vaccination resulted in the highest neutralizing titers against Omicron. Regeneron REGN10933 and REGN10987, and Lilly LY-CoV555 and LY-CoV016 monoclonal antibodies were ineffective against Omicron, while Sotrovimab was partially effective. The results highlight the benefit of a booster immunization in providing protection against Omicron but demonstrate the challenge to monoclonal antibody therapies.

Activated by interaction: Cytokines trigger immune responses when they bind to their cognate receptors. Class I cytokine receptors rely on the associated Janus kinase 2 (JAK2) to initiate signal transduction. There has been debate over whether activation involves ligand-induced dimerization of these receptors or ligand-induced conformational change of preformed dimers. Wilmes et al. imaged cytokine receptors in the plasma membranes of live human cells by single-molecule fluorescence microscopy and observed ligand-induced dimerization. They found that the JAK2 pseudokinase domains contribute to dimerization and that hyperactive JAK2 mutants promote dimerization, consistent with the model that dimerization triggers activation. Science , this issue p. 643

The most frequently mutated oncogene in cancer is KRAS, which uses alternative fourth exons to generate two gene products (KRAS4A and KRAS4B) that differ only in their C-terminal membrane-targeting region¹. Because oncogenic mutations occur in exons 2 or 3, two constitutively active KRAS proteins—each capable of transforming cells—are encoded when KRAS is activated by mutation². No functional distinctions among the splice variants have so far been established. Oncogenic KRAS alters the metabolism of tumour cells³ in several ways, including increased glucose uptake and glycolysis even in the presence of abundant oxygen⁴ (the Warburg effect). Whereas these metabolic effects of oncogenic KRAS have been explained by transcriptional upregulation of glucose transporters and glycolytic enzymes^3,4,5, it is not known whether there is direct regulation of metabolic enzymes. Here we report a direct, GTP-dependent interaction between KRAS4A and hexokinase 1 (HK1) that alters the activity of the kinase, and thereby establish that HK1 is an effector of KRAS4A. This interaction is unique to KRAS4A because the palmitoylation–depalmitoylation cycle of this RAS isoform enables colocalization with HK1 on the outer mitochondrial membrane. The expression of KRAS4A in cancer may drive unique metabolic vulnerabilities that can be exploited therapeutically.

KCa3.1 (also known as SK4 or IK1) is a mammalian intermediate-conductance potassium channel that plays a critical role in the activation of T cells, B cells, and mast cells, effluxing potassium ions to maintain a negative membrane potential for influxing calcium ions. KCa3.1 shares primary sequence similarity with three other (low-conductance) potassium channels: KCa2.1, KCa2.2, and KCa2.3 (also known as SK1–3). These four homotetrameric channels bind calmodulin (CaM) in the cytoplasmic region, and calcium binding to CaM triggers channel activation. Unique to KCa3.1, activation also requires phosphorylation of a single histidine residue, His358, in the cytoplasmic region, which relieves copper-mediated inhibition of the channel. Near the cytoplasmic C-terminus of KCa3.1 (and KCa2.1–2.3), secondary-structure analysis predicts the presence of a coiled-coil/heptad repeat. Here, we report the crystal structure of the C-terminal coiled-coil region of KCa3.1, which forms a parallel four-helix bundle, consistent with the tetrameric nature of the channel. Interestingly, the four copies of a histidine residue, His389, in an ‘a’ position within the heptad repeat, are observed to bind a copper ion along the four-fold axis of the bundle. These results suggest that His358, the inhibitory histidine in KCa3.1, might coordinate a copper ion through a similar binding mode.

JAK2 is a member of the Janus kinase (JAKs) family of non-receptor protein tyrosine kinases, which includes JAK1–3 and TYK2. JAKs serve as the cytoplasmic signaling components of cytokine receptors and are activated through cytokine-mediated trans-phosphorylation, which leads to receptor phosphorylation and recruitment and phosphorylation of signal transducer and activator of transcription (STAT) proteins. JAKs are unique among tyrosine kinases in that they possess a pseudokinase domain, which is just upstream of the C-terminal tyrosine kinase domain. A wealth of biochemical and clinical data have established that the pseudokinase domain of JAKs is crucial for maintaining a low basal (absence of cytokine) level of tyrosine kinase activity. In particular, gain-of-function mutations in the JAK genes, most frequently, V617F in the pseudokinase domain of JAK2, have been mapped in patients with blood disorders, including myeloproliferative neoplasms and leukemias. Recent structural and biochemical studies have begun to decipher the molecular mechanisms that maintain the basal, low-activity state of JAKs and that, via mutation, lead to constitutive activity and disease. This review will examine these mechanisms and describe how this knowledge could potentially inform drug development efforts aimed at obtaining a mutant (V617F)-selective inhibitor of JAK2.

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-ε (PKCε) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCε inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr¹¹⁶⁰ as a PKCε substrate in the functionally critical INSR kinase activation loop. We hypothesized that Thr¹¹⁶⁰ phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCε in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue Thr¹¹⁵⁰ (Insr^T1150A mice) were protected from high fat diet–induced hepatic insulin resistance. Insr^T1150A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR Thr¹¹⁶⁰ phosphorylation and provide further mechanistic links between PKCε and INSR in mediating NAFLD-induced hepatic insulin resistance.

KCa2.1, KCa2.2, KCa2.3 and KCa3.1 constitute a family of mammalian small- to intermediate-conductance potassium channels that are activated by calcium-calmodulin. KCa3.1 is unique among these four channels in that activation requires, in addition to calcium, phosphorylation of a single histidine residue (His358) in the cytoplasmic region, by nucleoside diphosphate kinase-B (NDPK-B). The mechanism by which KCa3.1 is activated by histidine phosphorylation is unknown. Histidine phosphorylation is well characterized in prokaryotes but poorly understood in eukaryotes. Here, we demonstrate that phosphorylation of His358 activates KCa3.1 by antagonizing copper-mediated inhibition of the channel. Furthermore, we show that activated CD4⁺ T cells deficient in intracellular copper exhibit increased KCa3.1 histidine phosphorylation and channel activity, leading to increased calcium flux and cytokine production. These findings reveal a novel regulatory mechanism for a mammalian potassium channel and for T-cell activation, and highlight a unique feature of histidine versus serine/threonine and tyrosine as a regulatory phosphorylation site.