The Activation Mechanism of the Insulin Receptor: A Structural Perspective.

The insulin receptor (IR) is a type II receptor tyrosine kinase that plays essential roles in metabolism, growth, and proliferation. Dysregulation of IR signaling is linked to many human diseases, such as diabetes and cancers. The resolution revolution in cryo-electron microscopy has led to the determination of several structures of IR with different numbers of bound insulin molecules in recent years, which have tremendously improved our understanding of how IR is activated by insulin. Here, we review the insulin-induced activation mechanism of IR, including (a) the detailed binding modes and functions of insulin at site 1 and site 2 and (b) the insulin-induced structural transitions that are required for IR activation. We highlight several other key aspects of the activation and regulation of IR signaling and discuss the remaining gaps in our understanding of the IR activation mechanism and potential avenues of future research.

TAM receptor signaling in immune homeostasis.

The TAM receptor tyrosine kinases (RTKs)-TYRO3, AXL, and MERTK-together with their cognate agonists GAS6 and PROS1 play an essential role in the resolution of inflammation. Deficiencies in TAM signaling have been associated with chronic inflammatory and autoimmune diseases. Three processes regulated by TAM signaling may contribute, either independently or collectively, to immune homeostasis: the negative regulation of the innate immune response, the phagocytosis of apoptotic cells, and the restoration of vascular integrity. Recent studies have also revealed the function of TAMs in infectious diseases and cancer. Here, we review the important milestones in the discovery of these RTKs and their ligands and the studies that underscore the functional importance of this signaling pathway in physiological immune settings and disease.

Mechanisms for Regulating and Organizing Receptor Signaling by Endocytosis.

Intricate relationships between endocytosis and cellular signaling, first recognized nearly 40 years ago through the study of tyrosine kinase growth factor receptors, are now known to exist for multiple receptor classes and to affect myriad physiological and developmental processes. This review summarizes our present understanding of how endocytosis orchestrates cellular signaling networks, with an emphasis on mechanistic underpinnings and focusing on two receptor classes-tyrosine kinase and G protein-coupled receptors-that have been investigated in particular detail. Together, these examples provide a useful survey of the current consensus, uncertainties, and controversies in this rapidly advancing area of cell biology.

A Network of Macrophages Supports Mitochondrial Homeostasis in the Heart.

Cardiomyocytes are subjected to the intense mechanical stress and metabolic demands of the beating heart. It is unclear whether these cells, which are long-lived and rarely renew, manage to preserve homeostasis on their own. While analyzing macrophages lodged within the healthy myocardium, we discovered that they actively took up material, including mitochondria, derived from cardiomyocytes. Cardiomyocytes ejected dysfunctional mitochondria and other cargo in dedicated membranous particles reminiscent of neural exophers, through a process driven by the cardiomyocyte's autophagy machinery that was enhanced during cardiac stress. Depletion of cardiac macrophages or deficiency in the phagocytic receptor Mertk resulted in defective elimination of mitochondria from the myocardial tissue, activation of the inflammasome, impaired autophagy, accumulation of anomalous mitochondria in cardiomyocytes, metabolic alterations, and ventricular dysfunction. Thus, we identify an immune-parenchymal pair in the murine heart that enables transfer of unfit material to preserve metabolic stability and organ function. VIDEO ABSTRACT.

The Global Phosphorylation Landscape of SARS-CoV-2 Infection.

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.

A Tumor-Agnostic NTRK (TRK) Inhibitor.

Larotrectinib is a small-molecule kinase inhibitor that targets NTRK fusions that occur in multiple types of cancer. Its FDA approval represents the first instance of a treatment indication being designated "tumor-agnostic" from the outset, being based on actionable genomic insights. To view this Bench to Bedside, open or download the PDF.

Microglia use TAM receptors to detect and engulf amyloid ß plaques.

Two microglial TAM receptor tyrosine kinases, Axl and Mer, have been linked to Alzheimer's disease, but their roles in disease have not been tested experimentally. We find that in Alzheimer's disease and its mouse models, induced expression of Axl and Mer in amyloid plaque-associated microglia was coupled to induced plaque decoration by the TAM ligand Gas6 and its co-ligand phosphatidylserine. In the APP/PS1 mouse model of Alzheimer's disease, genetic ablation of Axl and Mer resulted in microglia that were unable to normally detect, respond to, organize or phagocytose amyloid-ß plaques. These major deficits notwithstanding, TAM-deficient APP/PS1 mice developed fewer dense-core plaques than APP/PS1 mice with normal microglia. Our findings reveal that the TAM system is an essential mediator of microglial recognition and engulfment of amyloid plaques and that TAM-driven microglial phagocytosis does not inhibit, but rather promotes, dense-core plaque development.

The Dark Side of Cell Signaling: Positive Roles for Negative Regulators.

Cell signaling is dominated by analyzing positive responses to stimuli. Signal activation is balanced by negative regulators that are generally considered to terminate signaling. Rather than exerting only negative effects, however, many such regulators play important roles in enhancing cell-signaling control. Considering responses downstream of selected cell-surface receptors, we discuss how receptor internalization affects signaling specificity and how rapid kinase/phosphatase and GTP/GDP cycles increase responsiveness and allow kinetic proofreading in receptor signaling. We highlight the blurring of distinctions between positive and negative signals, recasting signal termination as the response to a switch-like transition into a new cellular state.

Zika Virus Causes Testis Damage and Leads to Male Infertility in Mice.

Zika virus (ZIKV) persists in the semen of male patients, a first for flavivirus infection. Here, we demonstrate that ZIKV can induce inflammation in the testis and epididymidis, but not in the prostate or seminal vesicle, and can lead to damaged testes after 60 days post-infection in mice. ZIKV induces innate immune responses in Leydig, Sertoli, and epididymal epithelial cells, resulting in the production of pro-inflammatory cytokines/chemokines. However, ZIKV does not induce a rapid and abundant cytokine production in peritubular cell and spermatogonia, suggesting that these cells are vulnerable for ZIKV infection and could be the potential repositories for ZIKV. Our study demonstrates a correlation between ZIKV and testis infection/damage and suggests that ZIKV infection, under certain circumstances, can eventually lead to male infertility.

Non-genomic and Immune Evolution of Melanoma Acquiring MAPKi Resistance.

Clinically acquired resistance to MAPK inhibitor (MAPKi) therapies for melanoma cannot be fully explained by genomic mechanisms and may be accompanied by co-evolution of intra-tumoral immunity. We sought to discover non-genomic mechanisms of acquired resistance and dynamic immune compositions by a comparative, transcriptomic-methylomic analysis of patient-matched melanoma tumors biopsied before therapy and during disease progression. Transcriptomic alterations across resistant tumors were highly recurrent, in contrast to mutations, and were frequently correlated with differential methylation of tumor cell-intrinsic CpG sites. We identified in the tumor cell compartment supra-physiologic c-MET up-expression, infra-physiologic LEF1 down-expression and YAP1 signature enrichment as drivers of acquired resistance. Importantly, high intra-tumoral cytolytic T cell inflammation prior to MAPKi therapy preceded CD8 T cell deficiency/exhaustion and loss of antigen presentation in half of disease-progressive melanomas, suggesting cross-resistance to salvage anti-PD-1/PD-L1 immunotherapy. Thus, melanoma acquires MAPKi resistance with highly dynamic and recurrent non-genomic alterations and co-evolving intra-tumoral immunity.

Modeling diverse genetic subtypes of lung adenocarcinoma with a next-generation alveolar type 2 organoid platform.

Lung cancer is the leading cause of cancer-related death worldwide. Lung adenocarcinoma (LUAD), the most common histological subtype, accounts for 40% of all cases. While existing genetically engineered mouse models (GEMMs) recapitulate the histological progression and transcriptional evolution of human LUAD, they are time-consuming and technically demanding. In contrast, cell line transplant models are fast and flexible, but these models fail to capture the full spectrum of disease progression. Organoid technologies provide a means to create next-generation cancer models that integrate the most advantageous features of autochthonous and transplant-based systems. However, robust and faithful LUAD organoid platforms are currently lacking. Here, we describe optimized conditions to continuously expand murine alveolar type 2 (AT2) cells, a prominent cell of origin for LUAD, in organoid culture. These organoids display canonical features of AT2 cells, including marker gene expression, the presence of lamellar bodies, and an ability to differentiate into the AT1 lineage. We used this system to develop flexible and versatile immunocompetent organoid-based models of KRAS, BRAF, and ALK mutant LUAD. Notably, organoid-based tumors display extensive burden and complete penetrance and are histopathologically indistinguishable from their autochthonous counterparts. Altogether, this organoid platform is a powerful, versatile new model system to study LUAD.

Oncogenic signaling of RTK fusions becomes more granular.

A spectrum of cancers arises from chromosomal translocations that fuse receptor tyrosine kinase domains to oligomerization domains from unrelated proteins. Tulpule et al. (2021) demonstrate that fusion proteins with the ability to assemble higher-order cytoplasmic protein granules can activate RAS signaling in a lipid membrane-independent manner.

Structural Insights into Pseudokinase Domains of Receptor Tyrosine Kinases.

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.

Phase separation enhances probability of receptor signalling and drug targeting.

The probability of a given receptor tyrosine kinase (RTK) triggering a defined cellular outcome is low because of the promiscuous nature of signalling, the randomness of molecular diffusion through the cell, and the ongoing nonfunctional submembrane signalling activity or noise. Signal transduction is therefore a 'numbers game', where enough cell surface receptors and effector proteins must initially be engaged to guarantee formation of a functional signalling complex against a background of redundant events. The presence of intracellular liquid-liquid phase separation (LLPS) at the plasma membrane provides a mechanism through which the probabilistic nature of signalling can be weighted in favour of the required, discrete cellular outcome and mutual exclusivity in signal initiation.

Wnt signaling regulates acetylcholine receptor translocation and synaptic plasticity in the adult nervous system.

The adult nervous system is plastic, allowing us to learn, remember, and forget. Experience-dependent plasticity occurs at synapses--the specialized points of contact between neurons where signaling occurs. However, the mechanisms that regulate the strength of synaptic signaling are not well understood. Here, we define a Wnt-signaling pathway that modifies synaptic strength in the adult nervous system by regulating the translocation of one class of acetylcholine receptors (AChRs) to synapses. In Caenorhabditis elegans, we show that mutations in CWN-2 (Wnt ligand), LIN-17 (Frizzled), CAM-1 (Ror receptor tyrosine kinase), or the downstream effector DSH-1 (disheveled) result in similar subsynaptic accumulations of ACR-16/α7 AChRs, a consequent reduction in synaptic current, and predictable behavioral defects. Photoconversion experiments revealed defective translocation of ACR-16/α7 to synapses in Wnt-signaling mutants. Using optogenetic nerve stimulation, we demonstrate activity-dependent synaptic plasticity and its dependence on ACR-16/α7 translocation mediated by Wnt signaling via LIN-17/CAM-1 heteromeric receptors.

Structural basis of cytokine-mediated activation of ALK family receptors.

Anaplastic lymphoma kinase (ALK)1 and the related leukocyte tyrosine kinase (LTK)2 are recently deorphanized receptor tyrosine kinases3. Together with their activating cytokines, ALKAL1 and ALKAL24-6 (also called FAM150A and FAM150B or AUGß and AUGα, respectively), they are involved in neural development7, cancer7-9 and autoimmune diseases10. Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain11, consistent with a metabolic role for Drosophila ALK12. Despite such functional pleiotropy and growing therapeutic relevance13,14, structural insights into ALK and LTK and their complexes with cognate cytokines have remained scarce. Here we show that the cytokine-binding segments of human ALK and LTK comprise a novel architectural chimera of a permuted TNF-like module that braces a glycine-rich subdomain featuring a hexagonal lattice of long polyglycine type II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicits similar dimeric assemblies with two-fold symmetry, that tent a single cytokine molecule proximal to the cell membrane. We show that the membrane-proximal EGF-like domain dictates the apparent cytokine preference of ALK. Assisted by these diverse structure-function findings, we propose a structural and mechanistic blueprint for complexes of ALK family receptors, and thereby extend the repertoire of ligand-mediated dimerization mechanisms adopted by receptor tyrosine kinases.

Mechanism of disease and therapeutic rescue of Dok7 congenital myasthenia.

Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM1,2. The most common disease-causing mutation (DOK71124_1127 dup) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7CM mice) and a mouse with point mutations in the two tyrosine residues (Dok72YF). We show that Dok7CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.

Structure-based investigation of a DNA aptamer targeting PTK7 reveals an intricate 3D fold guiding functional optimization.

DNA aptamers have emerged as novel molecular tools in disease theranostics owing to their high binding affinity and specificity for protein targets, which rely on their ability to fold into distinctive three-dimensional (3D) structures. However, delicate atomic interactions that shape the 3D structures are often ignored when designing and modeling aptamers, leading to inefficient functional optimization. Challenges persist in determining high-resolution aptamer-protein complex structures. Moreover, the experimentally determined 3D structures of DNA molecules with exquisite functions remain scarce. These factors impede our comprehension and optimization of some important DNA aptamers. Here, we performed a streamlined solution NMR-based structural investigation on the 41-nt sgc8c, a prominent DNA aptamer used to target membrane protein tyrosine kinase 7, for cancer theranostics. We show that sgc8c prefolds into an intricate three-way junction (3WJ) structure stabilized by long-range tertiary interactions and extensive base-base stackings. Delineated by NMR chemical shift perturbations, site-directed mutagenesis, and 3D structural information, we identified essential nucleotides constituting the key functional elements of sgc8c that are centralized at the core of 3WJ. Leveraging the well-established structure-function relationship, we efficiently engineered two sgc8c variants by modifying the apical loop and introducing L-DNA base pairs to simultaneously enhance thermostability, biostability, and binding affinity for both protein and cell targets, a feat not previously attained despite extensive efforts. This work showcases a simplified NMR-based approach to comprehend and optimize sgc8c without acquiring the complex structure, and offers principles for the sophisticated structure-function organization of DNA molecules.

ALK signaling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition.

High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (ALK), which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR (ataxia telangiectasia and Rad3-related)/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signaling leads to phosphorylation of ATR and CHK1, supporting an effective DNA damage response. The importance of ALK inhibition is supported by mouse data, in which ATRi monotreatment resulted in a robust initial response, but subsequent relapse, in contrast to a 14-d ALKi/ATRi combination treatment that resulted in a robust and sustained response. Finally, we show that the remarkable response to the 14-d combined ATR/ALK inhibition protocol reflects a robust differentiation response, reprogramming tumor cells to a neuronal/Schwann cell lineage identity. Our results identify an ability of ATR inhibition to promote NB differentiation and underscore the importance of further exploring combined ALK/ATR inhibition in NB, particularly in high-risk patient groups with oncogene-induced replication stress.

TAM receptors in phagocytosis: Beyond the mere internalization of particles.

TYRO3, AXL, and MERTK constitute the TAM family of receptor tyrosine kinases, activated by their ligands GAS6 and PROS1. TAMs are necessary for adult homeostasis in the immune, nervous, reproductive, skeletal, and vascular systems. Among additional cellular functions employed by TAMs, phagocytosis is central for tissue health. TAM receptors are dominant in providing phagocytes with the molecular machinery necessary to engulf diverse targets, including apoptotic cells, myelin debris, and portions of live cells in a phosphatidylserine-dependent manner. Simultaneously, TAMs drive the release of anti-inflammatory and tissue repair molecules. Disruption of the TAM-driven phagocytic pathway has detrimental consequences, resulting in autoimmunity, male infertility, blindness, and disrupted vascular integrity, and which is thought to contribute to neurodegenerative diseases. Although structurally and functionally redundant, the TAM receptors and ligands underlie complex signaling cascades, of which several key aspects are yet to be elucidated. We discuss similarities and differences between TAMs and other phagocytic pathways, highlight future directions and how TAMs can be harnessed therapeutically to modulate phagocytosis.