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Sex determination requires the commitment of bipotential gonads to either a testis or an ovarian fate. Gene deletion of the kinase Map3k4 results in gonadal sex reversal in XY mice, and transgenic re-expression of Map3k4 rescues the sex reversal phenotype. Map3k4 encodes a large, multi-functional protein possessing a kinase domain and several, additional protein–protein interaction domains. Although MAP3K4 plays a critical role in male gonadal sex determination, it is unknown if the kinase activity of MAP3K4 is required. Here, we use mice expressing full-length, kinase-inactive MAP3K4 from the endogenous Map3k4 locus to examine the requirement of MAP3K4 kinase activity in sex determination. Although homozygous kinase-inactivation of MAP3K4 (Map3k4KI/KI) is lethal, a small fraction survive to adulthood. We show Map3k4KI/KI adults exhibit a 4:1 female-biased sex ratio. Many adult Map3k4KI/KI phenotypic females have a Y chromosome. XY Map3k4KI/KI adults with sex reversal display female mating behavior, but do not give rise to offspring. Reproductive organs are overtly female, but there is a broad spectrum of ovarian phenotypes, including ovarian absence, primitive ovaries, reduced ovarian size, and ovaries having follicles in all stages of development. Further, XY Map3k4KI/KI adults are smaller than either male or female Map3k4WT/WT mice. Examination of the critical stage of gonadal sex determination at E11.5 shows that loss of MAP3K4 kinase activity results in the loss of Sry expression in XY Map3k4KI/KI embryos, indicating embryonic male gonadal sex reversal. Together, these findings demonstrate the essential role for kinase activity of MAP3K4 in male gonadal sex determination. Summary sentence Male gonadal sex determination requires the kinase activity of the multi-domain containing protein MAP3K4. Graphical Abstract

Cancer-specific metabolic alterations hyperactivate the kinase activity of the mammalian/mechanistic target of rapamycin (mTOR) for overcoming stressful environments. Rapalogs, which allosterically inhibit mTOR complex 1 (mTORC1), have been approved as anticancer agents. However, the immunosuppressive side effect of these compounds results in the promotion of tumor metastasis, thereby limiting their therapeutic efficacy. We first report a nonrapalog inhibitor, WRX606, identified by a hybrid strategy of in silico and in cell selections. Our studies showed that WRX606 formed a ternary complex with FK506-binding protein-12 (FKBP12) and FKBP-rapamycin-binding (FRB) domain of mTOR, resulting in the allosteric inhibition of mTORC1. WRX606 inhibited the phosphorylation of not only the ribosomal protein S6 kinase 1 (S6K1) but also eIF4E-binding protein-1 (4E-BP1). Hence, WRX606 efficiently suppressed tumor growth in mice without promotion of metastasis. These results suggest that WRX606 is a potent lead compound for developing anticancer drugs discovered by in silico and in cell methods.

The precise control of the nervous system function under the vitality of synapses is extremely critical. Efforts have been taken to explore the underlying cellular and molecular mechanisms for synapse formation. Cell adhesion molecules have been found important for synapse assembly in the brain. Many trans-adhesion complexes have been identified to modulate excitatory synapse formation. However, little is known about the synaptogenic mechanisms for inhibitory synapses. ErbB4 is a receptor tyrosine kinase enriched in interneurons. Here, we showed that overexpressing ErbB4 in HEK293T cells induced gephyrin or GABAAR α1 puncta in co-cultured primary hippocampal neurons. This induction of ErbB4 was independent of its kinase activity. K751M, a kinase-dead mutant of ErbB4, can also induce gephyrin or GABAAR α1 puncta in the co-culture system. We further constructed K751M knock-in mice and found that the homozygous were viable at birth and fertile without changes in gross brain structure. The number of interneurons and inhibitory synapses onto pyramidal neurons (PyNs) were comparable between K751M and wild-type mice but decreased in ErbB4-Null mice. Moreover, ErbB4 can interact in trans with Slitrk3, a transmembrane postsynaptic protein at inhibitory synapses, through the extracellular RLD domain of ErbB4. The deletion of RLD diminished the induction of gephyrin or GABAAR α1 puncta by ErbB4. Finally, disruption of ErbB4–Slitrk3 interaction through neutralization of Slitrk3 by secretable RLD decreased inhibitory synapses onto PyNs and impaired GABAergic transmission. These results identify that ErbB4, as a cell adhesion molecule, promotes inhibitory synapse formation onto PyNs by interacting with Slitrk3 and in a kinase-independent manner, providing an unexpected mechanism of ErbB4 in inhibitory synapse formation.

Class switch recombination (CSR) changes the effector functions of antibodies and is carried out by classical and alternative nonhomologous end joining (c-NHEJ and A-EJ) of repetitive switch (S) region double-strand breaks (DSBs). The master DNA damage response (DDR) kinase ataxia-telangiectasia mutated (ATM) is critical for CSR in part by suppressing S region DSB resection. However, whether another related DDR kinase ATM- and Rad3-related (ATR) plays similar role in CSR remains elusive. In this study, we investigated the requirement for ATR kinase activity on CSR in both c-NHEJ competent and deficient B cell lines with high-throughput sequencing of S-S junctions. We found that ATR kinase inhibition efficiently blocked both c-NHEJ- and A-EJ-mediated CSR without affecting germline transcription and activation-induced cytosine deaminase expression. In contrast to ATM, ATR does not suppress S region DSB resection and microhomology usage. In addition, ATR kinase inhibition did not affect Cas9-generated DSB end joining by either c-NHEJ and A-EJ. ATR kinase-inhibited stimulated B cells proliferate much slower than controls and exhibited altered cell cycle profile with increased G1 and G2/M phase cells. In summary, our data revealed a role for ATR in promoting both c-NHEJ- and A-EJ-mediated CSR through regulating cell proliferation upon damage without negatively influencing DSB end-joining features.

Gain-of-function mutations in leucine-rich kinase 2 (LRRK2) are associated with increased incidence of Parkinson disease (PD); thus, pharmacological inhibition of LRRK2 kinase activity is postulated as a disease-modifying treatment of PD. Histomorphological changes in lungs of nonhuman primates (NHPs) treated with small-molecule LRRK2 kinase inhibitors have brought the safety of this treatment approach into question. Although it remains unclear how LRRK2 kinase inhibition affects the lung, continued studies in NHPs prove to be both cost- and resource-prohibitive. To develop a tractable alternative animal model platform, we dosed male mice in-diet with the potent, highly selective LRRK2 kinase inhibitor MLi-2 and induced histomorphological changes in lung within 1 week. Oral bolus dosing of MLi-2 at a frequency modeled to provide steady-state exposure equivalent to that achieved with in-diet dosing induced type II pneumocyte vacuolation, suggesting pulmonary changes require sustained LRRK2 kinase inhibition. Treating mice with MLi-2 in-diet for up to 6 months resulted in type II pneumocyte vacuolation that progressed only modestly over time and was fully reversible after withdrawal of MLi-2. Immunohistochemical analysis of lung revealed a significant increase in prosurfactant protein C staining within type II pneumocytes. In the present study, we demonstrated the kinetics for onset, progression, and rapid reversibility of chronic LRRK2 kinase inhibitor effects on lung histomorphology in rodents and provide further evidence for the derisking of safety and tolerability concerns for chronic LRRK2 kinase inhibition in PD. SIGNIFICANCE STATEMENT We have defined a mouse model by which the on-target lung effects of leucine-rich kinase 2 (LRRK2) kinase inhibition can be monitored, whereas previous in vivo testing relied solely on nonhuman primates. Data serve to derisk long-term treatment with LRRK2 kinase inhibitors, as all lung changes were mild and readily reversible.

New findings What is the central question of the study? Is Vps34 a nutrient-sensitive activator of mTORC1 in human skeletal muscle? What is the main finding and its importance? We show that altering nutrient availability, via protein-carbohydrate feeding, does not increase Vps34 kinase activity in human skeletal muscle. Instead, feeding increased Vps34-mTORC1 co-localization in parallel to increased mTORC1 activity. These findings may have important implications in the understanding nutrient-induced mTORC1 activation in skeletal muscle via interaction with Vps34. Abstract The Class III PI3Kinase, Vps34, has recently been proposed as a nutrient sensor, essential for activation of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). We therefore investigated the effects of increasing nutrient availability through protein-carbohydrate (PRO-CHO) feeding on Vps34 kinase activity and cellular localization in human skeletal muscle. Eight young, healthy males (21 ± 0.5 yrs, 77.7 ± 9.9 kg, 25.9 ± 2.7 kg/m2 , mean ± SD) ingested a PRO-CHO beverage containing 20/44/1 g PRO/CHO/FAT respectively, with skeletal muscle biopsies obtained at baseline and 1 h and 3 h post-feeding. PRO-CHO feeding did not alter Vps34 kinase activity, but did stimulate Vps34 translocation toward the cell periphery (PRE (mean ± SD) - 0.273 ± 0.040, 1 h - 0.348 ± 0.061, Pearson's Coefficient (r)) where it co-localized with mTOR (PRE - 0.312 ± 0.040, 1 h - 0.348 ± 0.069, Pearson's Coefficient (r)). These alterations occurred in parallel to an increase in S6K1 kinase activity (941 ± 466% of PRE at 1 h post-feeding). Subsequent in vitro experiments in C2C12 and human primary myotubes displayed no effect of the Vps34-specific inhibitor SAR405 on mTORC1 signalling responses to elevated nutrient availability. Therefore, in summary, PRO-CHO ingestion does not increase Vps34 activity in human skeletal muscle, whilst pharmacological inhibition of Vps34 does not prevent nutrient stimulation of mTORC1 in vitro. However, PRO-CHO ingestion promotes Vps34 translocation to the cell periphery, enabling Vps34 to associate with mTOR. Therefore, our data suggests that interaction between Vps34 and mTOR, rather than changes in Vps34 activity per se may be involved in PRO-CHO activation of mTORC1 in human skeletal muscle.

As a member of the epidermal growth factor receptor (EGFR) family, ERBB3 plays an essential role in development and disease independent of inherently inactive kinase domain. Recently, ERBB3 has been found to bind to ATP and has catalytic activity in vitro. However, the biological function of ERBB3 kinase activity remains elusive in vivo. Here we have identified the physiological function of inactivated ERBB3 kinase activity by creating Erbb3‐K740M knockin mice in which ATP cannot bind to ERBB3. Unlike Erbb3 knockout mice, kinase‐inactive Erbb3K740M homozygous mice were born in Mendelian ratios and showed normal development. After dextran sulfate sodium‐induced colitis, the kinase‐inactive Erbb3 mutant mice showed normal recovery. However, the outgrowth of ileal organoids by neuregulin‐1 treatment was more attenuated in Erbb3 mutant mice than in WT mice. Moreover, in combination with the ApcMin mouse, the proportion of polyps less than 1 mm in diameter in mutant mice was higher than in control mice and an increase in the number of apoptotic cells was observed in polyps from mutant mice compared with polyps from control mice. Taken together, the ERBB3 kinase activity contributes to the outgrowth of ileal organoids and intestinal tumorigenesis, and the development of ERBB3 kinase inhibitors, including epidermal growth factor receptor family members, can be a potential way to target colorectal cancer., ERBB3 kinase activity contributes to the outgrowth of ileal organoids and intestinal tumorigenesis, and the development of ERBB3 kinase inhibitors, including epidermal growth factor receptor family members, can be a potential way to target colorectal cancer

Pathogenic variants in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified that increase the risk for developing Parkinson's disease in a dominantly inherited fashion. These pathogenic variants, of which G2019S is the most common, cause abnormally high kinase activity, and compounds that inhibit this activity are being pursued as potentially disease-modifying therapeutics. Because LRRK2 regulates important cellular processes, developing inhibitors that can selectively target the pathogenic variant while sparing normal LRRK2 activity could offer potential advantages in heterozygous carriers. We conducted a high-throughput screen and identified a single selective compound that preferentially inhibited G2019S-LRRK2. Optimization of this scaffold led to a series of novel, potent, and highly selective G2019S-LRRK2 inhibitors.

Ataxia telangiectasia mutated (ATM), a critical DNA damage sensor with protein kinase activity, is frequently altered in human cancers including mantle cell lymphoma. Loss of ATM protein is linked to accumulation of nonfunctional mitochondria and defective mitophagy in both murine thymocytes and in ataxia-telangiectasia cells. However, the mechanistic role of ATM kinase in cancer cell mitophagy is unknown. Here, we provide evidence that FCCP-induced mitophagy in mantle cell lymphoma and other cancer cell lines is dependent on ATM but independent of its kinase function. While Granta-519 mantle cell lymphoma cells possess single copy kinase-dead ATM and are resistant to FCCP-induced mitophagy, both Jeko-1 and Mino cells are ATMproficient and induce mitophagy. Stable knockdown of ATM in Jeko-1 and Mino cells conferred resistance to mitophagy and was associated with reduced ATP production, oxygen consumption, and increased mitochondrial reactive oxygen species. ATM interacts with the E3 ubiquitin ligase Parkin in a kinase-independent manner. Knockdown of ATM in HeLa cells resulted in proteasomal degradation of GFP-Parkin which was rescued by the proteasome inhibitor, MG132, suggesting that the ATMParkin interaction is important for Parkin stability. Neither loss of ATM kinase activity in primary B-cell lymphomas nor inhibition of ATM kinase in mantle cell lymphoma, ataxia-telangiectasia and HeLa cell lines mitigated FCCP- or CCCP-induced mitophagy suggesting that ATM kinase activity is dispensable for mitophagy. Malignant B-cell lymphomas without detectable ATM, Parkin, Pink1, and Parkin-UbSer65 phosphorylation were resistant to mitophagy, providing the first molecular evidence of the role of ATM in mitophagy in mantle cell lymphoma and other B-cell lymphomas.

Piezo1 is a mechanosensitive cation channel with widespread physiological importance; however, its role in the heart is poorly understood. Cardiac fibroblasts help preserve myocardial integrity and play a key role in regulating its repair and remodeling following stress or injury. Here, we investigated Piezo1 expression and function in cultured human and mouse cardiac fibroblasts. RT-PCR experiments confirmed that Piezo1 mRNA in cardiac fibroblasts is expressed at levels similar to those in endothelial cells. Results from the Fura-2 intracellular Ca2+ assay validated Piezo1 as a functional ion channel that is activated by its agonist, Yoda1. Yoda1-induced Ca2+ entry was inhibited by Piezo1 blockers (gadolinium and ruthenium red) and was reduced proportionally by siRNA-mediated Piezo1 knockdown or in murine Piezo1+/- cells. Results from cell-attached patch-clamp recordings on human cardiac fibroblasts established that they contain mechanically activated ion channels and that their pressure responses are reduced by the Piezo1 knockdown. Investigation of Yoda1 effects on selected remodeling genes indicated that Piezo1 activation increases both mRNA levels and protein secretion of interleukin-6 (IL-6), a pro-hypertrophic and profibrotic cytokine, in a Piezo1-dependent manner. Moreover, Piezo1 knockdown reduced basal IL-6 expression from cells cultured on collagen-coated softer substrates. Multiplex kinase activity profiling, combined with kinase inhibitor experiments and phospho-specific immunoblotting, established that Piezo1 activation stimulates IL-6 secretion via the p38 mitogen-activated protein kinase downstream of Ca2+ entry. In summary, cardiac fibroblasts express mechanically activated Piezo1 channels coupled to secretion of the paracrine signaling molecule IL-6. Piezo1 may therefore be important in regulating cardiac remodeling.

Pathologic inclusions composed of α-synuclein called Lewy pathology are hallmarks of Parkinson’s Disease (PD). Dominant inherited mutations in leucine rich repeat kinase 2 (LRRK2) are the most common genetic cause of PD. Lewy pathology is found in the majority of individuals with LRRK2-PD, particularly those with the G2019S-LRRK2 mutation. Lewy pathology in LRRK2-PD associates with increased non-motor symptoms such as cognitive deficits, anxiety, and orthostatic hypotension. Thus, understanding the relationship between LRRK2 and α-synuclein could be important for determining the mechanisms of non-motor symptoms. In PD models, expression of mutant LRRK2 reduces membrane localization of α-synuclein, and enhances formation of pathologic α-synuclein, particularly when synaptic activity is increased. α-Synuclein and LRRK2 both localize to the presynaptic terminal. LRRK2 plays a role in membrane traffic, including axonal transport, and therefore may influence α-synuclein synaptic localization. This study shows that LRRK2 kinase activity influences α-synuclein targeting to the presynaptic terminal. We used the selective LRRK2 kinase inhibitors, MLi-2 and PF-06685360 (PF-360) to determine the impact of reduced LRRK2 kinase activity on presynaptic localization of α-synuclein. Expansion microscopy (ExM) in primary hippocampal cultures and the mouse striatum, in vivo, was used to more precisely resolve the presynaptic localization of α-synuclein. Live imaging of axonal transport of α-synuclein-GFP was used to investigate the impact of LRRK2 kinase inhibition on α-synuclein axonal transport towards the presynaptic terminal. Reduced LRRK2 kinase activity increases α-synuclein overlap with presynaptic markers in primary neurons, and increases anterograde axonal transport of α-synuclein-GFP. In vivo, LRRK2 inhibition increases α-synuclein overlap with glutamatergic, cortico-striatal terminals, and dopaminergic nigral-striatal presynaptic terminals. The findings suggest that LRRK2 kinase activity plays a role in axonal transport, and presynaptic targeting of α-synuclein. These data provide potential mechanisms by which LRRK2-mediated perturbations of α-synuclein localization could cause pathology in both LRRK2-PD, and idiopathic PD. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-021-01283-7.

Cyclin-dependent kinases (Cdks) are generally known to be involved in controlling the cell cycle, but Cdk5 is a unique member of this protein family for being most active in post-mitotic neurons. Cdk5 is developmentally important in regulating neuronal migration, neurite outgrowth, and axon guidance. Cdk5 is enriched in synaptic membranes and is known to modulate synaptic activity. Postnatally, Cdk5 can also affect neuronal processes such as dopaminergic signaling and pain sensitivity. Dysregulated Cdk5, in contrast, has been linked to neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Despite primarily being implicated in neuronal development and activity, Cdk5 has lately been linked to non-neuronal functions including cancer cell growth, immune responses, and diabetes. Since Cdk5 activity is tightly regulated, a method for measuring its kinase activity is needed to fully understand the precise role of Cdk5 in developmental and disease processes. This article includes methods for detecting Cdk5 kinase activity in cultured cells or tissues, identifying new substrates, and screening for new kinase inhibitors. Furthermore, since Cdk5 shares homology and substrate specificity with Cdk1 and Cdk2, the Cdk5 kinase assay can be used, with modification, to measure the activity of other Cdks as well. © 2021 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Measuring Cdk5 activity from protein lysates Support Protocol 1: Immunoprecipitation of Cdk5 using Dynabeads Alternate Protocol: Non-radioactive protocols to measure Cdk5 kinase activity Support Protocol 2: Western blot analysis for the detection of Cdk5, p35, and p39 Support Protocol 3: Immunodetection analysis for Cdk5, p35, and p39 Support Protocol 4: Genetically engineered mice (+ and - controls) Basic Protocol 2: Identifying new Cdk5 substrates and kinase inhibitors.

The serine/threonine protein kinase Akt integrates diverse upstream inputs to regulate cell survival, growth, metabolism, migration, and differentiation. Mounting evidence suggests that Akt activity is differentially regulated depending on its subcellular location, which can include the plasma membrane, endomembrane, and nuclear compartment. This spatial control of Akt activity is critical for achieving signaling specificity and proper physiological functions, and deregulation of compartment-specific Akt signaling is implicated in various diseases, including cancer and diabetes. Understanding the spatial coordination of the signaling network centered around this key kinase and the underlying regulatory mechanisms requires precise tracking of Akt activity at distinct subcellular compartments within its native biological contexts. To address this challenge, new molecular tools are being developed, enabling us to directly interrogate the spatiotemporal regulation of Akt in living cells. These include, for instance, the newly developed genetically encodable fluorescent-protein-based Akt kinase activity reporter (AktAR2), which serves as a substrate surrogate of Akt kinase and translates Akt-specific phosphorylation into a quantifiable change in Förster resonance energy transfer (FRET). In addition, we developed the Akt substrate tandem occupancy peptide sponge (Akt-STOPS), which allows biochemical perturbation of subcellular Akt activity. Both molecular tools can be readily targeted to distinct subcellular localizations. Here, we describe a workflow to study Akt kinase activity at different subcellular locations in living cells. We provide a protocol for using genetically targeted AktAR2 and Akt-STOPS, along with fluorescence imaging in living NIH3T3 cells, to visualize and perturb, respectively, the activity of endogenous Akt kinase at different subcellular compartments. We further describe a protocol for using chemically inducible dimerization (CID) to control the plasma membrane-specific inhibition of Akt activity in real time. Lastly, we describe a protocol for maintaining NIH3T3 cells in culture, a cell line known to exhibit robust Akt activity. In all, this approach enables interrogation of spatiotemporal regulation and functions of Akt, as well as the intricate signaling networks in which it is embedded, at specific subcellular locations. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Visualizing and perturbing subcellular Akt kinase activity using AktAR and Akt-STOPS Basic Protocol 2: Using chemically inducible dimerization (CID) to control inhibition of Akt at the plasma membrane Support Protocol: Maintaining NIH3T3 cells in culture.

Breast cancer affects women globally; the majority of breast cancer-related mortalities are due to metastasis. Acquisition of a mesenchymal phenotype has been implicated in the progression of breast cancer cells to an invasive, metastatic state. Triple-negative breast cancer (TNBC) subtypes have high rates of metastases, recurrence, and have poorer prognoses compared to other breast cancer types, partially due to lack of commonly targeted receptors. Kinases have diverse and pivotal functions in metastasis in TNBC, and discovery of new kinase targets for TNBC is warranted. We previously used a screening approach to identify intermediate-synthesis nonpotent, nonselective small-molecule inhibitors from the Published Kinase Inhibitor Set that reversed the mesenchymal phenotype in TNBC cells. Two of these inhibitors (GSK346294A and GSK448459A) are structurally similar, but have unique kinase activity profiles and exhibited differential biologic effects on TNBC cells, specifically on epithelial-to-mesenchymal transition (EMT). Here, we further interrogate these effects and compare activity of these inhibitors on transwell migration, gene (qRT-PCR) and protein (western blot) expressions, and cancer stem cell-like behavior. We incorporated translational patient-derived xenograft models in these studies, and we focused on the lead inhibitor hit, GSK346294A, to demonstrate the utility of our comparative analysis as a screening modality to identify novel kinase targets and signaling pathways to pursue in TNBC. This study introduces a new method for discovering novel kinase targets that reverse the EMT phenotype; this screening approach can be applied to all cancer types and is not limited to breast cancer.

Dihydronicotinamide riboside (NRH) has been suggested to act as a precursor for the synthesis of NAD+, but the biochemical pathway converting it has been unknown. Here, we show that NRH can be converted into NAD+ via a salvage pathway in which adenosine kinase (ADK, also known as AK) acts as an NRH kinase. Using isotope-labelling approaches, we demonstrate that NRH is fully incorporated into NAD+, with NMNH acting as an intermediate. We further show that AK is enriched in fractions from cell lysates with NRH kinase activity, and that AK can convert NRH into NAD+. In cultured cells and mouse liver, pharmacological or genetic inhibition of AK blocks formation of reduced nicotinamide mononucleotide (NMNH) and inhibits NRH-stimulated NAD+ biosynthesis. Finally, we confirm the presence of endogenous NRH in the liver with metabolomics. Our findings establish NRH as a natural precursor of NAD+ and reveal a new route for NAD+ biosynthesis via an NRH salvage pathway involving AK.

Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase‐dependent apoptosis and caspase‐independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild‐type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections., Inhibiting RIP1 has benefits to skin inflammation but not certain viral infections.

A conditional knock-in mouse was generated in which the TAK1 catalytic subunit was largely replaced by the kinase-inactive TAK1[D175A] mutant in immune cells. The activation of p38α MAP kinase, c-Jun N-terminal kinases 1 and 2 (JNK1/2) and the canonical IKK complex induced by stimulation with several TLR-activating ligands was reduced in bone marrow-derived macrophages (BMDM) from TAK1[D175A] mice. TLR signalling in TAK1[D175A] BMDM was catalysed by the residual wild-type TAK1 in these cells because it was abolished by either of two structurally unrelated TAK1 inhibitors (NG25 and 5Z-7-oxozeaenol) whose off-target effects do not overlap. The secretion of inflammatory mediators and production of the mRNAs encoding these cytokines induced by TLR ligation was greatly reduced in peritoneal neutrophils or BMDM from TAK1[D175A] mice. The Pam3CSK4- or LPS-stimulated activation of MAP kinases and the canonical IKK complex, as well as cytokine secretion, was also abolished in TAK1 knock-out human THP1 monocytes or macrophages. The results establish that TAK1 protein kinase activity is required for TLR-dependent signalling and cytokine secretion in myeloid cells from mice. We discuss possible reasons why other investigators, studying myeloid mice with a conditional knock-out of TAK1 or a different conditional kinase-inactive knock-in of TAK1, reported TAK1 to be a negative regulator of LPS-signalling and cytokine production in mouse macrophages and neutrophils.

Bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator that is a therapeutic target in many cancers and inflammatory diseases. BRD4 plays important roles in transcription as an active kinase, which phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II), the proto-oncogene c-MYC, and transcription factors TAF7 and CDK9. BRD4 is also a passive scaffold that recruits transcription factors. Despite these well-established functions, there has been little characterization of BRD4’s biophysical properties or its kinase activity. We report here that the 156 kD mouse BRD4 exists in an extended dimeric conformation with a sedimentation coefficient of ∼6.7 S and a high frictional ratio. Deletion of the conserved B motif (aa 503–548) disrupts BRD4’s dimerization. BRD4 kinase activity maps to amino acids 351 to 598, which span bromodomain-2, the B motif, and the BID domain (BD2-B-BID) and contributes to the in vivo phosphorylation of its substrates. As further assessed by analytical ultracentrifugation, BRD4 directly binds purified Pol II CTD. Importantly, the conserved A motif of BRD4 is essential for phosphorylation of Pol II CTD, but not for phosphorylation of TAF7, mapping its binding site to the A motif. Peptides of the viral MLV integrase (MLVIN) protein and cellular histone lysine methyltransferase, NSD3, which have been shown by NMR to bind to the extra-terminal (ET) domain, also are phosphorylated by BRD4. Thus, BRD4 has multiple distinct substrate-binding sites and a common kinase domain. These results provide new insights into the structure and kinase function of BRD4.

FGF19 signaling through the FGFR4/β-klotho receptor complex has been shown to be a key driver of growth and survival in a subset of hepatocellular carcinomas, making selective FGFR4 inhibition an attractive treatment opportunity. A kinome-wide sequence alignment highlighted a poorly conserved cysteine residue within the FGFR4 ATP-binding site at position 552, two positions beyond the gate-keeper residue. Several strategies for targeting this cysteine to identify FGFR4 selective inhibitor starting points are summarized which made use of both rational and unbiased screening approaches. The optimization of a 2-formylquinoline amide hit series is described in which the aldehyde makes a hemithioacetal reversible-covalent interaction with cysteine 552. Key challenges addressed during the optimization are improving the FGFR4 potency, metabolic stability, and solubility leading ultimately to the highly selective first-in-class clinical candidate roblitinib.

The ability of regulatory T (Treg) cells to migrate into inflammatory sites is reduced in autoimmune diseases, including rheumatoid arthritis (RA). The reasons for impaired Treg cell migration remain largely unknown. We performed multiplex human kinase activity arrays to explore possible differences in the post-translational phosphorylation status of kinase related proteins that could account for altered Treg cell migration in RA. Results were verified by migration assays and Western blot analysis of CD4+ T cells from RA patients and from mice with collagen type II induced arthritis. Kinome profiling of CD4+ T cells from RA patients revealed significantly altered post-translational phosphorylation of kinase related proteins, including G-protein-signaling modulator 2 (GPSM2), protein tyrosine kinase 6 (PTK6) and vitronectin precursor (VTNC). These proteins have not been associated with RA until now. We found that GPSM2 expression is reduced in CD4+ T cells from RA patients and is significantly downregulated in experimental autoimmune arthritis following immunization of mice with collagen type II. Interestingly, GPSM2 acts as a promoter of Treg cell migration in healthy individuals. Treatment of RA patients with interleukin-6 receptor (IL-6R) blocking antibodies restores GPSM2 expression, thereby improving Treg cell migration. Our study highlights the potential of multiplex kinase activity arrays as a tool for the identification of RA-related proteins which could serve as targets for novel treatments.

Removal of apoptotic cells by phagocytes (also called efferocytosis) is a crucial process for tissue homeostasis. Professional phagocytes express a plethora of surface receptors enabling them to sense and engulf apoptotic cells, thus avoiding persistence of dead cells and cellular debris and their consequent effects. Dysregulation of efferocytosis is thought to lead to secondary necrosis and associated inflammation and immune activation. Efferocytosis in primarily murine macrophages and dendritic cells has been shown to require TAM RTKs, with MERTK and AXL being critical for clearance of apoptotic cells. The functional role of human orthologs, especially the exact contribution of each individual receptor is less well studied. Here we show that human macrophages differentiated in vitro from iPSC-derived precursor cells express both AXL and MERTK and engulf apoptotic cells. TAM RTK agonism by the natural ligand growth-arrest specific 6 (GAS6) significantly enhanced such efferocytosis. Using a newly-developed mouse model of kinase-dead MERTK, we demonstrate that MERTK kinase activity is essential for efferocytosis in peritoneal macrophages in vivo. Moreover, human iPSC-derived macrophages treated in vitro with blocking antibodies or small molecule inhibitors recapitulated this observation. Hence, our results highlight a conserved MERTK function between mice and humans, and the critical role of its kinase activity in homeostatic efferocytosis.

Variants in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with increased risk for familial and sporadic Parkinson’s disease (PD). Pathogenic variants in LRRK2, including the common variant G2019S, result in increased LRRK2 kinase activity, supporting the therapeutic potential of LRRK2 kinase inhibitors for PD. To better understand the role of LRRK2 in disease and to support the clinical development of LRRK2 inhibitors, quantitative and high-throughput assays to measure LRRK2 levels and activity are needed. We developed and applied such assays to measure the levels of LRRK2 as well as the phosphorylation of LRRK2 itself or one of its substrates, Rab10 (pT73 Rab10). We observed increased LRRK2 activity in various cellular models of disease, including iPSC-derived microglia, as well as in human subjects carrying the disease-linked variant LRRK2 G2019S. Capitalizing on the high-throughput and sensitive nature of these assays, we detected a significant reduction in LRRK2 activity in subjects carrying missense variants in LRRK2 associated with reduced disease risk. Finally, we optimized these assays to enable analysis of LRRK2 activity following inhibition in human peripheral blood mononuclear cells (PBMCs) and whole blood, demonstrating their potential utility as biomarkers to assess changes in LRRK2 expression and activity in the clinic.

Background: Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with familiar and sporadic Parkinson’s disease. LRRK2 modulates the autophagy-lysosome pathway (ALP), a clearance process subserving the quality control of cellular proteins and organelles. Since dysfunctional ALP might lead to α-synuclein accumulation and, hence, Parkinson’s disease, LRRK2 kinase modulation of ALP, its age-dependence and relation with pSer129 a-synuclein inclusions in striatal and nigral neurons were investigated in vivo. Methods: Striatal ALP markers were analyzed by Western blotting in 3, 12 and 20-month-old LRRK2 G2019S knock-in mice (bearing enhanced kinase activity), LRRK2 knock-out mice, LRRK2 D1994S knock-in (kinase-dead) mice and wild-type controls. The lysosomotropic agent chloroquine was used to investigate the autophagic flux in vivo. Quantitative Real-time PCR was used to quantify the transcript levels of key ALP genes. The activity of the lysosomal enzyme glucocerebrosidase was measured using enzymatic assay. Immunohistochemistry was used to co-localize LC3B puncta with pSer129 a-synuclein inclusion in striatal MAP-positive and nigral TH-positive neurons. Results: No genotype differences in macroautophagy and chaperone-mediated autophagy markers were observed at 3 months. Conversely, increase of LC3-I, p62, LAMP2 and GAPDH levels, decrease of p-mTOR levels and downregulation of mTOR and TFEB expression was observed in 12-month-old kinase-dead mice. The LC3-II/LC3-I ratio was reduced following administration of chloroquine, suggesting a defective autophagic flux. G2019S knock-in mice showed LAMP2 accumulation and downregulation of ALP key genes MAP1LC3B, LAMP2, mTOR, TFEB and GBA1. Subacute administration of the LRRK2 kinase inhibitor MLi-2 in wild-type and G2019S knock-in mice did not replicate the pattern of kinase-dead mice. Lysosomal glucocerebrosidase activity was increased in 3 and 12-month-old knock-out and kinase-dead mice, and GBA1 expression reduced in 12-month-old G2019S knock-in mice. Immunofluorescence revealed a dissociation between LC3B puncta accumulation and pSer129 a-synuclein inclusions in striatal neurons of kinase-dead and G2019S knock-in mice. Conclusions: We conclude that constitutive LRRK2 kinase silencing results in early deregulation of GCase activity followed by late impairment of macroautophagy and chaperone-mediated autophagy. In G2019S knock-in mice, pSer129 a-synuclein inclusions observed under basal conditions appear unrelated to autophagy impairment.

The X-linked CDKL5 gene codes for a kinase whose mutations have been associated with a suite of neurodevelopmental disorders generally characterized by early-onset epileptic encephalopathy and severe intellectual disability. The impact of these mutations on CDKL5 functions and brain development remain mainly unknown, although the importance of maintaining the catalytic activity is generally recognized. Since no cure exists for CDKL5 disorders, the demand for innovative therapies is a real emergency. The recent discovery that CDKL5 is dosage sensitive poses concerns on conventional protein and gene augmentative therapies. Thus, RNA-based therapeutic approaches might be preferred. We studied the efficacy of read-through therapy on CDKL5 premature termination codons (PTCs) that correspond roughly to 15% of all mutations. Our results provide the first demonstration that all tested CDKL5 nonsense mutations are efficiently suppressed by aminoglycoside drugs. The functional characterization of the restored full-length CDKL5 reveals that read-through proteins fully recover their subcellular localization, but only partially rescue their catalytic activity. Since read-through can cause amino acid substitution, CDKL5 patients carrying the PTC outside the catalytic domain might benefit more from a nonsense suppression therapy. Eventually, we demonstrate that non-aminoglycoside drugs, such as Ataluren (PTC124) and GJ072, are unable to induce read-through activity on CDKL5 PTCs. Although these drugs might be more effective in vivo, these results question the validity of the Ataluren phase 2 clinical trial that is currently ongoing on CDKL5 patients.

The role of type I diacylglycerol kinases (DGKs) in the regulation of insulin secretion was investigated in MIN6 β-cells. In intracellular Ca2+ concentration ([Ca2+]i) measurement experiments, 1 μM R59949, a type I DGK inhibitor, and 10 μM DiC8, a diacylglycerol (DAG) analog, amplified 22.2 mM glucose-induced [Ca2+]i oscillations in a protein kinase C (PKC)-dependent manner, whereas 10 μM R59949 and 100 μM DiC8 decreased [Ca2+]i independent of PKC. High concentrations of R59949 and DiC8 attenuated voltage-dependent Ca2+ channel currents. According to these results, 22.2 mM glucose-stimulated insulin secretion (GSIS) was potentiated by 1 μM R59949 but suppressed by 10 μM of the same. The DGKα inhibitor R59022 showed a similar dual effect. Conversely, DiC8 at 10 and 100 μM potentiated GSIS, although 100 μM DiC8 decreased [Ca2+]i. These results suggest that DAG accumulated through declined type I DGK activity shows a dual effect on insulin secretion depending on the degree of accumulation; a mild DAG accumulation induces a PKC-dependent stimulatory effect on insulin secretion, whereas an excessive DAG accumulation suppresses it in a PKC-independent manner, possibly via attenuation of VDCC activity. Keywords: Diacylglycerol, Diacylglycerol kinase, Insulin secretion, Intracellular Ca2+ concentration, Voltage-dependent Ca2+ channel

Isoalantolactone (IATL), a sesquiterpene lactone compound, possesses many pharmacological and biological activities, but its role in glioblastoma (GBM) treatment is still unknown. The aim of the current study was to investigate the antiglioma effects of IATL and to explore the underlying molecular mechanisms. In the current study, the biological functions of IATL were examined by MTT, cell migration, colony formation, and cell apoptosis assays. Confocal immunofluorescence techniques, chromatin immunoprecipitation, and pull‐down assays were used to explore the precise underlying molecular mechanisms. To examine IATL activity and the molecular mechanisms by which it inhibits glioma growth in vivo, we used a xenograft tumor mouse model. Furthermore, Western blotting was used to confirm the changes in protein expression after IATL treatment. According to the results, IATL inhibited IKKβ phosphorylation, thus inhibiting both the binding of NF‐κB to the cyclooxygenase 2 (COX‐2) promoter and the recruitment of p300 and eventually inhibiting COX‐2 expression. In addition, IATL induced glioma cell apoptosis by promoting the conversion of F‐actin to G‐actin, which in turn activates the cytochrome c (Cyt c) and caspase‐dependent apoptotic pathways. In the animal experiments, IATL reduced the size and weight of glioma tumors in xenograft mice and inhibited the expression of COX‐2 and phosphorylated NF‐κB p65 in the transplanted tumors. In conclusion, the current study indicated that IATL inhibited the expression of COX‐2 through the NF‐κB signaling pathway and induced the apoptosis of glioma cells by increasing actin transformation. These results suggested that IATL could be greatly effective in GBM treatment.

Skin fibrosis is mainly characterized by excessive collagen deposition. Studies have recently identified a number of flavonoids with variable structures that have the potency of inhibiting collagen synthesis and thus attenuating organ fibrosis. In this study, we found that flavones with 5, 7, 3′, 4′ hydroxy substitution reduced collagen expression most efficiently. Among those flavones, luteolin, quercetin, and myricetin were selected for follow-up. In vivo, the three compounds ameliorated skin fibrosis and reduced collagen deposition. Further analysis showed the compounds had significant inhibition on the proliferation, activation and contractile ability of dermal fibroblasts in vitro and in vivo. More importantly, we revealed that luteolin, quercetin, and myricetin selectively downregulated the phosphorylation of Smad2/3 in TGF-β/Smads signaling via binding to activin receptor-like kinase 5 (ALK5) and impairing its catalytic activity. We also found flavones with 5, 7, 3′, 4′ hydroxy substitution showed stronger affinity with ALK5 compared with other flavonoids. Herein, we identified at least in part the underlying molecular basis as well as the critical structures that contribute to the antifibrotic bioactivity of flavones, which might benefit drug design and modification.

Excessive neutrophil degranulation is a common feature of many inflammatory disorders, including alpha-1 antitrypsin (AAT) deficiency. Our group has demonstrated that phospholipid transfer protein (PLTP) prevents neutrophil degranulation but serine proteases, which AAT inhibits, cleave PLTP in diseased airways. We propose to identify if airway PLTP activity can be restored by AAT augmentation therapy and how PLTP subdues degranulation of neutrophils in AAT deficient subjects. Airway PLTP activity was lower in AAT deficient patients but elevated in the airways of patients on augmentation therapy. Functional AAT protein (from PiMM homozygotes) prevented PLTP cleavage unlike its mutated ZZ variant (PiZZ). PLTP lowered leukotriene B4 induced degranulation of primary, secondary and tertiary granules from neutrophils from both groups (n = 14/group). Neutrophils isolated from Pltp knockout mice have enhance neutrophil degranulation. Both AAT and PLTP reduced neutrophil degranulation and superoxide production, possibly though their inhibition of the Src tyrosine kinase, Hck. Src kinase inhibitors saracatinib and dasatinib reduced neutrophil degranulation and superoxide production. Therefore, AAT protects PLTP from proteolytic cleavage and both AAT and PLTP mediate degranulation, possibly via Hck tyrosine kinase inhibition. Deficiency of AAT could contribute to reduced lung PLTP activity and elevated neutrophil signaling associated with lung disease.

Mitochondrial fusion/fission dynamics plays a fundamental role in neuroprotection; however, there is still a severe lack of therapeutic targets for this biological process. Here, we found that the naturally derived small molecule echinacoside (ECH) significantly promotes mitochondrial fusion progression. ECH selectively binds to the previously uncharacterized casein kinase 2 (CK2) α′ subunit (CK2α′) as a direct cellular target, and genetic knockdown of CK2α′ abolishes ECH-mediated mitochondrial fusion. Mechanistically, ECH allosterically regulates CK2α′ conformation to recruit basic transcription factor 3 (BTF3) to form a binary protein complex. Then, the CK2α′/BTF3 complex facilitates β-catenin nuclear translocation to activate TCF/LEF transcription factors and stimulate transcription of the mitochondrial fusion gene Mfn2. Strikingly, in a mouse middle cerebral artery occlusion (MCAO) model, ECH administration was found to significantly improve cerebral injuries and behavioral deficits by enhancing Mfn2 expression in wild-type but not CK2α′+/− mice. Taken together, our findings reveal, for the first time, that CK2 is essential for promoting mitochondrial fusion in a Wnt/β-catenin-dependent manner and suggest that pharmacologically targeting CK2 is a promising therapeutic strategy for ischemic stroke.

Macrophages play a crucial role in host innate immune defense against infection and tissue injury. Macrophages are highly plastic cells and their subtypes have been characterized as M1 (also termed classically activated) and M2 (alternatively activated). Although the M1/M2 paradigm has been well documented, less is known regarding the role of macrophage activation/polarization in inflammation-associated necrotic cell death. To address this gap in current knowledge, we prepared bone marrow-derived macrophages, induced them to M1 or M2 subtypes, and then investigated the expression of necroptosis signaling molecules and macrophage subtype-dependent responses to different necroptosis inducers. We found that necroptosis effector mixed lineage kinase domain-like protein (MLKL) and the key necroptosis regulator Z-DNA/RNA binding protein 1 were predominantly induced in M1 but not M2 macrophages. Interestingly, the protein but not mRNA levels of receptor-interacting protein kinase-3 (RIPK3) were also upregulated in M1 macrophages. We further found that macrophage necrotic cell death, the releases of lactate dehydrogenase and dead cell proteases as well as MLKL phosphorylation at Ser345 in response to various necroptosis inducers were greatly augmented in M1 but not M2 macrophages, and the accelerated effects were blocked by two structurally distinct specific RIPK3 inhibitors GSK872 or GSK843. Thus, our findings demonstrate that M1 but not M2 subtypes of macrophages are more susceptible to inflammation-related lytic cell death in an RIPK3 kinase activity-dependent manner.

Backgrounds & Aims Alcohol-related liver disease (ALD) is a major cause of chronic liver disease worldwide with limited therapeutic options. Interleukin-1 receptor associated kinase 4 (IRAK4), the master kinase of Toll-like receptor (TLR)/IL-1R-mediated signalling activation, is considered a novel therapeutic target in inflammatory diseases, but has not been investigated in the context of ALD. Methods IRAK4 phosphorylation and IRAK1 protein were analysed in liver from alcohol-related hepatitis patients and healthy controls. IRAK4 kinase activity-inactive knock-in (Irak4 KI) mice and bone marrow chimeric mice were exposed to chronic ethanol-induced liver injury. IL-1β-induced IRAK4-mediated signalling and acute phase response were investigated in cultured hepatocytes. IRAK1/4 inhibitor was used to test the therapeutic potential for ethanol-induced liver injury in mice. Results Increased IRAK4 phosphorylation and reduced IRAK1 protein were found in livers of patients with alcoholic hepatitis. In the chronic ethanol-induced liver injury mouse model, hepatic inflammation and hepatocellular damage were attenuated in Irak4 KI mice. IRAK4 kinase activity promotes expression of acute phase proteins in response to ethanol exposure, including C-reactive protein and serum amyloid A1 (SAA1). SAA1 and IL-1β synergistically exacerbate ethanol-induced cell death ex vivo. Pharmacological blockage of IRAK4 kinase abrogated ethanol-induced liver injury, inflammation, steatosis, as well as acute phase gene expression and protein production in mice. Conclusions Our data elucidate the critical role of IRAK4 kinase activity in the pathogenesis of ethanol-induced liver injury in mice and provide preclinical validation for use of an IRAK1/4 inhibitor as a new potential therapeutic strategy for the treatment of ALD. Lay summary Herein, we have identified the role of IRAK4 kinase activity in the development of alcohol-induced liver injury in mice. Hepatocyte-specific IRAK4 is associated with an acute phase response and release of proinflammatory cytokines/chemokines, which synergistically exacerbate alcohol-induced hepatocyte cell death ex vivo. Pharmacological inhibition of IRAK4 kinase activity effectively attenuates alcohol-induced liver injury in mice and could have therapeutic implications.

Neuroblastoma is the most common extracranial solid tumor in childhood. Gain of chromosome 17q material is found in &gt, 60% of neuroblastoma tumors and is associated with poor patient prognosis. The NME1 gene is located in the 17q21.3 region, and high NME1 expression is correlated with poor neuroblastoma patient outcomes. However, the functional roles and signaling activity of NME1 in neuroblastoma cells and tumors are unknown. NME1 and NME2 have been shown to possess histidine (His) kinase activity. Using anti-1- and 3-pHis specific monoclonal antibodies and polyclonal anti-pH118 NME1/2 antibodies, we demonstrated the presence of pH118-NME1/2 and multiple additional pHis-containing proteins in all tested neuroblastoma cell lines and in xenograft neuroblastoma tumors, supporting the presence of histidine kinase activity in neuroblastoma cells and demonstrating the potential significance of histidine kinase signaling in neuroblastoma pathogenesis. We have also demonstrated associations between NME1 expression and neuroblastoma cell migration and differentiation. Our demonstration of NME1 histidine phosphorylation in neuroblastoma and of the potential role of NME1 in neuroblastoma cell migration and differentiation suggest a functional role for NME1 in neuroblastoma pathogenesis and open the possibility of identifying new therapeutic targets and developing novel approaches to neuroblastoma therapy.

Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion. REPORTING SUMMARY. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Signaling via the tropomyosin-related kinase receptor subtype B (TrkB) regulates neuromuscular transmission, and inhibition of TrkB kinase activity by 1NMPP1 in TrkBF616Amice worsens neuromuscular transmission failure (NMTF). We hypothesized that acute inhibition of TrkB kinase activity will impair the ability of the diaphragm muscle to produce maximal transdiaphragmatic pressure (Pdi) without impacting the ability to generate forces associated with ventilation, consistent with the greater susceptibility to NMTF in motor units responsible for higher-force nonventilatory behaviors. Adult male and female TrkBF616Amice were injected with 1NMPP1 ( n = 8) or vehicle (DMSO; n = 8) 1 h before Pdi measurements during eupneic breathing, hypoxia/hypercapnia (10% O2/5% CO2), tracheal occlusion, spontaneous deep breaths (“sighs”) and during maximal activation elicited by bilateral phrenic nerve stimulation. In the vehicle-treated group, Pdi increased from ~10 cmH2O during eupnea and hypoxia/hypercapnia, to ~35 cmH2O during sighs and tracheal occlusion, and to ~65 cm H2O during maximal stimulation. There was no effect of acute 1NMPP1 treatment on Pdi generated during most behaviors, except during maximal stimulation (~30% reduction; P < 0.05). This reduction in maximal Pdi is generally similar to the worsening of NMTF previously reported with TrkB kinase inhibition in rodents. Accordingly, impaired TrkB signaling limits the range of motor behaviors accomplished by the diaphragm muscle and may contribute to neuromuscular dysfunction, primarily by impacting fatigable, higher force-generating motor units.NEW & NOTEWORTHY TrkB signaling plays an important role in maintaining neuromuscular function in the diaphragm muscle and may be necessary to accomplish the various motor behaviors ranging from ventilation to expulsive, behaviors requiring near-maximal forces. This study shows that inhibition of TrkB kinase activity impairs maximal pressure generation by the diaphragm muscle, but the ability to generate the lower pressures required for ventilatory behaviors is not impacted.

In this issue, Malireddi et al. use a model of pathogen-induced priming and inhibition of TAK1 to demonstrate RIPK1 kinase activity–independent inflammasome activation and pyroptosis, apoptosis, and necroptosis that drive myeloid proliferation and sepsis in TAK1-deficient animals., RIPK1 kinase activity has been shown to be essential to driving pyroptosis, apoptosis, and necroptosis. However, here we show a kinase activity–independent role for RIPK1 in these processes using a model of TLR priming in a TAK1-deficient setting to mimic pathogen-induced priming and inhibition. TLR priming of TAK1-deficient macrophages triggered inflammasome activation, including the activation of caspase-8 and gasdermin D, and the recruitment of NLRP3 and ASC into a novel RIPK1 kinase activity–independent cell death complex to drive pyroptosis and apoptosis. Furthermore, we found fully functional RIPK1 kinase activity–independent necroptosis driven by the RIPK3–MLKL pathway in TAK1-deficient macrophages. In vivo, TAK1 inactivation resulted in RIPK3–caspase-8 signaling axis–driven myeloid proliferation and a severe sepsis-like syndrome. Overall, our study highlights a previously unknown mechanism for RIPK1 kinase activity–independent inflammasome activation and pyroptosis, apoptosis, and necroptosis (PANoptosis) that could be targeted for treatment of TAK1-associated myeloid proliferation and sepsis.

Inflammatory bowel disease (IBD) is a chronic and debilitating disorder that has few treatment options due to a lack of comprehensive understanding of its molecular pathogenesis. We used multiplexed mass spectrometry to collect high-content information on protein phosphorylation in two different mouse models of IBD. Because the biological function of the vast majority of phosphorylation sites remains unknown, we developed Substrate-based Kinase Activity Inference (SKAI), a methodology to infer kinase activity from phosphoproteomic data. This approach draws upon prior knowledge of kinase-substrate interactions to construct custom lists of kinases and their respective substrate sites, termed kinase-substrate sets that employ prior knowledge across organisms. This expansion as much as triples the amount of prior knowledge available. We then used these sets within the Gene Set Enrichment Analysis framework to infer kinase activity based on increased or decreased phosphorylation of its substrates in a dataset. When applied to the phosphoproteomic datasets from the two mouse models, SKAI predicted largely non-overlapping kinase activation profiles. These results suggest that chronic inflammation may arise through activation of largely divergent signaling networks. However, the one kinase inferred to be activated in both mouse models was mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2 or MK2), a serine/threonine kinase that functions downstream of p38 stress-activated mitogen-activated protein kinase. Treatment of mice with active colitis with ATI450, an orally bioavailable small molecule inhibitor of the MK2 pathway, reduced inflammatory signaling in the colon and alleviated the clinical and histological features of inflammation. These studies establish MK2 as a therapeutic target in IBD and identify ATI450 as a potential therapy for the disease.

Neural stem cells give rise to granule dentate neurons throughout life in the hippocampus. Upon activation, these stem cells generate fast proliferating progenitors that complete several rounds of divisions before differentiating into neurons. Although the mechanisms regulating the activation of stem cells have been intensively studied, little attention has been given so far to the intrinsic machinery allowing the expansion of the progenitor pool. The cell cycle protein Cdk6 positively regulates the proliferation of hippocampal progenitors, but the mechanism involved remains elusive. Whereas Cdk6 functions primarily as a cell cycle kinase, it can also act as transcriptional regulator in cancer cells and hematopoietic stem cells. Using mouse genetics, we show here that the function of Cdk6 in hippocampal neurogenesis relies specifically on its kinase activity. The present study also reveals a specific regulatory mechanism for Cdk6 in hippocampal progenitors. In contrast to the classical model of the cell cycle, we observe that the Cip/Kip family member p27, rather than the Ink4 family, negatively regulates Cdk6 in the adult hippocampus. Altogether, our data uncover a unique, cell type-specific regulatory mechanism controlling the expansion of hippocampal progenitors, where Cdk6 kinase activity is modulated by p27.

When cells with excess DNA, such as tetraploid cells, undergo cell division, it can contribute to cellular transformation via asymmetrical chromosome segregation-generated genetic diversity. Cell cycle progression of tetraploid cells is suppressed by large tumor suppressor 2 (LATS2) kinase-induced inhibitory phosphorylation of the transcriptional coactivator Yes-associated protein (YAP). We recently reported that the oncogene v-Src induces tetraploidy and promotes cell cycle progression of tetraploid cells by suppressing LATS2 activity. We explore here the mechanism by which v-Src suppresses LATS2 activity and the role of LATS2 in v-Src-expressing cells. LATS2 was directly phosphorylated by v-Src and the proto-oncogene c-Src, resulting in decreased LATS2 kinase activity. This kinase-deficient LATS2 accumulated in a YAP transcriptional activity-dependent manner, and knockdown of either LATS2 or the LATS2-binding partner moesin-ezrin-radixin-like protein (Merlin) accelerated v-Src-induced membrane bleb formation. Upon v-Src expression, the interaction of Merlin with LATS2 was increased possibly due to a decrease in Merlin phosphorylation at Ser518, the dephosphorylation of which is required for the open conformation of Merlin and interaction with LATS2. LATS2 was colocalized with Merlin at the plasma membrane in a manner that depends on the Merlin-binding region of LATS2. The bleb formation in v-Src-expressing and LATS2-knockdown cells was rescued by the reexpression of wild-type or kinase-dead LATS2 but not the LATS2 mutant lacking the Merlin-binding region. These results suggest that the kinase-deficient LATS2 plays a role with Merlin at the plasma membrane in the maintenance of cortical rigidity in v-Src-expressing cells, which may cause tumor suppression.