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Cisplatin, 5FU and docetaxel (TPF) are the most common chemotherapy regimen used for advanced OSCC. However, many cancer patients experience relapse, continued tumor growth, and spread due to drug resistance, which leads to treatment failure and metastatic disease. Here, using a CRISPR/Cas9 based kinome knockout screening, Misshapen-like kinase 1 (MINK1) is identified as an important mediator of 5FU resistance in OSCC. Analysis of clinical samples demonstrated significantly higher MINK1 expression in the tumor tissues of chemotherapy non-responder as compared to chemotherapy responders. The in-vitro and xenograft experiments indicate that knocking out MINK1 restores 5FU mediated cell death in chemoresistant OSCC. An antibody based phosphorylation array screen revealed MINK1 as a negative regulator of p53. Mechanistically, MINK1 modulates AKT phosphorylation at Ser473, which enables p-MDM2 (Ser 166) mediated degradation of p53. We also identified lestaurtinib as a potent inhibitor of MINK1 kinase activity. Lestaurtinib significantly induces 5FU mediated cell death in chemoresistant OSCC lines. The patient derived chemoresistant cell based xenograft data suggest that lestaurtinib restores 5FU sensitivity and facilitates a significant reduction of tumor burden. Overall, our study suggests that MINK1 is a major driver of 5FU resistance in OSCC. The novel combination of MINK1 inhibitor lestaurtinib and 5FU needs further clinical investigation in advanced OSCC. One sentence summary Lestaurtinib reverses 5FU sensitivity in drug resistant OSCC.

Loss of NF2 (merlin) has been suggested as a genetic cause of neurofibromatosis type 2 and malignant peripheral nerve sheath tumor (MPNST). Previously, we demonstrated that NF2 sustained TGFβ receptor 2 (TβR2) expression and reduction or loss of NF2 activated non-canonical TGFβ signaling, which reduced Raf kinase inhibitor protein (RKIP) expression via TβR1 kinase activity. Here, we show that a selective RKIP inducer (novel chemical, Nf18001) inhibits tumor growth and promotes schwannoma cell differentiation into mature Schwann cells under NF2-deficient conditions. In addition, Nf18001 is not cytotoxic to cells expressing NF2 and is not disturb canonical TGFβ signaling. Moreover, the novel chemical induces expression of SOX10, a marker of differentiated Schwann cells, and promotes nuclear export and degradation of SOX2, a stem cell factor. Treatment with Nf18001 inhibited tumor growth in an allograft model with mouse schwannoma cells. These results strongly suggest that selective RKIP inducers could be useful for the treatment of neurofibromatosis type 2 as well as NF2-deficient MPNST. Implications: This study identifies that a selective RKIP inducer inhibits tumor growth and promotes schwannoma cell differentiation under NF2-deficient conditions by reducing SOX2 and increasing SOX10 expression.

Background The incidence of bladder urothelial carcinoma (UC), a common malignancy of the urinary tract, is approximately three times higher in men than in women. High expression of the mitotic kinase BUB1 is associated with the occurrence and development of several cancers, although the relationship between BUB1 and bladder tumorigenesis remains unclear. Methods Using a microarray approach, we found increased BUB1 expression in human BCa. The association between BUB1 and STAT3 phosphorylation was determined through molecular and cell biological methods. We evaluated the impact of pharmacologic inhibition of BUB1 kinase activity on proliferation and BCa progression in vitro and in vivo. Results In this study, we found that BUB1 expression was increased in human bladder cancer (BCa). We further identified through a series of molecular and cell biological approaches that BUB1 interacted directly with STAT3 and mediated the phosphorylation of STAT3 at Ser727. In addition, the findings that pharmacologic inhibition of BUB1 kinase activity significantly suppressed BCa cell proliferation and the progression of bladder cancer in vitro and in vivo were further verified. Finally, we found that the BUB1/STAT3 complex promoted the transcription of STAT3 target genes and that depletion of BUB1 and mutation of the BUB1 kinase domain abrogated this transcriptional activity, further highlighting the critical role of kinase activity in the activation of STAT3 target genes. A pharmacological inhibitor of BUB1 (2OH-BNPP1) was able to significantly inhibit the growth of BCa cell xenografts. Conclusion This study showed that the BUB1 kinase drives the progression and proliferation of BCa by regulating the transcriptional activation of STAT3 signaling and may be an attractive candidate for therapeutic targeting in BCa.

Ras genes are frequently mutated in many cancer types; however, there are currently no conclusively effective anticancer drugs against Ras-induced cancer. Therefore, the downstream effectors of Ras signaling need to be identified for the development of promising novel therapeutic approaches. We previously reported that oncogenic Ras induced the expression of NF-HEV/IL-33, a member of the interleukin-1 family, and showed that intracellular IL-33 was required for oncogenic Ras-induced cellular transformation. In the present study, we demonstrated that the c-Mer proto-oncogene tyrosine kinase (MerTK), a receptor tyrosine kinase, played essential roles in oncogenic Ras/IL-33 signaling. The expression of MerTK was enhanced in transformed NIH-3T3 cells by the expression of oncogenic Ras, H-Ras (G12V), in an IL-33-dependent manner. In human colorectal cancer tissues, MerTK expression also correlated with IL-33 expression. The knockdown of IL-33 or MerTK effectively attenuated the migration of NIH-3T3 cells transformed by H-Ras (G12V) and A549, LoVo, and HCT116 cells harboring an oncogenic K-Ras mutation. Furthermore, the suppression of Ras-induced cell migration by the knockdown of IL-33 was rescued by the enforced expression of MerTK. The present results also revealed that MerTK was effectively phosphorylated in NIH-3T3 cells transformed by Ras (G12V). Ras signaling was essential for the tyrosine phosphorylation of MerTK, and the kinase activity of MerTK was indispensable for accelerating cell migration. Collectively, the present results reveal a novel role for MerTK in cancer malignancy, which may be utilized to develop novel therapeutic strategies that target Ras-transformed cells.

In vitro studies have established the prevalent theory that the mitochondrial kinase PINK1 protects neurodegeneration by removing damaged mitochondria in Parkinson’s disease (PD). However, difficulty in detecting endogenous PINK1 protein in rodent brains and cell lines has prevented the rigorous investigation of the in vivo role of PINK1. Here we report that PINK1 kinase form is selectively expressed in the human and monkey brains. CRISPR/Cas9-mediated deficiency of PINK1 causes similar neurodegeneration in the brains of fetal and adult monkeys as well as cultured monkey neurons without affecting mitochondrial protein expression and morphology. Importantly, PINK1 mutations in the primate brain and human cells reduce protein phosphorylation that is important for neuronal function and survival. Our findings suggest that PINK1 kinase activity rather than its mitochondrial function is essential for the neuronal survival in the primate brains and that its kinase dysfunction could be involved in the pathogenesis of PD.

During salinity stress, osmoregulatory processes in euryhaline fish need to modify for their survival, and glucose is the preferred mode of extra energy during such conditions. These organisms must have a proper mechanism to maintain glucose homeostasis during such modified osmoregulatory process across different body fluids. Hence, we studied high salinity effect on regulation of glucose homeostasis in Mozambique tilapia. The fish were induced to 15‰ salinity for 21 days. Glucose, glycogen, ion concentrations, Na+-K+-ATPase, pyruvate kinase, γ-amylase activities and GLUT mRNA expressions were investigated in liver, intestine, gill and white muscle tissues. At the end of experiment, Na+ ion concentrations, glucose content and activity of Na+-K+-ATPase especially in the gill and intestine were increased, while decrease in liver and gill glycogen content was seen. Lower concentration of glycogen decrease was observed in the intestine and white muscle of the treated group. High pyruvate kinase activity was noticed in liver and gill tissues that correlates with high Na+-K+-ATPase activity. Elevated γ-amylase activity was observed in the liver and intestine suggesting breakdown of glycogen; however, gill and white muscle did not show any increased activity. Increase in GLUT1 and GLUT4 mRNA expressions was observed especially in the gill and intestine, while increase in GLUT2 mRNA expressions was observed in the liver. Upregulations of GLUTs suggest higher influx of glucose into the cell for catabolism to provide energy and further to drive the enhanced osmoregulatory process. These findings suggest glucose homeostasis being regulated in Mozambique tilapia during salinity acclimation.

Objective To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. Methods Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. Results In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. Conclusion These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.

Purpose: DYRK1A syndrome is among the most frequent monogenic forms of intellectual disability (ID). We refined the molecular and clinical description of this disorder and developed tools to improve interpretation of missense variants, which remains a major challenge in human genetics. Methods: We reported clinical and molecular data for 50 individuals with ID harboring DYRK1A variants and developed (1) a specific DYRK1A clinical score; (2) amino acid conservation data generated from 100 DYRK1A sequences across different taxa; (3) in vitro overexpression assays to study level, cellular localization, and kinase activity of DYRK1A mutant proteins; and (4) a specific blood DNA methylation signature. Results: This integrative approach was successful to reclassify several variants as pathogenic. However, we questioned the involvement of some others, such as p.Thr588Asn, still reported as likely pathogenic, and showed it does not cause an obvious phenotype in mice. Conclusion: Our study demonstrated the need for caution when interpreting variants in DYRK1A, even those occurring de novo. The tools developed will be useful to interpret accurately the variants identified in the future in this gene. Graphic abstract: [Figure not available: see fulltext.]

Diabetic kidney disease (DKD) is one of the most common complications of diabetes and is clinically featured by progressive albuminuria, consequent to glomerular destruction that involves podocyte senescence. Burgeoning evidence suggests that ketosis, in particular β-hydroxybutyrate, exerts a beneficial effect on aging and on myriad metabolic or chronic diseases, including obesity, diabetes and chronic kidney diseases. Its effect on DKD is largely unknown. In vitro in podocytes exposed to a diabetic milieu, β-hydroxybutyrate treatment substantially mitigated cellular senescence and injury, as evidenced by reduced formation of γH2AX foci, reduced staining for senescence-associated-β-galactosidase activity, diminished expression of key mediators of senescence signaling like p16INK4A and p21, and preserved expression of synaptopodin. This beneficial action of β-hydroxybutyrate coincided with a reinforced transcription factor Nrf2 antioxidant response. Mechanistically, β-hydroxybutyrate inhibition of glycogen synthase kinase 3β (GSK3β), a convergent point for myriad signaling pathways regulating Nrf2 activity, seems to contribute. Indeed, trigonelline, a selective inhibitor of Nrf2, or ectopic expression of constitutively active mutant GSK3β abolished, whereas selective activation of Nrf2 was sufficient for the anti-senescent and podocyte protective effects of β-hydroxybutyrate. Moreover, molecular modeling and docking analysis revealed that β-hydroxybutyrate is able to directly target the ATP-binding pocket of GSK3β and thereby block its kinase activity. In murine models of streptozotocin-elicited DKD, β-hydroxybutyrate therapy inhibited GSK3β and reinforced Nrf2 activation in glomerular podocytes, resulting in lessened podocyte senescence and injury and improved diabetic glomerulopathy and albuminuria. Thus, our findings may pave the way for developing a β-hydroxybutyrate-based novel approach of therapeutic ketosis for treating DKD.

Colorectal carcinoma (CRC) is the second most deadly cancer worldwide. Therapies that take advantage of DNA repair defects have been explored in various tumors but not yet systematically in CRC. Here, we found that Diphosphoinositol Pentakisphosphate Kinase 2 (PPIP5K2), an inositol pyrophosphate kinase, was highly expressed in CRC and associated with a poor prognosis of CRC patients. In vitro and in vivo functional studies demonstrated that PPIP5K2 could promote the proliferation and migration ability of CRC cells independent of its inositol pyrophosphate kinase activity. Mechanically, S1006 dephosphorylation of PPIP5K2 could accelerate its dissociation with 14-3-3 in the cytoplasm, resulting in more nuclear distribution. Moreover, DNA damage treatments such as doxorubicin (DOX) or irradiation (IR) could induce nuclear translocation of PPIP5K2, which subsequently promoted homologous recombination (HR) repair by binding and recruiting RPA70 to the DNA damage site as a novel scaffold protein. Importantly, we verified that S1006 dephosphorylation of PPIP5K2 could significantly enhance the DNA repair ability of CRC cells through a series of DNA repair phenotype assays. In conclusion, PPIP5K2 is critical for enhancing the survival of CRC cells via facilitating DNA HR repair. Our findings revealed an unrecognized biological function and mechanism model of PPIP5K2 dependent on S1006 phosphorylation and provided a potential therapeutic target for CRC patients.

Background: Inherited mutations in the LRRK2 protein are common causes of Parkinson’s disease, but the mechanisms by which increased kinase activity of mutant LRRK2 leads to pathological events remain to be determined. In vitro assays (heterologous cell culture, phospho-protein mass spectrometry) suggest that several Rab proteins might be directly phosphorylated by LRRK2-G2019S. An in vivo screen of Rab expression in dopaminergic neurons in young adult Drosophila demonstrated a strong genetic interaction between LRRK2-G2019S and Rab10. Objective: To determine if Rab10 is necessary for LRRK2-induced pathophysiological responses in the neurons that control movement, vision, circadian activity, and memory. These four systems were chosen because they are modulated by dopaminergic neurons in both humans and flies. Methods: LRRK2-G2019S was expressed in Drosophila dopaminergic neurons and the effects of Rab10 depletion on Proboscis Extension, retinal neurophysiology, circadian activity pattern (‘sleep’), and courtship memory determined in aged flies. Results: Rab10 loss-of-function rescued LRRK2-G2019S induced bradykinesia and retinal signaling deficits. Rab10 knock-down, however, did not rescue the marked sleep phenotype which results from dopaminergic LRRK2-G2019S. Courtship memory is not affected by LRRK2, but is markedly improved by Rab10 depletion. Anatomically, both LRRK2-G2019S and Rab10 are seen in the cytoplasm and at the synaptic endings of dopaminergic neurons. Conclusion: We conclude that, in Drosophila dopaminergic neurons, Rab10 is involved in some, but not all, LRRK2-induced behavioral deficits. Therefore, variations in Rab expression may contribute to susceptibility of different dopaminergic nuclei to neurodegeneration seen in people with Parkinson’s disease.

Tyrosine kinase inhibitors of anaplastic lymphoma kinase (ALK-TKIs) including alectinib have been the standard therapy against ALK fusion gene-positive non–small cell lung cancers (NSCLCs). Many ALK fusion variants have been identified in NSCLCs, and the predominant variants are echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) variant 1 (V1), V2 and V3a/b. However, there have been conflicting reports on the clinical responses of these variants to ALK-TKIs, and there are few reports on other less common ALK variants. To examine the influence of ALK variants on the efficacy of ALK-TKIs, we analyzed the sensitivity to alectinib of eight types of ALK variant: three major variants (V1, V2 and V3a) and five less common variants (V4; kinesin family member 5-ALK; kinesin light chain 1-ALK; striatin, calmodulin-binding protein-ALK; and tropomyosin-receptor kinase fused gene-ALK). Analysis was done by cell-free kinase assays using the recombinant proteins and by cell, growth assays using murine Ba/F3 cells expressing ALK variants. The kinase activity of each recombinant protein was significantly inhibited by alectinib. Intracellular ALK phosphorylation levels and its downstream signaling mediators, STAT3 and ERK, were suppressed by alectinib in each ALK variant-expressing Ba/F3 cell. Each cellular proliferation was markedly inhibited by alectinib treatment. There was no significant difference in the IC50 values between cells, with a

MAP4K4 is a Ste20 member and reported to play important roles in various pathologies, including in cancer. However, the mechanism by which MAP4K4 promotes pancreatic cancer is not fully understood. It is suggested that MAP4K4 might function as a cancer promoter via specific downstream target(s) in an organ-specific manner. Here we identified MLK3 as a direct downstream target of MAP4K4. The MAP4K4 and MLK3 associates with each other, and MAP4K4 phosphorylates MLK3 on Thr738 and increases MLK3 kinase activity and downstream signaling. The phosphorylation of MLK3 by MAP4K4 promotes pancreatic cancer cell proliferation, migration, and colony formation. Moreover, MAP4K4 is overexpressed in human pancreatic tumors and directly correlates with the disease progression. The MAP4K4-specific pharmacological inhibitor, GNE-495, impedes pancreatic cancer cell growth, migration, induces cell death, and arrests cell cycle progression. Additionally, the GNE-495 reduced the tumor burden and extended survival of the KPC mice with pancreatic cancer. The MAP4K4 inhibitor also reduced MAP4K4 protein expression, tumor stroma, and induced cell death in murine pancreatic tumors. These findings collectively suggest that MLK3 phosphorylation by MAP4K4 promotes pancreatic cancer, and therefore therapies targeting MAP4K4 might alleviate the pancreatic cancer tumor burden in patients.

The nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain-containing 3 (NLRP3) inflammasome is essential in inflammation and inflammatory disorders. Phosphorylation at various sites on NLRP3 differentially regulates inflammasome activation. The Ser725 phosphorylation site on NLRP3 is depicted in multiple inflammasome activation scenarios, but the importance and regulation of this site has not been clarified. The present study revealed that the phosphorylation of Ser725 was an essential step for the priming of the NLRP3 inflammasome in macrophages. We also showed that Ser725 was directly phosphorylated by misshapen (Msn)/NIK-related kinase 1 (MINK1), depending on the direct interaction between MINK1 and the NLRP3 LRR domain. MINK1 deficiency reduced NLRP3 activation and suppressed inflammatory responses in mouse models of acute sepsis and peritonitis. Reactive oxygen species (ROS) upregulated the kinase activity of MINK1 and subsequently promoted inflammasome priming via NLRP3 Ser725 phosphorylation. Eliminating ROS suppressed NLRP3 activation and reduced sepsis and peritonitis symptoms in a MINK1-dependent manner. Altogether, our study reveals a direct regulation of the NLRP3 inflammasome by Msn family kinase MINK1 and suggests that modulation of MINK1 activity is a potential intervention strategy for inflammasome-related diseases.

Vacuolar protein sorting 35 (VPS35) regulates neurotransmitter receptor recycling from endosomes. A missense mutation (D620N) in VPS35 leads to autosomal-dominant, late-onset Parkinson’s disease. Here, we study the basic neurobiology of VPS35 and Parkinson’s disease mutation effects in the D620N knock-in mouse and the effect of leucine-rich repeat kinase 2 (LRRK2) inhibition on synaptic phenotypes. The study was conducted using a VPS35 D620N knock-in mouse that expresses VPS35 at endogenous levels. Protein levels, phosphorylation states, and binding ratios in brain lysates from knock-in mice and wild-type littermates were assayed by co-immunoprecipitation and western blot. Dendritic protein co-localization, AMPA receptor surface expression, synapse density, and glutamatergic synapse activity in primary cortical cultures from knock-in and wild-type littermates were assayed using immunocytochemistry and whole-cell patch clamp electrophysiology. In brain tissue, we confirm VPS35 forms complexes with LRRK2 and AMPA-type glutamate receptor GluA1 subunits, in addition to NMDA-type glutamate receptor GluN1 subunits and D2-type dopamine receptors. Receptor and LRRK2 binding was unaltered in D620N knock-in mice, but we confirm the mutation results in reduced binding of VPS35 with WASH complex member FAM21, and increases phosphorylation of the LRRK2 kinase substrate Rab10, which is reversed by LRRK2 kinase inhibition in vivo. In cultured cortical neurons from knock-in mice, pRab10 is also increased, and reversed by LRRK2 inhibition. The mutation also results in increased endosomal recycling protein cluster density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate transmission, and GluA1 surface expression. LRRK2 kinase inhibition, which reversed Rab10 hyper-phosphorylation, did not rescue elevated glutamate release or surface GluA1 expression in knock-in neurons, but did alter AMPAR traffic in wild-type cells. The results improve our understanding of the cell biology of VPS35, and the consequences of the D620N mutation in developing neuronal networks. Together the data support a chronic synaptopathy model for latent neurodegeneration, providing phenotypes and candidate pathophysiological stresses that may drive eventual transition to late-stage parkinsonism in VPS35 PD. The study demonstrates the VPS35 mutation has effects that are independent of ongoing LRRK2 kinase activity, and that LRRK2 kinase inhibition alters basal physiology of glutamate synapses in vitro. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-021-00848-w.

Doublecortin-like kinase 1 (DCLK1) is a Doublecortin family kinase involved in a range of brain development processes including cell migration, axon/dendrite growth, and synapse development. The Dclk1 gene potentially generates multiple splicing isoforms, but the detailed expression patterns in the brain as well as in vivo functions of each isoform are still incompletely understood. Here we assessed expression patterns of DCLK1 isoforms using multiple platforms including in silico, in situ, and in vitro datasets in the developing mouse brain, and show quantitative evidence that among the four DCLK1 isoforms, DCLK1-L and DCL are mainly expressed in the embryonic cortex whereas DCLK1-L and CPG16 become dominant compared to DCL and CARP in the postnatal cortex. We also provide compelling evidence that DCLK1 isoforms are distributed in the partially distinct brain regions in the embryonic and the postnatal stages. We further show that overexpression of DCLK1-L, but not the other isoforms, in neural progenitors causes severe migration defects in the cortex, and that the migration defects are dependent on the kinase activity of DCLK1-L. Our data thus uncover partially segregated localization of DCLK1 isoforms in the developing mouse brain and suggest different roles for distinct DCLK1 isoforms in the brain development and function.

Background Osteoarthritis (OA) and rheumatoid arthritis (RA) were two major joint diseases with similar clinical phenotypes. This study aimed to determine the mechanistic similarities and differences between OA and RA by integrated analysis of multiple gene expression data sets. Methods Microarray data sets of OA and RA were obtained from the Gene Expression Omnibus (GEO). By integrating multiple gene data sets, specific differentially expressed genes (DEGs) were identified. The Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and protein–protein interaction (PPI) network analysis of DEGs were conducted to determine hub genes and pathways. The “Cell Type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT)” algorithm was employed to evaluate the immune infiltration cells (IICs) profiles in OA and RA. Moreover, mouse models of RA and OA were established, and selected hub genes were verified in synovial tissues with quantitative polymerase chain reaction (qPCR). Results A total of 1116 DEGs were identified between OA and RA. GO functional enrichment analysis showed that DEGs were enriched in regulation of cell morphogenesis involved in differentiation, positive regulation of neuron differentiation, nuclear speck, RNA polymerase II transcription factor complex, protein serine/threonine kinase activity and proximal promoter sequence-specific DNA binding. KEGG pathway analysis showed that DEGs were enriched in EGFR tyrosine kinase inhibitor resistance, ubiquitin mediated proteolysis, FoxO signaling pathway and TGF-beta signaling pathway. Immune cell infiltration analysis identified 9 IICs with significantly different distributions between OA and RA samples. qPCR results showed that the expression levels of the hub genes (RPS6, RPS14, RPS25, RPL11, RPL27, SNRPE, EEF2 and RPL19) were significantly increased in OA samples compared to their counterparts in RA samples (P Conclusion This large-scale gene analyses provided new insights for disease-associated genes, molecular mechanisms as well as IICs profiles in OA and RA, which may offer a new direction for distinguishing diagnosis and treatment between OA and RA.

Regulation of epithelial cell death has emerged as a key mechanism controlling immune homeostasis in barrier surfaces. Necroptosis is a type of regulated necrotic cell death induced by receptor interacting protein kinase 3 (RIPK3) that has been shown to cause inflammatory pathologies in different tissues. The role of regulated cell death and particularly necroptosis in lung homeostasis and disease remains poorly understood. Here we show that mice with Airway Epithelial Cell (AEC)-specific deficiency of Fas-associated with death domain (FADD), an adapter essential for caspase-8 activation, developed exacerbated allergic airway inflammation in a mouse model of asthma induced by sensitization and challenge with house dust mite (HDM) extracts. Genetic inhibition of RIPK1 kinase activity by crossing to mice expressing kinase inactive RIPK1 as well as RIPK3 or MLKL deficiency prevented the development of exaggerated HDM-induced asthma pathology in FADDAEC-KO mice, suggesting that necroptosis of FADD-deficient AECs augmented the allergic immune response. These results reveal a role of AEC necroptosis in amplifying airway allergic inflammation and suggest that necroptosis could contribute to asthma exacerbations caused by respiratory virus infections inducing AEC death.

Cellular stress can induce cytoplasmic ribonucleoprotein complexes called stress granules that allow the cells to survive. Stress granules are also central to cellular responses to infections, in which they can act as platforms for viral sensing or modulate innate immune signaling through pattern recognition receptors. However, the effect of innate immune signaling on stress granules is poorly understood. In this study, we report that prior induction of innate immune signaling through TLRs inhibited stress granule assembly in a TLR ligand dose-dependent manner in murine bone marrow–derived macrophages. Time course analysis suggests that TLR stimulation can reverse stress granule assembly even after it has begun. Additionally, both MYD88- and TRIF-mediated TLR signaling inhibited stress granule assembly in response to endoplasmic reticulum stress in bone marrow–derived macrophages and the chemotherapeutic drug oxaliplatin in murine B16 melanoma cells. This inhibition was not due to a decrease in expression of the critical stress granule proteins G3BP1 and DDX3X and was independent of IRAK1/4, JNK, ERK and P38 kinase activity but dependent on IKK complex kinase activity. Overall, we have identified the TLR–IKK complex signaling axis as a regulator of stress granule assembly–disassembly dynamics, highlighting cross-talk between processes that are critical in health and disease.

The NLRP3 inflammasome mediates the production of proinflammatory cytokines and initiates inflammatory cell death. Although NLRP3 is essential for innate immunity, aberrant NLRP3 inflammasome activation contributes to a wide variety of inflammatory diseases. Understanding the pathways that control NLRP3 activation will help develop strategies to treat these diseases. Here we identify WNK1 as a negative regulator of the NLRP3 inflammasome. Macrophages deficient in WNK1 protein or kinase activity have increased NLRP3 activation and pyroptosis compared with control macrophages. Mice with conditional knockout of WNK1 in macrophages have increased IL-1β production in response to NLRP3 stimulation compared with control mice. Mechanistically, WNK1 tempers NLRP3 activation by balancing intracellular Cl– and K+ concentrations during NLRP3 activation. Collectively, this work shows that the WNK1 pathway has a critical function in suppressing NLRP3 activation and suggests that pharmacological inhibition of this pathway to treat hypertension might have negative clinical implications., The serine/threonine kinase WNK1 is an inhibitor of chloride efflux. Here the authors show that this inhibition is a means of negatively regulating the activation of the NLRP3 inflammasome in macrophages, leading to reduced inflammatory responses.

Receptor interaction protein kinase 1 (RIPK1) plays a diverse role in tumor necrosis factor α (TNFα) signalings. The ubiquitination of RIPK1 is essential for NF-κB activation, whereas its kinase activity promotes apoptosis and necroptosis. However, the mechanisms underlying have not been fully illuminated. Here we report that PH domain-containing family O member 2 (PLEKHO2) inhibits RIPK1-dependent cell death and is necessary for NF-κB activation in response to TNFα. Cells of PLKEHO2 deficiency are more susceptible to TNF-α induced apoptosis and necroptosis with increased RIPK1 activation, which is consistent with the observation that the susceptibility of PLEKHO2−/− cells is effectively prevented by treatment of RIPK1 kinase inhibitor. Moreover, PLEKHO2 deficient cells exhibit compromised RIPK1 ubiquitination and NF-κB activation in response to TNFα. Ultimately, PLEKHO2-deficient mice display greatly increased hepatotoxicity and lethality after TNFα-induced hepatitis. In summary, our study revealed that PLEKHO2 is a novel inhibitor of apoptosis and necroptosis, which plays a key role in regulating RIPK1 ubiquitination and activation

Background Increasing studies have reported that oncogenes regulate components of the immune system, suggesting that this is a mechanism for tumorigenesis. Aurora kinase A (AURKA), a serine/threonine kinase, is involved in cell mitosis and is essential for tumor cell proliferation, metastasis, and drug resistance. However, the mechanism by which AURKA is involved in immune response regulation is unclear. Therefore, this study aimed to investigate the role of AURKA in immune regulation in triple‐negative breast cancer (TNBC). Methods Peripheral blood mononuclear cells (PBMCs) were co‐cultured with TNBC cells. The xCELLigence Real‐Time Cell Analyzer‐MP system was used to detect the killing efficiency of immune cells on TNBC cells. The expression of immune effector molecules was tested by quantitative real‐time polymerase chain reaction (qRT‐PCR) to evaluate immune function. Furthermore, to validate AURKA‐regulated immune response in vivo, 4T1 murine breast cancer cell line with AURKA overexpression or downregulation was engrafted into BALB/c mice. The distribution and proportion of immune cells in tumors were further evaluated by immunohistochemistry and flow cytometry. Results Downregulation of AURKA in TNBC cells increased immune response by activating CD8+ T cell proliferation and activity. Nuclear rather than cytoplasmic AURKA‐derived programmed death‐ligand 1 (PD‐L1) expression was independent of its kinase activity. Mechanistic investigations showed that nuclear AURKA increased PD‐L1 expression via an MYC‐dependent pathway. PD‐L1 overexpression mostly reversed AURKA silencing‐induced expression of immune effector molecules, including interleukin‐ (IL‐2), interferon‐γ (IFN‐γ), and perforin. Moreover, AURKA expression was negatively correlated with the enrichment and activity of tumor‐infiltrating CD8+ T cells in 4T1 engrafted BALB/c mouse model. Conclusions Nuclear AURKA elevated PD‐L1 expression via an MYC‐dependent pathway and contributed to immune evasion in TNBC. Therapies targeting nuclear AURKA may restore immune responses against tumors., The present study showed that nuclear AURKA rather than its kinase activity induced PD‐L1 expression via a MYC‐dependent pathway. In addition, downregulation of AURKA led to increased CD8+ T cell infiltration and activation in vivo. Thus, our data provided evidences for the involvement of AURKA in immune evasion of triple‐negative breast cancer.

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.

Cue-elicited drug-craving is a cardinal feature of addiction that intensifies (incubates) during protracted withdrawal. In a rat model, these addiction-related behavioral pathologies are mediated, respectively, by time-dependent increases in PI3K/Akt1 signaling and reduced Group 1 metabotropic glutamate receptor (mGlu) expression, within the ventromedial prefrontal cortex (vmPFC). Herein, we examined the capacity of single oral dosing with everolimus, an FDA-approved inhibitor of the PI3K/Akt effector mTOR, to reduce incubated cocaine-craving and reverse incubation-associated changes in vmPFC kinase activity and mGlu expression. Rats were trained to lever-press for intravenous infusions of cocaine or delivery of sucrose pellets and then subjected to tests for cue-reinforced responding during early (3 days) or late (30–46 days) withdrawal. Rats were gavage-infused with everolimus (0–1.0 mg/kg), either prior to testing to examine for effects upon reinforcer-seeking behavior, or immediately following testing to probe effects upon the consolidation of extinction learning. Single oral dosing with everolimus dose-dependently blocked cocaine-seeking during late withdrawal and the effect lasted at least 24 h. No everolimus effects were observed for cue-elicited sucrose-seeking or cocaine-seeking in early withdrawal. In addition, everolimus treatment, following initial cue-testing, reduced subsequent cue hyper-responsivity exhibited observed during late withdrawal, arguing a facilitation of extinction memory consolidation. everolimus’ “anti-incubation” effect was associated with a reversal of withdrawal-induced changes in indices of PI3K/Akt1/mTOR activity, as well as Homer protein and mGlu1/5 expression, within the prelimbic (PL) subregion of the prefrontal cortex. Our results indicate mTOR inhibition as a viable strategy for interrupting heightened cocaine-craving and facilitating addiction recovery during protracted withdrawal.

Krabbe disease, an inherited leukodystrophy, is a sphingolipidosis caused by deficiency of β-galactocerebrosidase: it is characterized by myelin loss, and pathological activation of macrophage/microglia and astrocytes. To define driving pathogenic factors, we explored the expression repertoire of candidate neuroinflammatory genes: upregulation of receptor interacting protein kinase 1 (Ripk1) and disease-associated microglia (DAM) genes, including Cst7 and Ch25h, correlated with severity of Krabbe disease genetically modelled in the twitcher mouse. Upregulation of Ripk1 in Iba1/Mac2-positive microglia/macrophage associated with the pathognomic hypertrophic/globoid phenotype of this disease. Widespread accumulation of ubiquitinin1 in white and grey matter co-localised with p62. In Sandhoff disease, another sphingolipid disorder, neuroinflammation, accumulation of p62 and increased Ripk1 expression was observed. The upregulated DAM genes and macrophage/microglia expression of Ripk1 in the authentic model of Krabbe disease strongly resemble those reported in Alzheimer disease associating with disturbed autophagosomal/lysosomal homeostasis. Activation of this shared molecular repertoire, suggests the potential for therapeutic interdiction at a common activation step, irrespective of proximal causation. To clarify the role of Ripk1 in the pathogenesis of Krabbe disease, we first explored the contribution of its kinase function, by intercrossing twitcher and the K45A kinase-dead Ripk1 mouse and breeding to homozygosity. Genetic ablation of Ripk1 kinase activity neither altered the neuropathological features nor the survival of twitcher mice. We conclude that Ripk1 kinase-dependent inflammatory and degenerative capabilities play no instrumental role in Krabbe disease; however, putative kinase-independent functions of Ripk1 remain formally to be explored in its molecular pathogenesis.

Activated and promoter-bound heat-shock transcription factor 1 (HSF1) induces RNA polymerase II recruitment upon heat shock, and this is facilitated by the core Mediator in Drosophila and yeast. Another Mediator module, CDK8 kinase module (CKM), consisting of four subunits including MED12 and CDK8, plays a negative or positive role in the regulation of transcription; however, its involvement in HSF1-mediated transcription remains unclear. We herein demonstrated that HSF1 interacted with MED12 and recruited MED12 and CDK8 to the HSP70 promoter during heat shock in mammalian cells. The kinase activity of CDK8 (and its paralog CDK19) promoted HSP70 expression partly by phosphorylating HSF1-S326 and maintained proteostasis capacity. These results indicate an important role for CKM in the protection of cells against proteotoxic stress.

Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. Disseminated ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the maintenance and expansion of breast cancer stem cells (CSCs). Breast CSCs frequently exist as a minority population in therapy resistant tumors. In this study, we show that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. Seahorse metabolic assays demonstrated that cytoplasmic PELP1 influences cellular metabolism by increasing both glycolysis and mitochondrial respiration. PELP1 interacts with PFKFB3 and PFKFB4 proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB4 knockdown inhibited in vivo emergence of circulating tumor cell (CTC) populations in mammary intraductal (MIND) models. Application of PFKFB inhibitors in combination with ER targeted therapies blocked tumorsphere formation in multiple models of advanced breast cancer including tamoxifen (TamR) and paclitaxel (TaxR) resistant models, murine tumor cells, and ER+ patient-derived organoids (PDxO). Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cell populations that include CSCs and CTCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance.

AKT is involved in a number of key cellular processes including cell proliferation, apoptosis and metabolism. Hyperactivation of AKT is associated with many pathological conditions, particularly cancers. Emerging evidence indicates that arginine methylation is involved in modulating AKT signaling pathway. However, whether and how arginine methylation directly regulates AKT kinase activity remain unknown. Here we report that protein arginine methyltransferase 5 (PRMT5), but not other PRMTs, promotes AKT activation by catalyzing symmetric dimethylation of AKT1 at arginine 391 (R391). Mechanistically, AKT1-R391 methylation cooperates with phosphatidylinositol 3,4,5 trisphosphate (PIP3) to relieve the pleckstrin homology (PH)-in conformation, leading to AKT1 membrane translocation and subsequent activation by phosphoinositide-dependent kinase-1 (PDK1) and the mechanistic target of rapamycin complex 2 (mTORC2). As a result, deficiency in AKT1-R391 methylation significantly suppresses AKT1 kinase activity and tumorigenesis. Lastly, we show that PRMT5 inhibitor synergizes with AKT inhibitor or chemotherapeutic drugs to enhance cell death. Altogether, our study suggests that R391 methylation is an important step for AKT activation and its oncogenic function., Arginine methylation has been reported to regulate AKT-associated signalling. Here, the authors show that PRMT5-mediated arginine methylation promotes AKT activity and induces tumourigenesis.

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.

Purpose To expand the recent description of a new neurodevelopmental syndrome related to alterations in CDK19. Methods Individuals were identified through international collaboration. Functional studies included autophosphorylation assays for CDK19 Gly28Arg and Tyr32His variants and in vivo zebrafish assays of the CDK19(G28R) and CDK19(Y32H). Results We describe 11 unrelated individuals (age range: 9 months to 14 years) with de novo missense variants mapped to the kinase domain of CDK19, including two recurrent changes at residues Tyr32 and Gly28. In vitro autophosphorylation and substrate phosphorylation assays revealed that kinase activity of protein was lower for p.Gly28Arg and higher for p.Tyr32His substitutions compared with that of the wild-type protein. Injection of CDK19 messenger RNA (mRNA) with either the Tyr32His or the Gly28Arg variants using in vivo zebrafish model significantly increased fraction of embryos with morphological abnormalities. Overall, the phenotype of the now 14 individuals with CDK19-related disorder includes universal developmental delay and facial dysmorphism, hypotonia (79%), seizures (64%), ophthalmologic anomalies (64%), and autism/autistic traits (56%). Conclusion CDK19 de novo missense variants are responsible for a novel neurodevelopmental disorder. Both kinase assay and zebrafish experiments showed that the pathogenetic mechanism may be more diverse than previously thought.

Background and purpose α4β2 nicotinic acetylcholine receptor (nAChR), a subtype of the ligand-gated ion channel, is abundantly expressed in the brain and implicated in several neurological disorders. The endocytosis of nAChR plays important roles in the pathogenesis of neurological diseases, but its molecular mechanisms remain poorly understood. Experimental approach Loss-of-function approaches and mutants of α4β2 nAChR that display different endocytic properties were used to identify the cellular components and processes responsible for endocytosis. The signaling cascade that leads to endocytosis was deduced via protein interactions in predicted cellular components. The endocytosis of α4β2 nAChR was determined and crosschecked using an enzyme-linked immunosorbent assay and radioligand assay. Key results Endocytosis of α4β2 nAChR occurred through clathrin-mediated endocytosis in a dynamin-dependent manner. 14-3-3η-dependent Src-mediated phosphorylation of the nAChR α4 subunit at Y575 was required for nAChR endocytosis, and this occurred with the assistance of β-arrestin1 and G protein-coupled receptor (GPCR) kinase 2 (GRK2) without the need for kinase activity. Endocytosis triggered the mouse double minute 2 homolog-mediated ubiquitination and subsequent downregulation of α4β2 nAChR. Conclusions and implications α4β2 nAChR, an ionophore receptor, employs the metabotropic signaling pathway required for endocytosis, which leads to ubiquitination and downregulation. Further, GRK2 and β-arrestin1, representative players of GPCRs, are involved in the endocytosis of α4β2 nAChR via different mechanisms. Considering the functional and pathological implications of nAChR endocytosis, results obtained in this study are crucial for the progression of basic research and clinical investigations.

Lung cancer is the leading cause of cancer‐related deaths. LIM domain kinase (LIMK) 1 is a member of serine/threonine kinase family and highly expressed in various cancers. Luteolin, a polyphenolic plant flavonoid, has been reported to suppress tumour proliferation through inducing apoptosis and autophagy via MAPK activation in glioma. However, the mechanism of luteolin on suppressing lung cancer growth is still unclear. We found that luteolin targeted LIMK1 from the in silico screening and significantly inhibited the LIMK1 kinase activity, which was confirmed with pull‐down binding assay and computational docking models. Treatment with luteolin inhibited lung cancer cells anchorage‐independent colony growth and induced apoptosis and cell cycle arrest at G1 phase. Luteolin also decreased the expression of cyclin D1 and increased the levels of cleaved caspase‐3 by down‐regulating LIMK1 signalling related targets, including p‐LIMK and p‐cofilin. Furthermore, luteolin suppressed the lung cancer patient‐derived xenograft tumour growth by decreasing Ki‐67, p‐LIMK and p‐cofilin expression in vivo. Taken together, these results provide insight into the mechanism that underlies the anticancer effects of luteolin on lung cancer, which involved in down‐regulation of LIMK1 and its interaction with cofilin. It also provides valuable evidence for translation towards lung cancer clinical trials with luteolin.

G protein-coupled receptor kinase 5 (GRK5) regulates inflammatory responses via the nuclear factor-kappa B (NF-κB) pathway. This study investigated the functional involvement of GRK5 in the pathogenesis of inflammatory arthritis. Immunohistochemically, rheumatoid arthritis (RA) synovium had a significantly higher proportion of GRK5-positive cells in the synovial lining layer than healthy control synovium. Gene expression and NF-κB activation in lipopolysaccharide-stimulated human SW982 synovial cells were significantly suppressed by silencing of the GRK5 gene. Similarly, GRK5 kinase activity inhibition in human primary RA synovial cells attenuated gene expressions of inflammatory factors. In a murine model of collagen antibody-induced arthritis, arthritis scores and serum IL6 production of GRK5 knockout (GRK5-/-) mice were significantly lower than those of wild-type mice. Histologically, the degree of synovitis and cartilage degeneration in GRK5-/- mice was significantly lower than in wild-type mice. In in vitro analyses using activated murine macrophages and fibroblast-like synoviocytes, gene expression of inflammatory factors and p65 nuclear translocation were significantly lower in GRK5-/- mice compared to wild-type mice. In conclusion, our results suggested that GRK5 is deeply involved in the pathogenesis of inflammatory arthritis, therefore, GRK5 inhibition could be a potential therapeutic target for types of inflammatory arthritis such as RA.

The blood flow restoration of ischemic tissues causes myocardial injury. Dexmedetomidine (Dex) protects multi-organs against ischemia/reperfusion (I/R) injury. This study investigated the protective mechanism of Dex post-treatment in myocardial I/R injury. The rat model of myocardial I/R was established. The effects of Dex post-treatment on cardiac function and autophagy flow were observed. Dex attenuated myocardial I/R injury and reduced I/R-induced autophagy in rats. Dex weakened the interactions between Beclin1 and Vps34 and Beclin1 and Atg14L, thus downregulating Vps34 kinase activity. In vitro, the cardiomyocytes subjected to oxygen glucose deprivation/reoxygenation were treated with Dex and PI3K inhibitor LY294002. LY294002 attenuated the myocardial protective effect of DEX, indicating that Dex protected against cardiac I/R by activating the PI3K/Akt pathway. In conclusion, Dex upregulated the phosphorylation of Beclin1 at S295 site by activating the PI3K/Akt pathway and reduced the interactions of Atg14L-Beclin1-Vps34 complex, thus inhibiting autophagy and protecting against myocardial I/R injury.

Large tumor suppressor 1 (LATS1) is the key kinase controlling activation of Hippo signalling pathway. Post-translational modifications of LATS1 modulate its kinase activity. However, detailed mechanism underlying LATS1 stability and activation remains elusive. Here we report that LATS1 is acetylated by acetyltransferase CBP at K751 and is deacetylated by deacetylases SIRT3 and SIRT4. Acetylation at K751 stabilized LATS1 by decreasing LATS1 ubiquitination and inhibited LATS1 activation by reducing its phosphorylation. Mechanistically, LATS1 acetylation resulted in inhibition of YAP phosphorylation and degradation, leading to increased YAP nucleus translocation and promoted target gene expression. Functionally, LATS1-K751Q, the acetylation mimic mutant potentiated lung cancer cell migration, invasion and tumor growth, whereas LATS1-K751R, the acetylation deficient mutant inhibited these functions. Taken together, we demonstrated a previously unidentified post-translational modification of LATS1 that converts LATS1 from a tumor suppressor to a tumor promoter by suppression of Hippo signalling through acetylation of LATS1.

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

It is widely accepted that cellular processes are controlled by protein phosphorylation and has become increasingly clear that protein degradation, localization and conformation as well as protein-protein interaction are the examples of subsequent cellular events modulated by protein phosphorylation. Enamel matrix proteins belong to members of the secretory calcium binding phosphoprotein (SCPP) family clustered on chromosome 4q21, and most of the SCPP phosphoproteins have at least one S-X-E motifs (S; serine, X; any amino acid, E; glutamic acid). It has been reported that mutations in C4orf26 gene, located on chromosome 4q21, are associated with autosomal recessive type of Amelogenesis Imperfecta (AI), a hereditary condition that affects enamel formation/mineralization. The enamel phenotype observed in patients with C4orf26 mutations is hypomineralized and partially hypoplastic, indicating that C4orf26 protein may function at both secretory and maturation stages of amelogenesis. The previous in vitro study showed that the synthetic phosphorylated peptide based on C4orf26 protein sequence accelerates hydroxyapatite nucleation. Here we show the molecular cloning of Gm1045, mouse homologue of C4orf26, which has 2 splicing isoforms. Immunohistochemical analysis demonstrated that the immunolocalization of Gm1045 is mainly observed in enamel matrix in vivo. Our report is the first to show that FAM20C, the Golgi casein kinase, phosphorylates C4orf26 and Gm1045 in cell cultures. The extracellular localization of C4orf26/Gm1045 was regulated by FAM20C kinase activity. Thus, our data point out the biological importance of enamel matrix-kinase control of SCPP phosphoproteins and may have a broad impact on the regulation of amelogenesis and AI.

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.

Terminal regions of Drosophila embryos are patterned by signaling through ERK, which is genetically deregulated in multiple human diseases. Quantitative studies of terminal patterning have been recently used to investigate gain-of-function variants of human MEK1, encoding the MEK kinase that directly activates ERK by dual phosphorylation. Unexpectedly, several mutations reduced ERK activation by extracellular signals, possibly through a negative feedback triggered by signal-independent activity of the mutant variants. Here we present experimental evidence supporting this model. Using a MEK variant that combines a mutation within the negative regulatory region with alanine substitutions in the activation loop, we prove that pathogenic variants indeed acquire signal-independent kinase activity. We also demonstrate that signal-dependent activation of these variants is independent of kinase suppressor of Ras, a conserved adaptor that is indispensable for activation of normal MEK. Finally, we show that attenuation of ERK activation by extracellular signals stems from transcriptional induction of Mkp3, a dual specificity phosphatase that deactivates ERK by dephosphorylation. These findings in the Drosophila embryo highlight its power for investigating diverse effects of human disease mutations.

In multiple myeloma (MM), frequent mutations of NRAS, KRAS, or BRAF are found in up to 50% of newly diagnosed patients. The majority of the NRAS, KRAS, and BRAF mutations occur in hotspots causing constitutive activation of the corresponding proteins. Thus, targeting RAS mutation in MM will increase therapeutic efficiency and potentially overcome drug resistance. We identified germinal center kinase (GCK) as a novel therapeutic target in MM with RAS mutation. GCK knockdown (KD) in MM cells demonstrated in vitro and in vivo that silencing of GCK induces MM cell growth inhibition, associated with blocked MKK4/7-JNK phosphorylation and impaired degradation of IKZF1/3, BCL-6, and c-MYC. These effects were rescued by overexpression of a short hairpin RNA (shRNA)-resistant GCK, thereby excluding the potential off-target effects of GCK KD. In contrast, overexpression of shRNA-resistant GCK kinase-dead mutant (K45A) inhibited MM cell proliferation and failed to rescue the effects of GCK KD on MM growth inhibition, indicating that GCK kinase activity is critical for regulating MM cell proliferation and survival. Importantly, the higher sensitivity to GCK KD in RASMut cells suggests that targeting GCK is effective in MM, which harbors RAS mutations. In accordance with the effects of GCK KD, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 and led to MM cell proliferation inhibition accompanied by induction of apoptosis. Here, our data identify GCK as a novel target in RASMut MM cells, providing a rationale to treat RAS mutations in MM. Furthermore, GCK inhibitors might represent an alternative therapy to overcome immunomodulatory drug resistance in MM.

Ventilatory deficits are common in old age and may result from neuromuscular dysfunction. Signaling via the tropomyosin-related kinase receptor B (TrkB) regulates neuromuscular transmission and, in young mice, is important for the generation of transdiaphragmatic pressure (Pdi). Loss of TrkB signaling worsened neuromuscular transmission failure and reduced maximal Pdi, and these effects are similar to those observed in old age. Administration of TrkB agonists such as 7,8-dihydroxyflavone (7,8-DHF) improves neuromuscular transmission in young and old mice (18 mo; 75% survival). We hypothesized that TrkB signaling contributes to Pdi generation in old mice, particularly during maximal force behaviors. Old male and female TrkB(F616A) mice, with a mutation that induces 1NMPP1-mediated TrkB kinase inhibition, were randomly assigned to systemic treatment with vehicle, 7,8-DHF, or 1NMPP1 1 h before experiments. Pdi was measured during eupneic breathing (room air), hypoxia-hypercapnia (10% O(2)/5% CO(2)), tracheal occlusion, spontaneous deep breaths (“sighs”), and bilateral phrenic nerve stimulation (Pdi(max)). There were no differences in the Pdi amplitude across treatments during ventilatory behaviors (eupnea, hypoxia-hypercapnia, occlusion, or sigh). As expected, Pdi increased from eupnea and hypoxia-hypercapnia (∼7 cm H(2)O) to occlusion and sighs (∼25 cm H(2)O), with no differences across treatments. Pdi(max) was ∼50 cm H(2)O in the vehicle and 7,8-DHF groups and ∼40 cm H(2)O in the 1NMPP1 group (F(8,74) = 2; P = 0.02). Our results indicate that TrkB signaling is necessary for generating maximal forces by the diaphragm muscle in old mice and are consistent with aging effects of TrkB signaling on neuromuscular transmission. NEW & NOTEWORTHY TrkB signaling is necessary for generating maximal forces by the diaphragm muscle. In 19- to 21-mo-old TrkB(F616A) mice susceptible to 1NMPP1-induced inhibition of TrkB kinase activity, maximal Pdi generated by bilateral phrenic nerve stimulation was ∼20% lower after 1NMPP1 compared with vehicle-treated mice. Treatment with the TrkB agonist 7,8-dihydroxyflavone did not affect Pdi generation when compared with age-matched mice. Inhibition of TrkB kinase activity did not affect the forces generated during lower force behaviors in old age.

The endothelial adhesion protein E-selectin/CD62E is not required for leukocyte homing, unlike closely related family member P-selectin/CD62P. As transmigration through the endothelium is one of the first steps in generating a local immune response, we hypothesized that E-selectin may play additional roles in the early stages of immune activation. We found contact with E-selectin, but not P-selectin or vascular cell adhesion molecule 1 (CD106), induced phosphorylation of protein kinase B (AKT) and nuclear factor-κB in mouse bone marrow-derived macrophages (BMDMs) in vitro. This occurred within 15 min of E-selectin contact and was dependent on phosphatidylinositol-3 kinase activity. Binding to E-selectin activated downstream proteins including mammalian target of rapamycin, p70 ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1. Functionally, adhesion to E-selectin induced upregulation of CD86 expression and CCL2 secretion. We next asked whether contact with E-selectin impacts further BMDM stimulation. We found enhanced secretion of both interleukin (IL)-10 and CCL2, but not tumor necrosis factor or IL-6 in response to lipopolysaccharide (LPS) stimulation after adhesion to E-selectin. Importantly, adhesion to E-selectin did not polarize BMDMs to one type of response but enhanced both arginase activity and nitric oxide production following IL-4 or LPS stimulation, respectively. In cultured human monocytes, adhesion to E-selectin similarly induced phosphorylation of AKT. Finally, when E-selectin was blocked in vivo in mice, thioglycollate-elicited macrophages showed reduced CD86 expression, validating our in vitro studies. Our results imply functions for E-selectin beyond homing and suggest that E-selectin plays an early role in priming and amplifying innate immune responses.

Sharma and colleagues identify the kinase DAPK3 as a positive regulator of the STING-interferon-beta activation pathway. DAPK3 acts to modify E3 ubiquitin ligases that regulate STING K63-linked poly-ubiquitination. Evasion of host immunity is a hallmark of cancer; however, mechanisms linking oncogenic mutations and immune escape are incompletely understood. Through loss-of-function screening of 1,001 tumor suppressor genes, we identified death-associated protein kinase 3 (DAPK3) as a previously unrecognized driver of anti-tumor immunity through the stimulator of interferon genes (STING) pathway of cytosolic DNA sensing. Loss of DAPK3 expression or kinase activity impaired STING activation and interferon (IFN)-beta-stimulated gene induction. DAPK3 deficiency in IFN-beta-producing tumors drove rapid growth and reduced infiltration of CD103(+)CD8 alpha(+) dendritic cells and cytotoxic lymphocytes, attenuating the response to cancer chemo-immunotherapy. Mechanistically, DAPK3 coordinated post-translational modification of STING. In unstimulated cells, DAPK3 inhibited STING K48-linked poly-ubiquitination and proteasome-mediated degradation. After cGAMP stimulation, DAPK3 was required for STING K63-linked poly-ubiquitination and STING-TANK-binding kinase 1 interaction. Comprehensive phospho-proteomics uncovered a DAPK3-specific phospho-site on the E3 ligase LMO7, critical for LMO7-STING interaction and STING K63-linked poly-ubiquitination. Thus, DAPK3 is an essential kinase for STING activation that drives tumor-intrinsic innate immunity and tumor immune surveillance.

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.

The resistance of chronic myeloid leukaemia (CML) to tyrosine kinase inhibitors (TKIs) remains a significant clinical problem. Targeting alternative pathways, such as protein prenylation, is known to be effective in overcoming resistance. Simvastatin inhibits 3-hydroxy-3-methylglutaryl-CoA reductase (a key enzyme in isoprenoid-regulation), thereby inhibiting prenylation. We demonstrate that simvastatin alone effectively inhibits proliferation in a panel of TKI-resistant CML cell lines, regardless of mechanism of resistance. We further show that the combination of nilotinib and simvastatin synergistically kills CML cells via an increase in apoptosis and decrease in prosurvival proteins and cellular proliferation. Mechanistically, simvastatin inhibits protein prenylation as shown by increased levels of unprenylated Ras and rescue experiments with mevalonate resulted in abrogation of synergism. The combination also leads to an increase in the intracellular uptake and retention of radio-labelled nilotinib, which further enhances the inhibition of Bcr-Abl kinase activity. In primary CML samples, this combination inhibits clonogenicity in both imatinib-naive and resistant cells. Such combinatorial effects provide the basis for utilising these Food and Drug Administration-approved drugs as a potential clinical approach in overcoming resistance and improving CML treatment.

Protein tyrosine kinase 6 (PTK6; also called Brk) is overexpressed in 86% of patients with breast cancer; high PTK6 expression predicts poor outcome. We reported PTK6 induction by HIF/GR complexes in response to either cellular or host stress. However, PTK6-driven signaling events in the context of triple-negative breast cancer (TNBC) remain undefined. In a mouse model of TNBC, manipulation of PTK6 levels (i.e., via knock-out or add-back) had little effect on primary tumor volume, but altered lung metastasis. To delineate the mechanisms of PTK6 downstream signaling, we created kinase-dead (KM) and kinase-intact domain structure mutants of PTK6 via in-frame deletions of the N-terminal SH3 or SH2 domains. While the PTK6 kinase domain contributed to soft-agar colony formation, PTK6 kinase activity was entirely dispensable for cell migration. Specifically, TNBC models expressing a PTK6 variant lacking the SH2 domain (SH2-del PTK6) were unresponsive to growth factor–stimulated cell motility relative to SH3-del, KM, or wild-type PTK6 controls. Reverse-phase protein array revealed that while intact PTK6 mediates spheroid formation via p38 MAPK signaling, the SH2 domain of PTK6 limits this biology, and instead mediates TNBC cell motility via activation of the RhoA and/or AhR signaling pathways. Inhibition of RhoA and/or AhR blocked TNBC cell migration as well as the branching/invasive morphology of PTK6+/AhR+ primary breast tumor tissue organoids. Inhibition of RhoA also enhanced paclitaxel cytotoxicity in TNBC cells, including in a taxane-refractory TNBC model. Implications: The SH2-domain of PTK6 is a potent effector of advanced cancer phenotypes in TNBC via RhoA and AhR, identified herein as novel therapeutic targets in PTK6+ breast tumors.

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.

Background & Aims Gastric dysfunction in the elderly may cause reduced food intake, frailty, and increased mortality. The pacemaker and neuromodulator cells interstitial cells of Cajal (ICC) decline with age in humans, and their loss contributes to gastric dysfunction in progeric klotho mice hypomorphic for the anti-aging Klotho protein. The mechanisms of ICC depletion remain unclear. Klotho attenuates Wnt (wingless-type MMTV integration site) signaling. Here, we examined whether unopposed Wnt signaling could underlie aging-associated ICC loss by up-regulating transformation related protein TRP53 in ICC stem cells (ICC-SC). Methods Mice aged 1–107 weeks, klotho mice, APCΔ468 mice with overactive Wnt signaling, mouse ICC-SC, and human gastric smooth muscles were studied by RNA sequencing, reverse transcription–polymerase chain reaction, immunoblots, immunofluorescence, histochemistry, flow cytometry, and methyltetrazolium, ethynyl/bromodeoxyuridine incorporation, and ex-vivo gastric compliance assays. Cells were manipulated pharmacologically and by gene overexpression and RNA interference. Results The klotho and aged mice showed similar ICC loss and impaired gastric compliance. ICC-SC decline preceded ICC depletion. Canonical Wnt signaling and TRP53 increased in gastric muscles of klotho and aged mice and middle-aged humans. Overstimulated canonical Wnt signaling increased DNA damage response and TRP53 and reduced ICC-SC self-renewal and gastric ICC. TRP53 induction persistently inhibited G1/S and G2/M cell cycle phase transitions without activating apoptosis, autophagy, cellular quiescence, or canonical markers/mediators of senescence. G1/S block reflected increased cyclin-dependent kinase inhibitor 1B and reduced cyclin D1 from reduced extracellular signal-regulated kinase activity. Conclusions Increased Wnt signaling causes age-related ICC loss by up-regulating TRP53, which induces persistent ICC-SC cell cycle arrest without up-regulating canonical senescence markers., Graphical abstract

Cervical cancer is a common gynecologic cancer and a frequent cause of death. In this study, we investigated the role of MELK (maternal embryonic leucine zipper kinase) in cervical cancer. We found that HPV 18 E6/E7 promoted MELK expression by activating E2F1. MELK knockdown blocked cancer cells growth. Furthermore, we used MELK-8A to inhibit the kinase activity of MELK and caused the G2/M phase arrest of cancer cells. Under the treatment of inhibitors, Hela cells formed multipolar spindles and eventually underwent apoptosis. We also found that MELK is involved in protein translation and folding during cell division through the MELK interactome and the temporal proteomic analysis under inhibition with MELK-8A. Altogether, these results suggest that MELK may play a vital role in cancer cell proliferation and indicate a potential therapeutic target for cervical cancer.

Double-stranded RNA dependent kinase R (PKR) is originally identified as an intracellular sensor of viral infection, but its role in bacterial infection remains largely unknown. Here we report that PKR was an important regulator of antibacterial immunity in sepsis. Genetic deletion of PKR or pharmacological inhibition of its kinase activity markedly increased bacterial loads, organ injury, and mortality in polymicrobial infection induced by cecal ligation and puncture (CLP). In contrast, PKR deficiency or inhibition did not affect bacterial loads, organ injury, or mortality when mice were systemically challenged with Escherichia coli, an abundant microbe in the gastrointestinal tract. PKR deficiency or inhibition markedly decreased the release of interleukin (IL)-1β after CLP. Defect in IL-1 signaling phenocopied PKR deficiency or inhibition in CLP-induced bacterial sepsis. Taken together, these findings identified a critical role of the PKR signaling pathway in antibacterial immunity.