Kinase-dependent Mechanism Research Articles

Intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase-dependent mechanism.

The Parkinson's disease (PD)-linked protein Leucine-Rich Repeat Kinase 2 (LRRK2) consists of seven domains, including a kinase and a Roc G domain. Despite the availability of several high-resolution structures, the dynamic regulation of its unique intramolecular domain stack is nevertheless still not well understood. By in-depth biochemical analysis, assessing the Michaelis-Menten kinetics of the Roc G domain, we have confirmed that LRRK2 has, similar to other Roco protein family members, a KM value of LRRK2 that lies within the range of the physiological GTP concentrations within the cell. Furthermore, the R1441G PD variant located within a mutational hotspot in the Roc domain showed an increased catalytic efficiency. In contrast, the most common PD variant G2019S, located in the kinase domain, showed an increased KM and reduced catalytic efficiency, suggesting a negative feedback mechanism from the kinase domain to the G domain. Autophosphorylation of the G1+2 residue (T1343) in the Roc P-loop motif is critical for this phosphoregulation of both the KM and the kcat values of the Roc-catalyzed GTP hydrolysis, most likely by changing the monomer-dimer equilibrium. The LRRK2 T1343A variant has a similar increased kinase activity in cells compared to G2019S and the double mutant T1343A/G2019S has no further increased activity, suggesting that T1343 is crucial for the negative feedback in the LRRK2 signaling cascade. Together, our data reveal a novel intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase-dependent mechanism. Interestingly, PD mutants differently change the kinetics of the GTPase cycle, which might in part explain the difference in penetrance of these mutations in PD patients.

Anti-EGFR aptamer exhibits direct anti-cancer effects in NSCLC cells harboring EGFR L858R mutations

Non-small cell lung cancer (NSCLC) adenocarcinoma (LUAD) is a leading cause of death worldwide. Activating mutations in the tyrosine kinase domain of the oncogene epidermal growth factor receptor (EGFR) are responsible for ~10–50% of all LUAD cases. Although tyrosine kinase inhibitors (TKIs) have been effective in prolonging patient survival and quality of life, acquired resistance and disease progression are inevitable, presenting a clear unmet need for alternative or adjuvant therapeutics. Here we show that an anti-EGFR aptamer (EGFRapt) decreases viability and tumor growth of LUAD cell lines harboring the L858R ± T790M mutation in EGFR. Additionally, we elucidate the mechanism by which EGFRapt exerts these effects by monitoring cellular processes associated with kinase-dependent and kinase-independent mechanisms. Overall, these data establish that EGFRapt has direct anti-cancer activity in mutant EGFR positive LUAD via targetable mechanisms that are independent of existing approaches, and they provide a foundation for further development of nucleic acid-based therapies that target EGFR.

Neutrophil-specific Shp1 loss results in lethal pulmonary hemorrhage in mouse models of acute lung injury.

The acute respiratory distress syndrome (ARDS) is associated with significant morbidity and mortality and neutrophils are critical to its pathogenesis. Neutrophil activation is closely regulated by inhibitory tyrosine phosphatases including Src homology region 2 domain containing phosphatase-1 (Shp1). Here, we report that loss of neutrophil Shp1 in mice produced hyperinflammation and lethal pulmonary hemorrhage in sterile inflammation and pathogen-induced models of acute lung injury (ALI) through a Syk kinase-dependent mechanism. We observed large intravascular neutrophil clusters, perivascular inflammation, and excessive neutrophil extracellular traps in neutrophil-specific Shp1 knockout mice suggesting an underlying mechanism for the observed pulmonary hemorrhage. Targeted immunomodulation through the administration of a Shp1 activator (SC43) reduced agonist-induced reactive oxygen species in vitro and ameliorated ALI-induced alveolar neutrophilia and NETs in vivo. We propose that the pharmacologic activation of Shp1 has the potential to fine-tune neutrophil hyperinflammation that is central to the pathogenesis of ARDS.

Non-Receptor Tyrosine Kinases: Their Structure and Mechanistic Role in Tumor Progression and Resistance.

Protein tyrosine kinases (PTKs) function as key molecules in the signaling pathways in addition to their impact as a therapeutic target for the treatment of many human diseases, including cancer. PTKs are characterized by their ability to phosphorylate serine, threonine, or tyrosine residues and can thereby rapidly and reversibly alter the function of their protein substrates in the form of significant changes in protein confirmation and affinity for their interaction with protein partners to drive cellular functions under normal and pathological conditions. PTKs are classified into two groups: one of which represents tyrosine kinases, while the other one includes the members of the serine/threonine kinases. The group of tyrosine kinases is subdivided into subgroups: one of them includes the member of receptor tyrosine kinases (RTKs), while the other subgroup includes the member of non-receptor tyrosine kinases (NRTKs). Both these kinase groups function as an "on" or "off" switch in many cellular functions. NRTKs are enzymes which are overexpressed and activated in many cancer types and regulate variable cellular functions in response to extracellular signaling-dependent mechanisms. NRTK-mediated different cellular functions are regulated by kinase-dependent and kinase-independent mechanisms either in the cytoplasm or in the nucleus. Thus, targeting NRTKs is of great interest to improve the treatment strategy of different tumor types. This review deals with the structure and mechanistic role of NRTKs in tumor progression and resistance and their importance as therapeutic targets in tumor therapy.

Loss of neutrophil Shp1 produces hemorrhagic and lethal acute lung injury.

The acute respiratory distress syndrome (ARDS) is associated with significant morbidity and mortality and neutrophils are critical to its pathogenesis. Neutrophil activation is closely regulated by inhibitory tyrosine phosphatases including Src homology region 2 domain containing phosphatase-1 (Shp1). Here, we report that loss of neutrophil Shp1 in mice produced hyperinflammation and lethal pulmonary hemorrhage in sterile inflammation and pathogen-induced models of acute lung injury (ALI) through a Syk kinase-dependent mechanism. We observed large intravascular neutrophil clusters, perivascular inflammation, and excessive neutrophil extracellular traps in neutrophil-specific Shp1 knockout mice suggesting an underlying mechanism for the observed pulmonary hemorrhage. Targeted immunomodulation through the administration of a Shp1 activator (SC43) reduced agonist-induced reactive oxygen species in vitro and ameliorated ALI-induced alveolar neutrophilia and NETs in vivo. We propose that the pharmacologic activation of Shp1 has the potential to fine-tune neutrophil hyperinflammation that is central to the pathogenesis of ARDS.

Abstract P2050: Mixed Lineage Kinase 3 Kinase Function Opposes Left Ventricular Hypertrophy And Transcriptional Activity Of Nuclear Factor Of Activated T Cells In The Cardiac Myocyte.

Background: LV hypertrophy (LVH), dysfunction, and remodeling contribute to heart failure (HF). The signaling molecule Mixed Lineage Kinase 3 (MLK3) opposes stress-induced LV dysfunction and remodeling in a kinase-dependent, blood pressure-independent fashion. However, the required downstream MLK3 kinase effectors in the cardiac myocyte (CM) which oppose LVH and remodeling remain poorly understood. The Nuclear Factor of Activated T Cells (NFAT) promotes pathological remodeling, but its regulation in the CM by MLK3 has not been investigated. We hypothesized that MLK3 opposes NFAT in the CM through kinase-dependent mechanisms. Method: In reporter mice with a CM-specific luciferase transgene under the control of NFAT response elements (αMHC-NFAT-Luc mice), we administered the MLK3 inhibitor URMC-099 (10 mg/kg twice daily) or vehicle for 5 days (n=7 vehicle, 8 URMC-099) and 14 days (n=9 vehicle, 12 URMC-099). We measured cardiac chamber masses, performed immunoblotting for MLK3, and measured myocardial luciferase fluorescence as a readout for CM NFAT transcriptional activity. Result: At 5 days, URMC-099 had no effect on LVH or cardiac hypertrophy and did not induce NFAT activation. However, URMC-099 treatment reduced LV MLK3 protein expression (normalized to β-actin) compared with vehicle-treated littermates. After 14-day administration, URMC-099 induced significant increases in LV and heart mass, normalized to tibia length (TL), (LV/TL males: 4.21 ± 0.03 mg/mm vehicle vs 5.20 ± 0.10 mg/mm URMC-099; p<0.01; females: 3.73 ± 0.14 mg/mm vehicle vs 4.30 ± 0.13 mg/mm URMC-099; p<0.01). LV tissue from URMC-099-treated mice displayed a 7-fold increase in luciferase fluorescence (p<0.001), compared with vehicle-treated, indicating increased NFAT activation by URMC-099. Conclusion: Systemic MLK3 inhibition with URMC-099 causes reduced MLK3 expression, LVH, and increased CM NFAT activity. Because URMC-099 does not raise blood pressure, our findings support that MLK3 functions as a tonic brake on NFAT activation in the CM and opposes cardiac hypertrophy through kinase-dependent mechanisms.

A glucose-sensing mechanism with glucose transporter 1 and pyruvate kinase in the area postrema regulates hepatic glucose production in rats

The area postrema (AP) of the brain is exposed to circulating metabolites and hormones. However, whether AP detects glucose changes to exert biological responses remains unknown. Its neighboring nuclei, the nucleus tractus solitarius (NTS), responds to acute glucose infusion by inhibiting hepatic glucose production, but the mechanism also remains elusive. Herein, we characterized AP and NTS glucose-sensing mechanisms. Infusion of glucose into the AP, like the NTS, of chow rats suppressed glucose production during the pancreatic (basal insulin)-euglycemic clamps. Glucose transporter 1 or pyruvate kinase lentiviral-mediated knockdown in the AP negated AP glucose infusion to lower glucose production, while the glucoregulatory effect of NTS glucose infusion was also negated by knocking down glucose transporter 1 or pyruvate kinase in the NTS. Furthermore, we determined that high-fat (HF) feeding disrupts glucose infusion to lower glucose production in association with a modest reduction in the expression of glucose transporter 1, but not pyruvate kinase, in the AP and NTS. However, pyruvate dehydrogenase activator dichloroacetate infusion into the AP or NTS that enhanced downstream pyruvate metabolism and recapitulated the glucoregulatory effect of glucose in chow rats still failed to lower glucose production in HF rats. We discovered that a glucose transporter 1- and pyruvate kinase-dependent glucose-sensing mechanism in the AP (as well as the NTS) lowers glucose production in chow rats and that HF disrupts the glucose-sensing mechanism that is downstream of pyruvate metabolism in the AP and NTS. These findings highlight the role of AP and NTS in mediating glucose to regulate hepatic glucose production.

Autophagy Reduces the Degradation and Promotes Membrane Localization of Occludin to Enhance the Intestinal Epithelial Tight Junction Barrier against Paracellular Macromolecule Flux.

Functional loss of the gut epithelium's paracellular tight junction [TJ] barrier and defective autophagy are factors potentiating inflammatory bowel disease [IBD]. Previously, we showed the role of autophagy in enhancing the intestinal TJ barrier via pore-forming claudin-2 degradation. How autophagy regulates the TJ barrier-forming proteins remains unknown. Here, we investigated the role of autophagy in the regulation of occludin, a principal TJ component involved in TJ barrier enhancement. Autophagy induction using pharmacological activators and nutrient starvation increased total occludin levels in intestinal epithelial cells, mouse colonocytes and human colonoids. Autophagy induction enriched membrane occludin levels and reduced paracellular permeability of macromolecules. Autophagy-mediated TJ barrier enhancement was contingent on the presence of occludin as OCLN-/- nullified its TJ barrier-enhancing effect against macromolecular flux. Autophagy inhibited the constitutive degradation of occludin by preventing its caveolar endocytosis from the membrane and protected against inflammation-induced TJ barrier loss. Autophagy enhanced the phosphorylation of ERK-1/2 and inhibition of these kinases in Caco-2 cells and human colonic mucosa prevented the macromolecular barrier-enhancing effects of autophagy. In vivo, autophagy induction by rapamycin enhanced occludin levels in wild-type mouse intestines and protected against lipopolysaccharide- and tumour necrosis factor-α-induced TJ barrier loss. Disruption of autophagy with acute Atg7 knockout in adult mice decreased intestinal occludin levels, increasing baseline colonic TJ permeability and exacerbating the effect of experimental colitis. Our data suggest a novel role of autophagy in promoting the intestinal TJ barrier by increasing occludin levels in an ERK1/2 mitogen-activated protein kinase-dependent mechanism.

Impact of ultra-small silver nanoparticles of 2 nm (AgNP2) on neutrophil biology: AgNP2 alter the actin cytoskeleton and induce karyorrhexis by a mitogen-activated protein kinase-dependent mechanism in vitro and transitorily attract neutrophils in vivo

Silver (Ag) is known as an antibacterial agent and there is a growing interest to use silver nanoparticles (AgNPs) in a variety of medical applications and other sectors. Some studies reported that one of the undesired effects of AgNPs is inflammation and that these NPs can alter the biology of neutrophils. Since it is commonly accepted that the more NPs are small, the more toxic they are the aim of this study was to determine the impact of ultra-small silver nanoparticles of 2 nm (AgNP2) on the biology of neutrophils, key player cells in inflammation. We report that AgNP2 are potent neutrophil activators as they rapidly induce actin polymerization and dismantling the actin network. Although AgNP2 are not necrotic for neutrophils and do not induce ROS production, kinetic studies reveal that AgNP2 are rapid inducer of apoptosis. Pyknosis (mainly 1–2 large nuclear dots) was observed after only 1h of treatment followed by karyorrhexis (several small dots) and by a complete nuclear dissolution leading to anuclear neutrophils after 6h. These observations are not associated with the release of silver ions since treatment of neutrophils with 1–50 μg/ml AgNO3 (as a source of Ag+) did not induce any apparent changes. AgNP2 induce p38 and Erk-1/2 mitogen-activated protein kinase (MAPK) and although karyorrhexis was markedly reversed by MAPK inhibitors, the cell nuclei remain with a pyknotic-like phenotype but do not return to the characteristic polylobed nucleus. Using the murine air pouch model of inflammation AgNP2 were found to induce a neutrophil influx. Our data indicate that AgNP2 are potent neutrophil activators targeting the actin cytoskeleton and the mechanism involved for inducing apoptosis is rapid, complex, and partially includes MAPK pathways. Therefore, the ultra-small AgNP2 are more potent than larger ones for inducing apoptosis and they can transitorily attract neutrophils in vivo.

The integrin ligand SVEP1 regulates GPCR-mediated vasoconstriction via integrins α9β1 and α4β1.

Vascular tone is regulated by the relative contractile state of vascular smooth muscle cells (VSMCs). Several integrins directly modulate VSMC contraction by regulating calcium influx through L-type voltage-gated Ca2+ channels (VGCCs). Genetic variants in ITGA9, which encodes the α9 subunit of integrin α9β1, and SVEP1, a ligand for integrin α9β1, associate with elevated blood pressure; however, neither SVEP1 nor integrin α9β1 has reported roles in vasoregulation. We determined whether SVEP1 and integrin α9β1 can regulate VSMC contraction. SVEP1 and integrin binding were confirmed by immunoprecipitation and cell binding assays. Human induced pluripotent stem cell-derived VSMCs were used in in vitro [Ca2+ ]i studies, and aortas from a Svep1+/- knockout mouse model were used in wire myography to measure vessel contraction. We confirmed the ligation of SVEP1 to integrin α9β1 and additionally found SVEP1 to directly bind to integrin α4β1. Inhibition of SVEP1, integrin α4β1 or α9β1 significantly enhanced [Ca2+ ]i levels in isolated VSMCs to Gαq/11 -vasoconstrictors. This response was confirmed in whole vessels where a greater contraction to U46619 was seen in vessels from Svep1+/- mice compared to littermate controls or when integrin α4β1 or α9β1 was inhibited. Inhibition studies suggested that this effect was mediated via VGCCs, PKC and Rho A/Rho kinase dependent mechanisms. Our studies reveal a novel role for SVEP1 and the integrins α4β1 and α9β1 in reducing VSMC contractility. This could provide an explanation for the genetic associations with blood pressure risk at the SVEP1 and ITGA9 loci.

A40 IDENTIFYING NOVEL ROLES FOR TLR2 SIGNALING IN THE INTESTINAL EPITHELIUM USING ORGANOIDS

Abstract Background Inflammatory bowel disease (IBD) is thought to result from an imbalance between protective and damaging immune responses to the resident microbiota and other luminal antigens. Increasing evidence suggests that toll-like-receptor (TLR)-mediated innate immune dysfunction contributes to the pathogenesis of IBD. Among the receptors in the TLR family, TLR2 and its signalling pathway appears to play an important protective role in the gastrointestinal (GI) tract. Notably, our group previously discovered that Tlr2 expression by nonhematopoietic cells played an important protective role in the Citrobacter rodentium model of infectious colitis. Aims To determine if intestinal epithelial cells (IECs) control Tlr2 dependent tissue protective responses, we sought to characterize the role of Tlr2 signalling in intestinal organoids. In addition, to better define the role of Tlr2 during C. rodentium infection, we developed a novel in vitro model of C. rodentium infection using organoid-derived monolayers. Methods Organoids were derived from the colonic tissue of wild type (C57BL/6J) and Tlr2 deficient (-/-) mice and then stimulated with Tlr2 agonists, and their responses were evaluated by qPCR, ELISA, Western blot and immunostaining. In addition, 2D monolayers were grown from organoids, and infected with C. rodentium to explore whether Tlr2 signaling regulates other protective IEC functions such as barrier proteins and cell death in response to noxious stimuli. Results Stimulation of WT, but not Tlr2-/- mouse orgnaoids led to increased transcription of chemokine and cytokine genes Ccl20, Mcp-1, Cxcl1 and Tnf-α. This was accompanied by increased protein secretion through a NF-κB and p38 MAP kinase dependent mechanism. Interestingly, organoids derived from the distal colon displayed stronger Tlr2 responses than organoids from the proximal colon. During C. rodentium infection of monolayers, Tlr2 signaling had no effect on the distribution of tight junction proteins such as ZO-1 or Claudin3, however, Tlr2 did regulate levels of IEC apoptosis upon C. rodentium infection. Conclusions Our study demonstrates that colonic organoids express functional Tlr2, which upon stimulation, leads to pro-inflammatory responses as well as control over cell death. Our novel C. rodentium infection model enables us to further study the role of IEC in promoting host defense during bacterial infection. Futher work will examine how interactions with Tlr2 dependent cytokines (ie. IL-6, IL-22) impact IEC responses to C. rodentium. Funding Agencies CCC, CIHRCH.I.L.D

CD44: a potential therapeutic target in chronic myeloid leukemia.

Development of tyrosine kinase inhibitors (TKIs) achieved great success in the treatment of chronic phase chronic myeloid leukemia (CML). However, patients with CML still relapse without taking TKIs and cases in the accelerated phase or aggressive blast crisis rarely achieved deep response to TKIs. Drug resistance and persistence of leukemia stem cell (LSC) remain great challenges. BCR-ABL kinase dependent or independent mechanism of action are still far from being understood. To achieve a stable deep molecular response and treatment-free remission, finding new targets, eliminating LSC, reducing recurrence and improving prognosis are problems urgently to be solved. It is revealed that tumor microenvironment is crucial for survival, invasion and metastasis of tumor cells. As an adhesion molecule, CD44, a single-chain transmembrane glycoprotein, is not only being identified as a marker for cancer stem cells, but also plays a crucial role in microenvironmental communication and transmitting intracellular signaling for cell proliferation, differentiation, migration, and contributes to tumorigenesis. In this review, we focus on current data relevant to CD44, and outline CD44 structure, the regulation of CD44, functional properties of CD44 in survival, resistance, CML stem cells as well as the potential CD44-targeting therapy for CML management.

Oxidative Stress Induces a Breakdown of the Cytoskeleton and Tight Junctions of the Corneal Endothelial Cells.

Purpose: To investigate the impact of oxidative stress, which is a hallmark of Fuchs dystrophy, on the barrier function of the corneal endothelial cells. Methods: Experiments were carried out with cultured bovine and porcine corneal endothelial cells. For oxidative stress, cells were supplemented with riboflavin (Rf) and exposed to UV-A (15-30 min) to induce Type-1 photochemical reactions that release H2O2. The effect of the stress on the barrier function was assayed by transendothelial electrical resistance (TER) measurement. In addition, the associated changes in the organization of the microtubules, perijunctional actomyosin ring (PAMR), and ZO-1 were evaluated by immunocytochemistry, which was also repeated after direct exposure to H2O2 (100 μM, 1 h). Results: Exposure to H2O2 led to the disassembly of microtubules and the destruction of PAMR. In parallel, the contiguous locus of ZO-1 was disrupted, marking a loss of barrier integrity. Accordingly, a sustained loss in TER was induced when cells in the Rf-supplemented medium were exposed to UV-A. However, the addition of catalase (7,000 U/mL) to rapidly decompose H2O2 limited the loss in TER. Furthermore, the adverse effects on microtubules, PAMR, and ZO-1 were suppressed by including catalase, ascorbic acid (1 mM; 30 min), or pretreatment with p38 MAP kinase inhibitor (SB-203580; 10 μM, 1 h). Conclusions: Acute oxidative stress induces microtubule disassembly by a p38 MAP kinase-dependent mechanism, leading to the destruction of PAMR and loss of barrier function. The response to oxidative stress is reminiscent of the (TNF-α)-induced breakdown of barrier failure in the corneal endothelium.

A-769662 stimulates the differentiation of bone marrow-derived mesenchymal stem cells into osteoblasts via AMP-activated protein kinase-dependent mechanism.

AMP-activated protein kinase (AMPK) signaling shows an important role in energy metabolism and has recently been involved in osteogenic and adipogenic differentiation. In this study we aimed to investigate the role of AMPK activator, A-769662, in regulating the differentiation of mesenchymal stem cells derived from bone marrow (BMSCs) into osteoblastic and adipocytic cell lineage. The effect of A-769662 on osteogenesis was assessed by quantitative alkaline phosphatase (ALP) activity, matrix mineralization stained with Alizarin red, and gene expression analysis by quantitative polymerase chain reaction (qPCR). Adipogenesis was determined by Oil Red O staining for fat droplets and qPCR analysis of adipogenic markers. A-769662 activated the phosphorylation of AMPKα1 during the osteogenesis of mBMSCs as revealed by western blot analysis. A-769662 promoted the early stage of the commitment of mouse (m) BMSCs differentiation into osteoblasts, while inhibiting their differentiation into adipocytes in a dose-dependent manner. The effects of A-769662 on stimulating osteogenesis and inhibiting adipogenesis of mBMSCs were significantly eliminated in the presence of either AMPKα1 siRNA or Compound C, an inhibitor of AMPK pathway. In conclusion, we identified A-769662 as a new compound that promotes the commitment of BMSCs into osteoblasts versus adipocytes via AMPK-dependent mechanism. Thus our data show A-769662 as a potential osteo-anabolic drug for treatment of osteoporosis.

DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma

DYRK1A, one of the dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs), plays an important role in various biological processes by regulating downstream targets via kinase-dependent and independent mechanisms. Here, we report a novel role of DYRK1A in maintaining tumor growth and stemness of oral/oropharyngeal squamous cell carcinoma (OSCC) cells. Deletion of DYRK1A from OSCC cells abrogated their in vivo tumorigenicity and self-renewal capacity, the key features of cancer stem-like cells (CSCs; also referred to as tumor-initiating cells). The DYRK1A deletion also induced the suppression of CSC populations and properties, such as migration ability and chemoresistance. Conversely, ectopic expression of DYRK1A in OSCC cells augmented their CSC phenotype. Among five DYRK members (DYRK1A, 1B, 2, 3, and 4), DYRK1A is the most dominantly expressed kinase, and its expression is upregulated in OSCC compared to normal oral epithelial cells. More importantly, DYRK1A was highly enriched in various CSC-enriched OSCC populations compared to their corresponding non-CSC populations, indicating its pivotal role in cancer progression and stemness. Further, our study revealed that fibroblast growth factor 2 (FGF2) is a key regulator in the DYRK1A-mediated CSC regulation. Functional studies demonstrated that the loss of DYRK1A inhibits CSC phenotype via reduction of FGF2. Overexpression of DYRK1A promotes CSC phenotype via upregulation of FGF2. Our study delineates a novel mechanism of cancer stemness regulation by DYRK1A-FGF2 axis in OSCC. Thus, inhibition of DYRK1A would lead to a potential novel therapeutic option for targeting CSCs in OSCC.

Punching Holes in Cellular Membranes: Biology and Evolution of Gasdermins.

The gasdermin (GSDM) family has evolved as six gene clusters (GSDMA-E and Pejvakin, PJVK), and GSDM proteins are characterized by a unique N-terminal domain (N-GSDM). With the exception of PJVK, the N-GSDM domain is capable of executing plasma membrane permeabilization. Depending on the cell death modality, several protease- and kinase-dependent mechanisms directly regulate the activity of GSDME and GSDMD, the two most widely expressed and best-studied GSDMs. We provide an overview of all GSDMs in terms of biological function, tissue expression, activation, regulation, and structure. In-depth phylogenetic analysis reveals that GSDM genes show many gene duplications and deletions, suggesting that strong evolutionary forces and a unique position of the PJVK gene are associated with the occurrence of complex inner-ear development in vertebrates.

Regulation of OATP1B1 Function by Tyrosine Kinase-mediated Phosphorylation.

OATP1B1 (SLCO1B1) is the most abundant and pharmacologically relevant uptake transporter in the liver and a key mediator of xenobiotic clearance. However, the regulatory mechanisms that determine OATP1B1 activity remain uncertain, and as a result, unexpected drug-drug interactions involving OATP1B1 substrates continue to be reported, including several involving tyrosine kinase inhibitors (TKI). OATP1B1-mediated activity in overexpressing HEK293 cells and hepatocytes was assessed in the presence of FDA-approved TKIs, while rosuvastatin pharmacokinetics in the presence of an OATP1B1 inhibiting TKI were measured in vivo. Tyrosine phosphorylation of OATP1B1 was determined by LC/MS-MS-based proteomics and transport function was measured following exposure to siRNAs targeting 779 different kinases. Twenty-nine of 46 FDA-approved TKIs studied significantly inhibit OATP1B1 function. Inhibition of OATP1B1 by TKIs, such as nilotinib, is predominantly noncompetitive, can increase systemic concentrations of rosuvastatin in vivo, and is associated with reduced phosphorylation of OATP1B1 at tyrosine residue 645. Using genetic screens and functional validation studies, the Src kinase LYN was identified as a potential regulator of OATP1B1 activity that is highly sensitive to inhibition by various TKIs at clinically relevant concentrations. A novel kinase-dependent posttranslational mechanism of OATP1B1 activation was identified and interference with this process by TKIs can influence the elimination of a broad range of xenobiotic substrates.

Evidence for a Novel Key Regulatory Step in the Triglyceride Synthetic Pathway of Human Adipose Tissue

The triglyceride (TG) synthetic pathway is believed to be regulated at the level of the rate-limiting enzyme acyl-CoA:1,2-diacylglycerol O-acyltransferase (DGAT). Recent reports using rat hepatic tissue suggest a kinase-dependent mechanism for the regulation of DGAT activity. To examine this process further, the present study investigates the regulatory mechanisms involved in the modulation of DGAT in human adipocytes. Adipocytes were fractionated into a microsomal fraction containing DGAT and a cytosolic fraction containing a putative regulatory kinase. DGAT activity was determined bymeasuring the incorporation of 14C-oleoyl-CoA into TG with exogenously supplied 1,2-dioleoyl-sn-glycerol. Kinase activity was assayed by addition of the cytosolic fraction in the presence of Mg2+ and ATP. The results indicate a significant inhibition of human adipose tissue DGAT activity by as much as 43% (avg: 17.5% ± 10.4%, p < 0.01) via a mechanism consistent with a phosphorylation event. Partial purification of the putative cytosolic kinase was achieved by multidimensional chromatography. This study thus provides evidence for a novel and key regulatory step in the human TG biosynthetic pathway. Further research is necessary to determine whether the model outlined here is a physiologic conduit through which extracellular hormones exert a regulatory influence on TG synthesis.

CDK4/6 Dependence of Cyclin D1-Driven Parathyroid Neoplasia in Transgenic Mice.

The protein product of the cyclin D1 oncogene functions by activating partner cyclin-dependent kinases (cdk)4 or cdk6 to phosphorylate, thereby inactivating, the retinoblastoma protein pRB. Nonclassical, cdk-independent, functions of cyclin D1 have been described but their role in cyclin D1-driven neoplasia, with attendant implications for recently approved cdk4/6 chemotherapeutic inhibitors, requires further examination. We investigated whether cyclin D1's role in parathyroid tumorigenesis in vivo is effected primarily through kinase-dependent or kinase-independent mechanisms. Using a mouse model of cyclin D1-driven parathyroid tumorigenesis (PTH-D1), we generated new transgenic lines harboring a mutant cyclin D1 (KE) that is unable to activate its partner kinases. While this kinase-dead KE mutant effectively drove mammary tumorigenesis in an analogous model, parathyroid-overexpressed cyclin D1 KE mice did not develop the characteristic biochemical hyperparathyroidism or parathyroid hypercellularity of PTH-D1 mice. These results strongly suggest that in parathyroid cells, cyclin D1 drives tumorigenesis predominantly through cdk-dependent mechanisms, in marked contrast with the cdk-independence of cyclin D1-driven mouse mammary cancer. These findings highlight crucial tissue-specific mechanistic differences in cyclin D1-driven tumorigenesis, suggest that parathyroid/endocrine cells may be more tumorigenically vulnerable to acquired genetic perturbations in cdk-mediated proliferative control than other tissues, and carry important considerations for therapeutic intervention.

Of Mosaicism and Mechanisms: How JAK1 Goes Awry

Personalized medicines require understanding the molecular causes of disease. In this issue of Immunity, Gruber etal. reveal that a gain-of-function JAK1 genetic variant results in a mutant protein with mosaic expression that drives multi-organ immune dysregulation via kinase dependent and independent mechanisms. The work highlights how biochemistry can inform therapies to resolve complex immune disorders.