Tyrosine Kinase Fyn Research Articles

Pyk2 is a Novel Tau Tyrosine Kinase that is Regulated by the Tyrosine Kinase Fyn

The protein tyrosine kinase Pyk2 is encoded by PTK2B, a novel Alzheimer’s disease (AD) susceptibility variant, with the PTK2B risk allele being associated with increased mRNA levels, suggestive of increased Pyk2 levels. However, the role of Pyk2, a member of the focal adhesion kinase (FAK) family, in AD pathology and its regulation are largely unknown. To address this, we generated mice with neuronal expression of human Pyk2. Because we had previously reported an association of Pyk2 and hyperphosphorylated tau (a hallmark feature of AD) in human tau transgenic pR5 mice, we also generated Pyk2/tau double-transgenic mice, which exhibit increased tyrosine phosphorylation and accumulation of tau. We further demonstrated that Pyk2 colocalizes, interacts with, and phosphorylates tau in vivo and in vitro. Importantly, although Pyk2 interacts with the established tyrosine-directed tau kinase Fyn, we identified an increased Pyk2 activity in mice which constitutively overexpress Fyn (FynCA), and a decreased activity in mice lacking Fyn (FynKO). Together, our study reveals a novel role for Pyk2 as a direct tyrosine kinase of tau that is active downstream of Fyn. Our analysis may enhance the understanding of how the PTK2B risk allele contributes to tauopathy.

Identification of phosphorylation sites and binding pockets for modulation of NaV 1.5 channel by Fyn tyrosine kinase.

Cardiac sodium channel NaV 1.5 is the predominant form of sodium channels in cardiomyocytes, which exists as a macromolecular complex and interacts with multiple protein partners. Fyn kinase is one of the interacting proteins which colocalize, phosphorylate and modulate the NaV 1.5 channel. To elaborate this interaction we created expression vectors for the N-terminal, intracellular loop, and C-terminal regions of the NaV 1.5 channel, to express in HEK-293 cells. By co-immunoprecipitation and anti-phosphotyrosine blotting, we identified proline-rich binding sites for Fyn kinase in the N-terminal, IC-loopi-ii and C-terminal. After binding, Fyn kinase phosphorylates tyrosine residues present in the N- and C-terminal, which produce a depolarizing shift of 7 mV in fast inactivation. The functional relevance of these binding and phosphorylation sites was further underpinned by creating full length mutants masking these sites sequentially. An activation and inactivation curves were recorded with or without co-expressed Fyn kinase which indicates that phosphorylation of tyrosine residues at positions 68, 87, 112 in the N-terminal and at positions 1811 and 1889 in the C-terminal creates a depolarizing shift in fast inactivation of NaV 1.5 channel.

Integration of Distinct ShcA Signaling Complexes Promotes Breast Tumor Growth and Tyrosine Kinase Inhibitor Resistance.

The commonality between most phospho-tyrosine signaling networks is their shared use of adaptor proteins to transduce mitogenic signals. ShcA (SHC1) is one such adaptor protein that employs two phospho-tyrosine binding domains (PTB and SH2) and key phospho-tyrosine residues to promote mammary tumorigenesis. Receptor tyrosine kinases (RTK), such as ErbB2, bind the ShcA PTB domain to promote breast tumorigenesis by engaging Grb2 downstream of the ShcA tyrosine phosphorylation sites to activate AKT/mTOR signaling. However, breast tumors also rely on the ShcA PTB domain to bind numerous negative regulators that limit activation of secondary mitogenic signaling networks. This study examines the role of PTB-independent ShcA pools in controlling breast tumor growth and resistance to tyrosine kinase inhibitors. We demonstrate that PTB-independent ShcA complexes predominately rely on the ShcA SH2 domain to activate multiple Src family kinases (SFK), including Src and Fyn, in ErbB2-positive breast cancers. Using genetic and pharmacologic approaches, we show that PTB-independent ShcA complexes augment mammary tumorigenesis by increasing the activity of the Src and Fyn tyrosine kinases in an SH2-dependent manner. This bifurcation of signaling complexes from distinct ShcA pools transduces non-redundant signals that integrate the AKT/mTOR and SFK pathways to cooperatively increase breast tumor growth and resistance to tyrosine kinase inhibitors, including lapatinib and PP2. This study mechanistically dissects how the interplay between diverse intracellular ShcA complexes impacts the tyrosine kinome to affect breast tumorigenesis.Implications: The ShcA adaptor, within distinct signaling complexes, impacts tyrosine kinase signaling, breast tumor growth, and resistance to tyrosine kinase inhibitors. Mol Cancer Res; 16(5); 894-908. ©2018 AACR.

The phospholipase A2 pathway controls a synaptic cholesterol ester cycle and synapse damage.

The cellular prion protein (PrPC) acts as a scaffold protein that organises signalling complexes. In synaptosomes, the aggregation of PrPC by amyloid-β (Aβ) oligomers attracts and activates cytoplasmic phospholipase A2 (cPLA2), leading to synapse degeneration. The signalling platform is dependent on cholesterol released from cholesterol esters by cholesterol ester hydrolases (CEHs). The activation of cPLA2 requires cholesterol released from cholesterol esters by cholesterol ester hydrolases (CEHs), enzymes dependent upon platelet activating factor (PAF) released by activated cPLA2 This demonstrates a positive feedback system in which activated cPLA2 increased cholesterol concentrations, which in turn facilitated cPLA2 activation. PAF was also required for the incorporation of the tyrosine kinase Fyn and cyclooxygenase (COX)-2 into Aβ-PrPC-cPLA2 complexes. As a failure to deactivate signalling complexes can lead to pathology, the mechanisms involved in their dispersal were studied. PAF facilitated the incorporation of acyl-coenzyme A:cholesterol acyltransferase (ACAT)-1 into Aβ-PrPC-cPLA2-COX-2-Fyn complexes. The esterification of cholesterol reduced cholesterol concentrations, causing dispersal of Aβ-PrPC-cPLA2-COX-2-Fyn complexes and the cessation of signalling. This study identifies PAF as a key mediator regulating the cholesterol ester cycle, activation of cPLA2 and COX-2 within synapses, and synapse damage.

Tyrosine kinase Fyn promotes osteoarthritis by activating the β-catenin pathway

ObjectivesTo investigate the role of tyrosine kinase Fyn in the development of osteoarthritis (OA) and the underlying mechanisms, and to define whether targeting Fyn could prevent OA in mice.MethodsCartilage samples...

Decrease of cocaine, but not heroin, self-administration and relapse by the tyrosine kinase inhibitor masitinib in male Sprague Dawley rats

RationaleAccumulating evidence shows that cocaine, and also heroin, influence several tyrosine kinases, expressed in neurons and in non-neuronal populations such as microglia, astrocytes and mast-cells. Drug-induced activation of mast cells both triggers inflammatory processes in the brain mediated by the glial cells they activate, and facilitates histamine release which may directly influence the dopamine system. Thus, by triggering the activation and degranulation of mast cells dependent on the tyrosine kinase c-kit and Fyn, the latter being also involved in NMDA-dependent synaptic plasticity, cocaine and heroin may indirectly influence the neural mechanisms that mediate their reinforcing properties. Masitinib, a novel tyrosine kinase inhibitor with high selectivity for c-Kit, Fyn and Lyn, may alter the aberrant consequences of the activation of these tyrosine kinases by cocaine and heroin.ObjectiveWe investigated in rats the effect of a chronic oral treatment with masitinib (20 mg/kg) on the reinforcing and motivational properties of self-administered cocaine (250 μg/infusion) and heroin (40 μg/infusion).MethodsThree different cohorts of rats were trained instrumentally to respond for cocaine, heroin or food under continuous reinforcement. In each group, we assessed the influence of chronic daily treatment with masitinib on the maintenance of instrumental responding and intake and the motivation for the reinforcer. Thus, masitinib and vehicle-treated rats were challenged to adapt to high behavioural demand, to respond under a progressive ratio schedule of reinforcement and to reinstate instrumental responding after extinction and/or abstinence.ResultsMasitinib selectively decreased cocaine intake, the motivation for cocaine and the subsequent propensity to respond for cocaine under extinction, while having no effect on instrumental responding for heroin or food.ConclusionThe present findings suggest masitinib, a drug with proven efficacy in CNS disorders, could represent a novel treatment for cocaine addiction provided its influence on the reinforcing and incentive properties of the drug is confirmed.

Altered Intracellular Calcium Homeostasis Underlying Enhanced Glutamatergic Transmission in Striatal-Enriched Tyrosine Phosphatase (STEP) Knockout Mice.

The striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase involved in synaptic transmission. The current hypothesis on STEP function holds that it opposes synaptic strengthening by dephosphorylating and inactivating key neuronal proteins involved in synaptic plasticity and intracellular signaling, such as the MAP kinases ERK1/2 and p38, as well as the tyrosine kinase Fyn. Although STEP has a predominant role at the post-synaptic level, it is also expressed in nerve terminals. To better investigate its physiological role at the presynaptic level, we functionally investigated brain synaptosomes and autaptic hippocampal neurons from STEP knockout (KO) mice. Synaptosomes purified from mutant mice were characterized by an increased basal and evoked glutamate release compared with wild-type animals. Under resting conditions, STEP KO synaptosomes displayed increased cytosolic Ca2+ levels accompanied by an enhanced basal activity of Ca2+/calmodulin-dependent protein kinase type II (CaMKII) and hyperphosphorylation of synapsin I at CaMKII sites. Moreover, STEP KO hippocampal neurons exhibit an increase of excitatory synaptic strength attributable to an increased size of the readily releasable pool of synaptic vesicles. These results provide new evidence that STEP plays an important role at nerve terminals in the regulation of Ca2+ homeostasis and neurotransmitter release.

Photocarcinogenesis and Skin Cancer Prevention Strategies: An Update.

UV radiation is acknowledged as the primary cause of photocarcinogenesis and therefore contributes to the development of skin cancer entities such as squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and melanoma. Typical DNA photoproducts and indirect DNA damage caused by reactive oxygen species are the result of UV radiation. UV-induced DNA damage is repaired by nucleotide excision repair, which consequently counteracts the development of mutations and skin carcinogenesis. Tumour-suppressor genes are inactivated by mutation and growth-promoting pathways are activated leading to disruption of cell-cycle progression. Depending on the skin cancer entity, some genes are more frequently affected than others. In BCC mutations in Patched or Smoothened are common and affect the Sonic hedgehog pathway. In SCC, cell regulator protein p53 (TP53) mutations are prevalent, as well as mutations of the epidermal growth factor receptor (EGFR), cyclin-dependent kinase 2A (CDKN2A), Rat sarcoma (RAS), or the tyrosine kinase Fyn (FYN). UV-induced mutations in TP53 and CDKN2A are frequent in melanoma. UV-induced inflammatory processes also facilitate photocarcinogenesis. Recent studies showed a connection between photocarcinogenesis and citrus consumption, phytochemicals, alcohol consumption, hormone replacement therapy, as well as oral contraceptive use. Preventative measures include adequate use of sun protection and skin cancer screening at regular intervals, as well as the use of chemopreventative agents.

Nav1.7 is phosphorylated by Fyn tyrosine kinase which modulates channel expression and gating in a cell type-dependent manner.

Voltage-gated sodium channel Nav1.7 is a key molecule in nociception, and its dysfunction has been associated with various pain disorders. Here, we investigated the regulation of Nav1.7 biophysical properties by Fyn, an Src family tyrosine kinase. Nav1.7 was coexpressed with either constitutively active (FynCA) or dominant negative (FynDN) variants of Fyn kinase. FynCA elevated protein expression and tyrosine phosphorylation of Nav1.7 channels. Site-directed mutagenesis analysis identified two tyrosine residues (Y1470 and Y1471) located within the Nav1.7 DIII-DIV linker (L3) as phosphorylation sites of Fyn. Whole-cell recordings revealed that FynCA evoked larger changes in Nav1.7 biophysical properties when expressed in ND7/23 cells than in Human Embryonic Kidney (HEK) 293 cells, suggesting a cell type-specific modulation of Nav1.7 by Fyn kinase. In HEK 293 cells, substitution of both tyrosine residues with phenylalanine dramatically reduced current amplitude of mutant channels, which was partially rescued by expressing mutant channels in ND7/23 cells. Phenylalanine substitution showed little effect on FynCA-induced changes in Nav1.7 activation and inactivation, suggesting additional modifications in the channel or modulation by interaction with extrinsic factor(s). Our study demonstrates that Nav1.7 is a substrate for Fyn kinase, and the effect of the channel phosphorylation depends on the cell background. Fyn-mediated modulation of Nav1.7 may regulate DRG neuron excitability and contribute to pain perception. Whether this interaction could serve as a target for developing new pain therapeutics requires future study.

Binding investigation and preliminary optimisation of the 3-amino-1,2,4-triazin-5(2H)-one core for the development of new Fyn inhibitors

Fyn tyrosine kinase inhibitors are considered potential therapeutic agents for a variety of human cancers. Furthermore, the involvement of Fyn kinase in signalling pathways that lead to severe pathologies, such as Alzheimer’s and Parkinson’s diseases, has also been demonstrated. In this study, starting from 3-(benzo[d][1,3]dioxol-5-ylamino)-6-methyl-1,2,4-triazin-5(2H)-one (VS6), a hit compound that showed a micromolar inhibition of Fyn (IC50 = 4.8 μM), we computationally investigated the binding interactions of the 3-amino-1,2,4-triazin-5(2H)-one scaffold and started a preliminary hit to lead optimisation. This analysis led us to confirm the hypothesised binding mode of VS6 and to identify a new derivative that is about 6-fold more active than VS6 (compound 3, IC50 = 0.76 μM).

Tyrosine kinase Fyn regulates iNOS expression in LPS-stimulated astrocytes via modulation of ERK phosphorylation

The high concentrations of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in activated glial cells in response to neuroinflammatory stimuli have neurotoxic effects on the brain. At basal levels, iNOS expression is low, and proinflammatory stimuli induce iNOS expression in astrocytes, microglia, and oligodendrocytes. Fyn, a non-receptor tyrosine kinase, regulates iNOS expression in several types of immune cells. However, its role in stimulated astrocytes is less clear. In this study, we investigated the role of Fyn in the regulation of lipopolysaccharide (LPS)-induced iNOS expression in astrocytes from mice and rats. Intracerebroventricular LPS injections in cortical regions enhanced iNOS mRNA and protein levels, which were increased in Fyn-deficient mice. Accordingly, LPS-induced nitrite production was enhanced in primary astrocytes cultured from Fyn-deficient mice or rats. Similar results were observed in cultured astrocytes after the siRNA-induced knockdown of Fyn expression. Finally, we observed increased LPS-induced extracellular signal-regulated protein kinase (ERK) activation in Fyn-deficient astrocytes. These results suggested that Fyn has a regulatory role in iNOS expression in astrocytes during neuroinflammatory responses.

Upregulation of Tyrosine Kinase FYN in Human Thyroid Carcinoma: Role in Modulating Tumor Cell Proliferation, Invasion, and Migration.

Tyrosine kinase FYN-a member of the SRC family of kinases-is associated with mediating mitogenic signals and regulating cell cycle entry, growth, and proliferation. It was hypothesized that FYN is upregulated in thyroid carcinoma, which plays a critical role in tumorigenesis. FYN expression level in thyroid carcinoma tissue and tumor cell lines was determined. Also, the effects of FYN on thyroid cancer (TC) growth, signaling, cell cycle, and apoptosis were evaluated in vitro. FYN was knocked down in thyroid cancer cells (TPC-1) by siRNA to investigate the effects on cell proliferation, invasion, and migration. First, FYN was upregulated both in thyroid carcinoma tissue and in tumor cell lines. Loss of FYN by siRNA weakened proliferation, invasion, and migration of PTC-1 cells. Furthermore, it was demonstrated that knockdown of FYN can inhibit the G0/G1 phase of cell cycle. It was also observed that reduced expression of FYN could increase the level of NF-κB/P65 and IκBα. This study was the first to demonstrate critical positive regulation of thyroid tumorigenesis by FYN, which could be a potential target gene for thyroid carcinoma treatment. In addition, findings from this study highlighted the potential role of FYN inhibitors in TC therapy.

Simultaneous induction of HSP70 expression, and degranulation, in IgE/Ag-stimulated or extracellular HSP70-stimulated mast cells.

In mast cells, induction of HSP70 expression during antigen stimulation has not been reported. Mouse bone marrow-derived mast cells (BMMC) were stimulated with IgE/Ag or HSP70. Induction of HSP70 expression and signaling protein phosphorylation were evaluated by immunoblotting. HSP70 expression is induced in BMMC at an early stage of IgE/Ag-dependent stimulation, some of which is released from the cells in a granule-associated form. Induction of HSP70 expression was also observed with an IgE/Ag-stimulated human basophilic cell line, indicating that the phenomenon is not restricted to mouse BMMC. The induction of HSP70 expression, and its release, followed a similar time course to that of degranulation. Released HSP70 seems to be responsible for degranulation and production of eicosanoids, at least in part, because a neutralizing anti-HSP70 antibody mitigated these activities and because exogenous HSP70 not only induced immediate degranulation followed by autocrine HSP70 expression but also enhanced degranulation in IgE/Ag-stimulated BMMC. Extracellular HSP70 was found to induce phosphorylation of linker for activation of T cells (LAT) and a series of downstream signaling molecules in BMMC. We further found that Fyn, Lyn, and spleen tyrosine kinase (Syk), which are known to concern LAT phosphorylation in IgE/Ag-stimulated BMMC, were not phosphorylated in HSP70-stimulated BMMC, whereas lymphocyte-specific protein tyrosine kinase (Lck) was phosphorylated. FcεRI stimulation in BMMC and basophils induces HSP70 expression and its release. Extracellular HSP70 induces degranulation and mediator release via phosphorylation of LAT.

SY13-2 - Tyrosine Kinase Fyn Is a Novel Regulator of NADPH Oxidase 4 in Cardiac Remodeling

SY13-2 - Tyrosine Kinase Fyn Is a Novel Regulator of NADPH Oxidase 4 in Cardiac Remodeling

Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum.

The tyrosine kinase Fyn plays an important role in synaptic plasticity, learning, and memory. Here we report that Fyn is activated in response to 15 min D1 receptor (D1R) but not D2 receptor (D2R) stimulation specifically in the dorsomedial striatum (DMS) of mice but not in the other substriatal regions, the dorsolateral striatum (DLS), and the nucleus accumbens (NAc). Once activated Fyn phosphorylates its substrate GluN2B, and we show that GluN2B is phosphorylated only in the DMS but not in the other striatal regions. Striatal neurons are divided into D1R expressing medium spiny neurons (MSNs) and D2R expressing MSNs. Thus, to explore the cell-type specificity of this signaling pathway in the DMS, we developed a Cre-dependent Flip Excision (FLEX) approach to knockdown Fyn in D1R MSNs or D2R MSNs, and proved that the D1R-dependent Fyn activation is localized to DMS D1R MSNs. Importantly, we provide evidence to suggest that the differential association of Fyn and GluN2B with the scaffolding RACK1 is due to the differential localization of Fyn in lipid rafts.Our data further suggest that the differential cholesterol content in the three striatal regions may determine the region specificity of this signaling pathway. Together, our data show that the D1R-dependent Fyn/GluN2B pathway is selectively activated in D1R expressing MSNs in the DMS, and that the brain region specificity of pathway depends on the molecular and cellular compartmentalization of Fyn and GluN2B.

Striatal-enriched Tyrosine Protein Phosphatase (STEP) in the Mechanisms of Depressive Disorders.

Striatal-enriched tyrosine protein phosphatase (STEP) is expressed mainly in the brain. Its dysregulation is associated with Alzheimer's and Huntington's diseases, schizophrenia, fragile X syndrome, drug abuse and stroke/ischemia. However, an association between STEP and depressive disorders is still obscure. The review discusses the theoretical foundations and experimental facts concerning possible relationship between STEP dysregulation and depression risk. STEP dephosphorylates and inactivates several key neuronal signaling proteins such as extracellular signal-regulating kinase 1 and 2 (ERK1/2), stress activated protein kinases p38, the Src family tyrosine kinases Fyn, Pyk2, NMDA and AMPA glutamate receptors. The inactivation of these proteins decreases the expression of brain derived neurotrophic factor (BDNF) necessary for neurogenesis and neuronal survival. The deficit of BDNF results in progressive degeneration of neurons in the hippocampus and cortex and increases depression risk. At the same time, a STEP inhibitor, 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (TC-2153), increases BDNF expression in the hippocampus and attenuated the depressivelike behavior in mice. Thus, STEP is involved in the mechanism of depressive disorders and it is a promising molecular target for atypical antidepressant drugs of new generation.

The Kinase Fyn As a Novel Intermediate in L-DOPA-Induced Dyskinesia in Parkinson's Disease.

Dopamine replacement therapy with L-DOPA is the treatment of choice for Parkinson's disease; however, its long-term use is frequently associated with L-DOPA-induced dyskinesia (LID). Many molecules have been implicated in the development of LID, and several of these have been proposed as potential therapeutic targets. However, to date, none of these molecules have demonstrated full clinical efficacy, either because they lie downstream of dopaminergic signaling, or due to adverse side effects. Therefore, discovering new strategies to reduce LID in Parkinson's disease remains a major challenge. Here, we have explored the tyrosine kinase Fyn, as a novel intermediate molecule in the development of LID. Fyn, a member of the Src kinase family, is located in the postsynaptic density, where it regulates phosphorylation of the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor in response to dopamine D1 receptor stimulation. We have used Fyn knockout and wild-type mice, lesioned with 6-hydroxydopamine and chronically treated with L-DOPA, to investigate the role of Fyn in the induction of LID. We found that mice lacking Fyn displayed reduced LID, ΔFosB accumulation and NR2B phosphorylation compared to wild-type control mice. Pre-administration of saracatinib (AZD0530), an inhibitor of Fyn activity, also significantly reduced LID in dyskinetic wild-type mice. These results support that Fyn has a critical role in the molecular pathways affected during the development of LID and identify Fyn as a novel potential therapeutic target for the management of dyskinesia in Parkinson's disease.

Abstract 463: The Orphan G Protein-Coupled Receptor C5B is Regulated by Hyperglycemia and Activates Pro-Inflammatory Signaling in Endothelial Cells Via the Tyrosine Kinase Fyn

Background: Atherosclerosis is driven by an inflammatory process of the vascular wall in which endothelial cells (EC) are decisively involved. The orphan G protein-coupled receptor GPRC5B activates inflammatory pathways in adipocytes and regulates the glucose and lipid metabolism in Drosophila melanogaster . Hypothesis: We hypothesize that GPRC5B is involved in inflammatory signaling of EC, particularly in hyperglycemia. Methods & Results: Stimulation of mouse brain endothelial cells (bEnd.3) and primary human umbilical vein endothelial cells (HUVEC) with high glucose concentration (25 mM), Tumor Necrosis Factor α (TNFα 0,1 μg/ml) or Lipopolysaccharides (LPS 1 μg/ml), respectively, leads to a significant upregulation of GPRC5B mRNA and protein level compared to unstimulated cells (mRNA / protein: gluc +254% / +86%, p<0.001; TNFα +203% / +124%, p<0.001; LPS +195%, p<0.05, n=6). Similar results are obtained in HUVECs after stimulation with high glucose and cytokines. Overexpression of GPRC5B in EC induced a significant increase in Interleukin 6 (IL6), Intercellular Adhesion Molecule 1 (ICAM1) and Vascular Cell Adhesion Molecule 1 (VCAM1) mRNA and protein expression. Furthermore, activation of NFκB (luciferase reporter assay) and phosphorylation of extracellular signal–regulated kinases 1/2 (Erk 1/2) were significantly increased compared to controls (mRNA: IL6 +42%, p<0.01; ICAM1 +37%, p<0.01; VCAM1 +40%, p<0.001, n=8. Protein: ICAM1 +48%, p<0.05; VCAM1 +52%, p<0.05; pErk 1/2 +67%, p<0.01, n=5. NFκB: +165%, p<0.001, n=18). Conversely, the siRNA-mediated GPRC5B knockdown resulted in diminished expression of IL6 and VCAM1 as well as decreased NFκB activity compared to controls (IL6 -55%, p<0.05, n=3, NFκB -36%, p<0.05, n=6) . The knockdown results were confirmed in HUVECs. Co-immunoprecipitations in EC revealed the tyrosine kinase Fyn as interaction partner of GPRC5B. Conclusion: Our data suggest that GPRC5B is not only regulated by glucose and pro-inflammatory cytokines but also activates pro-inflammatory and pro-atherogenic pathways in mouse and human endothelial cells. Therefore, GPRC5B might play an important role in hyperglycemia-accelerated vascular inflammation and pathogenesis of atherosclerosis.

Protein Tyrosine Phosphatase δ Mediates the Sema3A-Induced Cortical Basal Dendritic Arborization through the Activation of Fyn Tyrosine Kinase.

Leukocyte common antigen-related (LAR) class protein tyrosine phosphatases (PTPs) are critical for axonal guidance; however, their relation to specific guidance cues is poorly defined. We here show that PTP-3, a LAR homolog in Caenorhabditis elegans, is involved in axon guidance regulated by Semaphorin-2A-signaling. PTPδ, one of the vertebrate LAR class PTPs, participates in the Semaphorin-3A (Sema3A)-induced growth cone collapse response of primary cultured dorsal root ganglion neurons from Mus musculus embryos. In vivo, however, the contribution of PTPδ in Sema3A-regualted axon guidance was minimal. Instead, PTPδ played a major role in Sema3A-dependent cortical dendritic growth. Ptpδ-/- and Sema3a-/- mutant mice exhibited poor arborization of basal dendrites of cortical layer V neurons. This phenotype was observed in both male and female mutants. The double-heterozygous mutants, Ptpδ+/-; Sema3a+/-, also showed a similar phenotype, indicating the genetic interaction. In Ptpδ-/- brains, Fyn and Src kinases were hyperphosphorylated at their C-terminal Tyr527 residues. Sema3A-stimulation induced dephosphorylation of Tyr527 in the dendrites of wild-type cortical neurons but not of Ptpδ-/- Arborization of cortical basal dendrites was reduced in Fyn-/- as well as in Ptpδ+/-; Fyn+/- double-heterozygous mutants. Collectively, PTPδ mediates Sema3A-signaling through the activation of Fyn by C-terminal dephosphorylation.SIGNIFICANCE STATEMENT The relation of leukocyte common antigen-related (LAR) class protein tyrosine phosphatases (PTPs) and specific axon guidance cues is poorly defined. We show that PTP-3, a LAR homolog in Caenorhabditis elegans, participates in Sema2A-regulated axon guidance. PTPδ, a member of vertebrate LAR class PTPs, is involved in Sema3A-regulated cortical dendritic growth. In Sema3A signaling, PTPδ activates Fyn and Src kinases by dephosphorylating their C-terminal Tyr residues. This is the first evidence showing that LAR class PTPs participate in Semaphorin signaling in vivo.

Phosphorylation of tau at Y18, but not tau-fyn binding, is required for tau to modulate NMDA receptor-dependent excitotoxicity in primary neuronal culture

BackgroundHyperexcitability of neuronal networks can lead to excessive release of the excitatory neurotransmitter glutamate, which in turn can cause neuronal damage by overactivating NMDA-type glutamate receptors and related signaling pathways. This process (excitotoxicity) has been implicated in the pathogenesis of many neurological conditions, ranging from childhood epilepsies to stroke and neurodegenerative disorders such as Alzheimer’s disease (AD). Reducing neuronal levels of the microtubule-associated protein tau counteracts network hyperexcitability of diverse causes, but whether this strategy can also diminish downstream excitotoxicity is less clear.MethodsWe established a cell-based assay to quantify excitotoxicity in primary cultures of mouse hippocampal neurons and investigated the role of tau in exicitotoxicity by modulating neuronal tau expression through genetic ablation or transduction with lentiviral vectors expressing anti-tau shRNA or constructs encoding wildtype versus mutant mouse tau.ResultsWe demonstrate that shRNA-mediated knockdown of tau reduces glutamate-induced, NMDA receptor-dependent Ca2+ influx and neurotoxicity in neurons from wildtype mice. Conversely, expression of wildtype mouse tau enhances Ca2+ influx and excitotoxicity in tau-deficient (Mapt−/−) neurons. Reconstituting tau expression in Mapt−/− neurons with mutant forms of tau reveals that the tau-related enhancement of Ca2+ influx and excitotoxicity depend on the phosphorylation of tau at tyrosine 18 (pY18), which is mediated by the tyrosine kinase Fyn. These effects are most evident at pathologically elevated concentrations of glutamate, do not involve GluN2B–containing NMDA receptors, and do not require binding of Fyn to tau’s major interacting PxxP motif or of tau to microtubules.ConclusionsAlthough tau has been implicated in diverse neurological diseases, its most pathogenic forms remain to be defined. Our study suggests that reducing the formation or level of pY18-tau can counteract excitotoxicity by diminishing NMDA receptor-dependent Ca2+ influx.