Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration.

mRNA transport in neurons requires formation of transport granules containing many protein components, and subsequent alterations in phosphorylation status can release transcripts for translation. Further, mutations in a structurally disordered domain of the transport granule protein hnRNPA2 increase its aggregation and cause hereditary proteinopathy of neurons, myocytes, and bone. We examine in vitro hnRNPA2 granule component phase separation, partitioning specificity, assembly/disassembly, and the link to neurodegeneration. Transport granule components hnRNPF and ch-TOG interact weakly with hnRNPA2 yet partition specifically into liquid phase droplets with the low complexity domain (LC) of hnRNPA2, but not FUS LC. In vitro hnRNPA2 tyrosine phosphorylation reduces hnRNPA2 phase separation, prevents partitioning of hnRNPF and ch-TOG into hnRNPA2 LC droplets, and decreases aggregation of hnRNPA2 disease variants. The expression of chimeric hnRNPA2 D290V in Caenorhabditis elegans results in stress-induced glutamatergic neurodegeneration; this neurodegeneration is rescued by loss of tdp-1, suggesting gain-of-function toxicity. The expression of Fyn, a tyrosine kinase that phosphorylates hnRNPA2, reduces neurodegeneration associated with chimeric hnRNPA2 D290V. These data suggest a model where phosphorylation alters LC interaction specificity, aggregation, and toxicity.

Suppression of protein tyrosine phosphatase N23 predisposes to breast tumorigenesis via activation of FYN kinase.

Disruption of the balanced modulation of reversible tyrosine phosphorylation has been implicated in the etiology of various human cancers, including breast cancer. Protein Tyrosine Phosphatase N23 (PTPN23) resides in chromosomal region 3p21.3, which is hemizygously or homozygously lost in some breast cancer patients. In a loss-of-function PTPome screen, our laboratory identified PTPN23 as a suppressor of cell motility and invasion in mammary epithelial and breast cancer cells. Now, our TCGA (The Cancer Genome Atlas) database analyses illustrate a correlation between low PTPN23 expression and poor survival in breast cancers of various subtypes. Therefore, we investigated the tumor-suppressive function of PTPN23 in an orthotopic transplantation mouse model. Suppression of PTPN23 in Comma 1Dß cells induced breast tumors within 56 wk. In PTPN23-depleted tumors, we detected hyperphosphorylation of the autophosphorylation site tyrosine in the SRC family kinase (SFK) FYN as well as Tyr142 in ß-catenin. We validated the underlying mechanism of PTPN23 function in breast tumorigenesis as that of a key phosphatase that normally suppresses the activity of FYN in two different models. We demonstrated that tumor outgrowth from PTPN23-deficient BT474 cells was suppressed in a xenograft model in vivo upon treatment with AZD0530, an SFK inhibitor. Furthermore, double knockout of FYN and PTPN23 via CRISPR/CAS9 also attenuated tumor outgrowth from PTPN23 knockout Cal51 cells. Overall, this mechanistic analysis of the tumor-suppressive function of PTPN23 in breast cancer supports the identification of FYN as a therapeutic target for breast tumors with heterozygous or homozygous loss of PTPN23.

Cleavage of VAMP2/3 Affects Oligodendrocyte Lineage Development in the Developing Mouse Spinal Cord.

In the developing and adult CNS, new oligodendrocytes (OLs) are generated from a population of cells known as oligodendrocyte precursor cells (OPCs). As they begin to differentiate, OPCs undergo a series of highly regulated changes to morphology, gene expression, and membrane organization. This stage represents a critical bottleneck in oligodendrogliogenesis, and the regulatory program that guides it is still not fully understood. Here, we show that in vivo toxin-mediated cleavage of the vesicle associated SNARE proteins VAMP2/3 in the OL lineage of both male and female mice impairs the ability of early OLs to mature into functional, myelinating OLs. In the developing mouse spinal cord, many VAMP2/3-cleaved OLs appeared to stall in the premyelinating, early OL stage, resulting in an overall loss of both myelin density and OL number. The Src kinase Fyn, a key regulator of oligodendrogliogenesis and myelination, is highly expressed among premyelinating OLs, but its expression decreases as OLs mature. We found that OLs with cleaved VAMP2/3 in the spinal cord white matter showed significantly higher expression of Fyn compared with neighboring control cells, potentially because of an extended premyelinating stage. Overall, our results show that functional VAMP2/3 in OL lineage cells is essential for proper myelin formation and plays a major role in controlling the maturation and terminal differentiation of premyelinating OLs.SIGNIFICANCE STATEMENT The production of mature oligodendrocytes (OLs) is essential for CNS myelination during development, myelin remodeling in adulthood, and remyelination following injury or in demyelinating disease. Before myelin sheath formation, newly formed OLs undergo a series of highly regulated changes during a stage of their development known as the premyelinating, or early OL stage. This stage acts as a critical checkpoint in OL development, and much is still unknown about the dynamic regulatory processes involved. In this study, we show that VAMP2/3, SNARE proteins involved in vesicular trafficking and secretion play an essential role in regulating premyelinating OL development and are required for healthy myelination in the developing mouse spinal cord.

Metastasis suppressor 1 interacts with protein tyrosine phosphatase receptor-δ to regulate adipogenesis.

Adipogenesis is a finely controlled process and its dysfunction may contribute to metabolic disorders such as obesity. Metastasis suppressor 1 (MTSS1) is a player in tumorigenesis and metastasis of various types of cancers. To date, it is not known whether and how MTSS1 plays a role in adipocyte differentiation. In the current study, we found that MTSS1 was upregulated during adipogenic differentiation of established mesenchymal cell lines and primary cultured bone marrow stromal cells. Gain-of-function and loss-of-function experiments uncovered that MTSS1 facilitated adipocyte differentiation from mesenchymal progenitor cells. Mechanistic explorations revealed that MTSS1 bound and interacted with FYN, a member of Src family of tyrosine kinases (SFKs), and protein tyrosine phosphatase receptor-δ (PTPRD). We demonstrated that PTPRD was capable of inducing the differentiation of adipocytes. Overexpression of PTPRD attenuated the impaired adipogenesis induced by the siRNA targeting MTSS1. Both MTSS1 and PTPRD activated SFKs by suppressing the phosphorylation of SFKs at Tyr530 and inducing the phosphorylation of FYN at Tyr419. Further investigation showed that MTSS1 and PTPRD were able to activate FYN. Collectively, our study has for the first time unraveled that MTSS1 plays a role in adipocyte differentiation in vitro through interacting with PTPRD and thereby activating SFKs such as FYN tyrosine kinase.

Paxillin participates in the sphingosylphosphorylcholine-induced abnormal contraction of vascular smooth muscle by regulating Rho-kinase activation.

BACKGROUND: The Ca2+-independent contraction of vascular smooth muscle is a leading cause of cardiovascular and cerebrovascular spasms. In the previous study, we demonstrated the involvement of Src family protein tyrosine kinase Fyn and Rho-kinase in the sphingosylphosphorylcholine (SPC)-induced abnormal and Ca2+-independent contraction of vascular smooth muscle, but the specific mechanism has not been completely clarified. METHODS: Paxillin knockdown human coronary artery smooth muscle cells (CASMCs) and smooth muscle-specific paxillin knockout mice were generated by using paxillin shRNA and the tamoxifen-inducible Cre-LoxP system, respectively. CASMCs contraction was observed by time-lapse recording. The vessel contractility was measured by using a myography assay. Fyn, Rho-kinase, and myosin light chain activation were assessed by immunoprecipitation and western blotting. The paxillin expression and actin stress fibers were visualized by histological analysis and immunofluorescent staining. RESULTS: The SPC-induced abnormal contraction was inhibited in paxillin knockdown CASMCs and arteries of paxillin knockout mice, indicating that paxillin is involved in this abnormal contraction. Further study showed that paxillin knockdown inhibited the SPC-induced Rho-kinase activation without affecting Fyn activation. In addition, paxillin knockdown significantly inhibited the SPC-induced actin stress fiber formation and myosin light chain phosphorylation. These results suggest that paxillin, as an upstream molecule of Rho-kinase, is involved in the SPC-induced abnormal contraction of vascular smooth muscle. CONCLUSIONS: The present study demonstrated that paxillin participates in the SPC-induced abnormal vascular smooth muscle contraction by regulating Rho-kinase activation. Video Abstract.

Fyn, Blk, and Lyn kinase inhibitors: A mini-review on medicinal attributes, research progress, and future insights.

Fyn, Blk, and Lyn are part of a group of proteins called Src family kinases. They are crucial in controlling cell communication and their response to the growth, changes, and immune system. Blocking these proteins with inhibitors can be a way to treat diseases where these proteins are too active. The primary mode of action of these inhibitors is to inhibit the phosphorylation of Fyn, Blk, and Lyn receptors, which in turn affects how signals pass within the cells. This review shows the structural and functional aspects of Fyn, Blk, and Lyn kinases, highlighting the significance of their dysregulation in diseases such as cancer and autoimmune disorders. The discussion encompasses the design strategies, SAR analysis, and chemical characteristics of effective inhibitors, shedding light on their specificity and potency. Furthermore, it explores the progress of clinical trials of these inhibitors, emphasizing their potential therapeutic applications.

In silico screening of Fyn kinase inhibitors using classification-based QSAR model, molecular docking, molecular dynamics and ADME study.

This study aimed to use a computational approach that combined the classification-based QSAR model, molecular docking, ADME studies, and molecular dynamics (MD) to identify potential inhibitors of Fyn kinase. First, a robust classification model was developed from a dataset of 1,078 compounds with known Fyn kinase inhibitory activity, using the XGBoost algorithm. After that, molecular docking was performed between potential compounds identified from the QSAR model and Fyn kinase to assess their binding strengths and key interactions, followed by MD simulations. ADME studies were additionally conducted to preliminarily evaluate the pharmacokinetics and drug-like characteristics of these compounds. The results showed that our obtained model exhibited good predictive performance with an accuracy of 0.95 on the test set, affirming its reliability in identifying potent Fyn kinase inhibitors. Through the application of this model in conjunction with molecular docking and ADME studies, nine compounds were identified as potential Fyn kinase inhibitors, including 208 (ZINC70708110), 728 (ZINC8792432), 734 (ZINC8792187), 736 (ZINC8792350), 738 (ZINC8792286), 739 (ZINC8792309), 817 (ZINC33901069), 852 (ZINC20759145), and 1227 (ZINC100006936). MD simulations further demonstrated that the four most promising compounds, 728, 734, 736, and 852 exhibited stable binding with Fyn kinase during the simulation process. Additionally, a web-based platform ( https://fynkinase.streamlit.app/ ) has been developed to streamline the screening process. This platform enables users to predict the activity of their substances of interest on Fyn kinase from their SMILES, using our classification-based QSAR model and molecular docking.

Computational discovery of novel FYN kinase inhibitors: a cheminformatics and machine learning-driven approach to targeted cancer and neurodegenerative therapy.

In this study, we explored the potential of novel inhibitors for FYN kinase, a critical target in cancer and neurodegenerative disorders, by integrating advanced cheminformatics, machine learning, and molecular simulation techniques. Our approach involved analyzing key interactions for FYN inhibition using established multi-kinase inhibitors such as Staurosporine, Dasatinib, and Saracatinib. We utilized ECFP4 circular fingerprints and the t-SNE machine learning algorithm to compare molecular similarities between FDA-approved drugs and known clinical trial inhibitors. This led to the identification of potential inhibitors, including Afatinib, Copanlisib, and Vandetanib. Using the DrugSpaceX platform, we generated a vast library of 72,196 analogues from these leads, which after careful refinement, resulted in 6008 promising candidates. Subsequent clustering identified 48 analogues with significant similarity to known inhibitors. Notably, two candidates derived from Vandetanib, DE27123047 and DE27123035, exhibited strong docking affinities and stable binding in molecular dynamics simulations. These candidates showed high potential as effective FYN kinase inhibitors, as evidenced by MMGBSA calculations and MCE-18 scores exceeding 50. Additionally, our exploration into their molecular architecture revealed potential modification sites on the quinazolin-4-amine scaffold, suggesting opportunities for strategic alterations to enhance activity and optimize ADME properties. Our research is a pioneering effort in drug discovery, unveiling novel candidates for FYN inhibition and demonstrating the efficacy of a multi-layered computational strategy. The molecular insights gained provide a pathway for strategic refinements and future experimental validations, setting a new direction in targeted drug development against diseases involving FYN kinase.

Peptide aptamer targeting Aß-PrP-Fyn axis reduces Alzheimer's disease pathologies in 5XFAD transgenic mouse model.

Currently, no effective therapeutics exist for the treatment of incurable neurodegenerative diseases such as Alzheimer's disease (AD). The cellular prion protein (PrPC) acts as a high-affinity receptor for amyloid beta oligomers (AßO), a main neurotoxic species mediating AD pathology. The interaction of AßO with PrPC subsequently activates Fyn tyrosine kinase and neuroinflammation. Herein, we used our previously developed peptide aptamer 8 (PA8) binding to PrPC as a therapeutic to target the AßO-PrP-Fyn axis and prevent its associated pathologies. Our in vitro results indicated that PA8 prevents the binding of AßO with PrPC and reduces AßO-induced neurotoxicity in mouse neuroblastoma N2a cells and primary hippocampal neurons. Next, we performed in vivo experiments using the transgenic 5XFAD mouse model of AD. The 5XFAD mice were treated with PA8 and its scaffold protein thioredoxin A (Trx) at a 14.4 µg/day dosage for 12 weeks by intraventricular infusion through Alzet® osmotic pumps. We observed that treatment with PA8 improves learning and memory functions of 5XFAD mice as compared to Trx-treated 5XFAD mice. We found that PA8 treatment significantly reduces AßO levels and Aß plaques in the brain tissue of 5XFAD mice. Interestingly, PA8 significantly reduces AßO-PrP interaction and its downstream signaling such as phosphorylation of Fyn kinase, reactive gliosis as well as apoptotic neurodegeneration in the 5XFAD mice compared to Trx-treated 5XFAD mice. Collectively, our results demonstrate that treatment with PA8 targeting the AßO-PrP-Fyn axis is a promising and novel approach to prevent and treat AD.

A new paradigm for regulation of protein phosphatase 2A function via Src and Fyn kinase-mediated tyrosine phosphorylation.

Protein phosphatase 2A (PP2A) is a major phospho-Ser/Thr phosphatase and a key regulator of cellular signal transduction pathways. While PP2A dysfunction has been linked to human cancer and neurodegenerative disorders such as Alzheimer's disease (AD), PP2A regulation remains relatively poorly understood. It has been reported that the PP2A catalytic subunit (PP2Ac) is inactivated by a single phosphorylation at the Tyr307 residue by tyrosine kinases such as v-Src. However, multiple mass spectrometry studies have revealed the existence of other putative PP2Ac phosphorylation sites in response to activation of Src and Fyn, two major Src family kinases (SFKs). Here, using PP2Ac phosphomutants and novel phosphosite-specific PP2Ac antibodies, we show that cellular pools of PP2Ac are instead phosphorylated on both Tyr127 and Tyr284 upon Src activation, and on Tyr284 following Fyn activation. We found these phosphorylation events enhanced the interaction of PP2Ac with SFKs. In addition, we reveal SFK-mediated phosphorylation of PP2Ac at Y284 promotes dissociation of the regulatory Bα subunit, altering PP2A substrate specificity; the phosphodeficient Y127/284F and Y284F PP2Ac mutants prevented SFK-mediated phosphorylation of Tau at the CP13 (pSer202) epitope, a pathological hallmark of AD, and SFK-dependent activation of ERK, a major growth regulatory kinase upregulated in many cancers. Our findings demonstrate a novel PP2A regulatory mechanism that challenges the existing dogma on the inhibition of PP2A catalytic activity by Tyr307 phosphorylation. We propose dysregulation of SFK signaling in cancer and AD can lead to alterations in PP2A phosphorylation and subsequent deregulation of key PP2A substrates, including ERK and Tau.

The Rho guanine nucleotide exchange factor PLEKHG1 is activated by interaction with and phosphorylation by Src family kinase member FYN.

Rho family small GTPases (Rho) regulate various cell motility processes by spatiotemporally controlling the actin cytoskeleton. Some Rho-specific guanine nucleotide exchange factors (RhoGEFs) are regulated via tyrosine phosphorylation by Src family tyrosine kinase (SFK). We also previously reported that PLEKHG2, a RhoGEF for the GTPases Rac1 and Cdc42, is tyrosine-phosphorylated by SRC. However, the details of the mechanisms by which SFK regulates RhoGEFs are not well understood. In this study, we found for the first time that PLEKHG1, which has very high homology to the Dbl and pleckstrin homology domains of PLEKHG2, activates Cdc42 following activation by FYN, a member of the SFK family. We also show that this activation of PLEKHG1 by FYN requires interaction between these two proteins and FYN-induced tyrosine phosphorylation of PLEKHG1. We also found that the region containing the Src homology 3 and Src homology 2 domains of FYN is required for this interaction. Finally, we demonstrated that tyrosine phosphorylation of Tyr-720 and Tyr-801 in PLEKHG1 is important for the activation of PLEKHG1. These results suggest that FYN is a regulator of PLEKHG1 and may regulate cell morphology through Rho signaling via the interaction with and tyrosine phosphorylation of PLEKHG1.

Aberrant palmitoylation caused by a ZDHHC21 mutation contributes to pathophysiology of Alzheimer's disease.

BACKGROUND: The identification of pathogenic mutations in Alzheimer's disease (AD) causal genes led to a better understanding of the pathobiology of AD. Familial Alzheimer's disease (FAD) is known to be associated with mutations in the APP, PSEN1, and PSEN2 genes involved in Aß production; however, these genetic defects occur in only about 10-20% of FAD cases, and more genes and new mechanism causing FAD remain largely obscure. METHODS: We performed exome sequencing on family members with a FAD pedigree and identified gene variant ZDHHC21 p.T209S. A ZDHHC21T209S/T209S knock-in mouse model was then generated using CRISPR/Cas9. The Morris water navigation task was then used to examine spatial learning and memory. The involvement of aberrant palmitoylation of FYN tyrosine kinase and APP in AD pathology was evaluated using biochemical methods and immunostaining. Aß and tau pathophysiology was evaluated using ELISA, biochemical methods, and immunostaining. Field recordings of synaptic long-term potentiation were obtained to examine synaptic plasticity. The density of synapses and dendritic branches was quantified using electron microscopy and Golgi staining. RESULTS: We identified a variant (c.999A > T, p.T209S) of ZDHHC21 gene in a Han Chinese family. The proband presented marked cognitive impairment at 55 years of age (Mini-Mental State Examination score = 5, Clinical Dementia Rating = 3). Considerable Aß retention was observed in the bilateral frontal, parietal, and lateral temporal cortices. The novel heterozygous missense mutation (p.T209S) was detected in all family members with AD and was not present in those unaffected, indicating cosegregation. ZDHHC21T209S/T209S mice exhibited cognitive impairment and synaptic dysfunction, suggesting the strong pathogenicity of the mutation. The ZDHHC21 p.T209S mutation significantly enhanced FYN palmitoylation, causing overactivation of NMDAR2B, inducing increased neuronal sensitivity to excitotoxicity leading to further synaptic dysfunction and neuronal loss. The palmitoylation of APP was also increased in ZDHHC21T209S/T209S mice, possibly contributing to Aß production. Palmitoyltransferase inhibitors reversed synaptic function impairment. CONCLUSIONS: ZDHHC21 p.T209S is a novel, candidate causal gene mutation in a Chinese FAD pedigree. Our discoveries strongly suggest that aberrant protein palmitoylation mediated by ZDHHC21 mutations is a new pathogenic mechanism of AD, warranting further investigations for the development of therapeutic interventions.

FYN: emerging biological roles and potential therapeutic targets in cancer.

Src family protein kinases (SFKs) play a key role in cell adhesion, invasion, proliferation, survival, apoptosis, and angiogenesis during tumor development. In humans, SFKs consists of eight family members with similar structure and function. There is a high level of overexpression or hyperactivity of SFKs in tumor, and they play an important role in multiple signaling pathways involved in tumorigenesis. FYN is a member of the SFKs that regulate normal cellular processes. Additionally, FYN is highly expressed in many cancers and promotes cancer growth and metastasis through diverse biological functions such as cell growth, apoptosis, and motility migration, as well as the development of drug resistance in many tumors. Moreover, FYN is involved in the regulation of multiple cancer-related signaling pathways, including interactions with ERK, COX-2, STAT5, MET and AKT. FYN is therefore an attractive therapeutic target for various tumor types, and suppressing FYN can improve the prognosis and prolong the life of patients. The purpose of this review is to provide an overview of FYN's structure, expression, upstream regulators, downstream substrate molecules, and biological functions in tumors.

Methyl donor supplementation reduces phospho-Tau, Fyn and demethylated protein phosphatase 2A levels and mitigates learning and motor deficits in a mouse model of tauopathy.

BACKGROUND: Reduced folate status and elevated levels of circulating homocysteine are modifiable risk factors for cognitive decline and dementia. Disturbances in one-carbon metabolism are associated with the pathological accumulation of phosphorylated tau, a hallmark feature of prevalent dementia, including Alzheimer's disease and subgroups of frontotemporal dementia. METHODS: Here, using transgenic TAU58/2 mouse models of human tauopathy, we tested whether dietary supplementation with L-methylfolate (the active folate form), choline and betaine can reduce tau phosphorylation and associated behavioural phenotypes. RESULTS: TAU58/2 mice fed with the methyl donor-enriched diet showed reduced phosphorylation of tau at the pathological S202 (CP13) and S396/S404 (PHF-1) epitopes and alleviation of associated motor and learning deficits. Compared with mice on the control diet, the decrease in cortical phosphorylated tau levels in mice fed with the methyl donor-enriched diet was associated with enhanced methylation of protein phosphatase 2A, the major brain tau Ser/Thr phosphatase. It also correlated with a reduction in protein levels of Fyn, a tau tyrosine kinase that plays a central role in mediating pathological tau-induced neurodegeneration. Conversely, Fyn expression levels were increased in mice with deficiencies in folate metabolism. CONCLUSIONS: Our findings provide the first experimental evidence that boosting one-carbon metabolism with L-methylfolate, choline and betaine can mitigate key pathological, learning and motor deficits in a tauopathy mouse model. They give support to using a combination of methyl donors as a preventive or disease-modifying strategy for tauopathies.

Deficiency of the Src homology phosphatase 2 in podocytes is associated with renoprotective effects in mice under hyperglycemia.

Diabetic nephropathy (DN) is a significant complication of diabetes and the leading cause of end-stage renal disease. Hyperglycemia-induced dysfunction of the glomerular podocytes is a major contributor to the deterioration of renal function in DN. Previously, we demonstrated that podocyte-specific disruption of the Src homology phosphatase 2 (Shp2) ameliorated lipopolysaccharide-induced renal injury. This study aims to evaluate the contribution of Shp2 to podocyte function under hyperglycemia and explore the molecular underpinnings. We report elevated Shp2 in the E11 podocyte cell line under high glucose and the kidney under streptozotocin- and high-fat diet-induced hyperglycemia. Consistently, Shp2 disruption in podocytes was associated with partial renoprotective effects under hyperglycemia, as evidenced by the preserved renal function. At the molecular level, Shp2 deficiency was associated with altered renal insulin signaling and diminished hyperglycemia-induced renal endoplasmic reticulum stress, inflammation, and fibrosis. Additionally, Shp2 knockdown in E11 podocytes mimicked the in vivo deficiency of this phosphatase and ameliorated the deleterious impact of high glucose, whereas Shp2 reconstitution reversed these effects. Moreover, Shp2 deficiency attenuated high glucose-induced E11 podocyte migration. Further, we identified the protein tyrosine kinase FYN as a putative mediator of Shp2 signaling in podocytes under high glucose. Collectively, these findings suggest that Shp2 inactivation may afford protection to podocytes under hyperglycemia and highlight this phosphatase as a potential target to ameliorate glomerular dysfunction in DN.

Fyn-Mediated Paxillin Tyrosine 31 Phosphorylation Regulates Migration and Invasion of Breast Cancer Cells.

Metastasis is the leading cause of death in breast cancer patients due to the lack of effective therapies. Elevated levels of paxillin expression have been observed in various cancer types, with tyrosine phosphorylation shown to play a critical role in driving cancer cell migration. However, the specific impact of the distinct tyrosine phosphorylation events of paxillin in the progression of breast cancer remains to be fully elucidated. Here, we found that paxillin overexpression in breast cancer tissue is associated with a patient's poor prognosis. Paxillin knockdown inhibited the migration and invasion of breast cancer cells. Furthermore, the phosphorylation of paxillin tyrosine residue 31 (Tyr31) was significantly increased upon the TGF-ß1-induced migration and invasion of breast cancer cells. Inhibiting Fyn activity or silencing Fyn decreases paxillin Tyr31 phosphorylation. The wild-type and constitutively active Fyn directly phosphorylate paxillin Tyr31 in an in vitro system, indicating that Fyn directly phosphorylates paxillin Tyr31. Additionally, the non-phosphorylatable mutant of paxillin at Tyr31 reduces actin stress fiber formation, migration, and invasion of breast cancer cells. Taken together, our results provide direct evidence that Fyn-mediated paxillin Tyr31 phosphorylation is required for breast cancer migration and invasion, suggesting that targeting paxillin Tyr31 phosphorylation could be a potential therapeutic strategy for mitigating breast cancer metastasis.

Exploring BenzylethoxyAryl Urea Scaffolds for Multitarget Immunomodulation Therapies.

Thirteen benzylethoxyaryl ureas have been synthesized and biologically evaluated as multitarget inhibitors of VEGFR-2 and PD-L1 proteins to overcome resistance phenomena offered by cancer. The antiproliferative activity of these molecules on several tumor cell lines (HT-29 and A549), on the endothelial cell line HMEC-1, on immune cells (Jurkat T) and on the non-tumor cell line HEK-293 has been determined. Selective indexes (SI) have been also determined and compounds bearing p-substituted phenyl urea unit together with a diaryl carbamate exhibited high SI values. Further studies on these selected compounds to determine their potential as small molecule immune potentiators (SMIPs) and as antitumor agents have been performed. From these studies, we have concluded that the designed ureas have good tumor antiangiogenic properties, exhibit good inhibition of CD11b expression, and regulate pathways involved in CD8 T-cell activity. These properties suggest that these compounds could be potentially useful in the development of new cancer immune treatments.

Disturbances in PP2A methylation and one-carbon metabolism compromise Fyn distribution, neuritogenesis, and APP regulation.

The nonreceptor protein tyrosine kinase Fyn and protein Ser/Thr phosphatase 2A (PP2A) are major multifunctional signaling molecules. Deregulation of Fyn and altered PP2A methylation are implicated in cancer and Alzheimer's disease (AD). Here, we tested the hypothesis that the methylation state of PP2A catalytic subunit, which influences PP2A subunit composition and substrate specificity, can affect Fyn regulation and function. Using Neuro-2a (N2a) neuroblastoma cell models, we first show that methylated PP2A holoenzymes containing the Bα subunit coimmunoprecipitate and copurify with Fyn in membrane rafts. PP2A methylation status regulates Fyn distribution and Fyn-dependent neuritogenesis, likely in part by affecting actin dynamics. A methylation-incompetent PP2A mutant fails to interact with Fyn. It perturbs the normal partitioning of Fyn and amyloid precursor protein (APP) in membrane microdomains, which governs Fyn function and APP processing. This correlates with enhanced amyloidogenic cleavage of APP, a hallmark of AD pathogenesis. Conversely, enhanced PP2A methylation promotes the nonamyloidogenic cleavage of APP in a Fyn-dependent manner. Disturbances in one-carbon metabolic pathways that control cellular methylation are associated with AD and cancer. Notably, they induce a parallel loss of membrane-associated methylated PP2A and Fyn enzymes in N2a cells and acute mouse brain slices. One-carbon metabolism also modulates Fyn-dependent process outgrowth in N2a cells. Thus, our findings identify a novel methylation-dependent PP2A/Fyn signaling module. They highlight the underestimated importance of cross talks between essential metabolic pathways and signaling scaffolds that are involved in normal cell homeostasis and currently being targeted for cancer and AD treatment.

Fyn and Lyn gene polymorphisms impact the risk of thyroid cancer.

Thyroid cancer is the most common malignancy of the endocrine glands, and during last couple of decades, its incidence has risen alarmingly, across the globe. Etiology of thyroid cancer is still debatable. There are a few worth mentioning risk factors which contribute to initiation of abnormalities in thyroid gland leading to cancer. Genetic instability is major risk factors in thyroid carcinogenesis. Among the genetic factors, the Src family of genes (Src, Yes1, Fyn and Lyn) have been implicated in many cancers but there is little data regarding the association of these (Src, Yes1, Fyn and Lyn) genes with thyroid carcinogenesis. Fyn and Lyn genes of Src family found engaged in proliferation, migration, invasion, angiogenesis, and metastasis in different cancers. This study was planned to examine the effect of Fyn and Lyn SNPs on thyroid cancer risk in Pakistani population in 500 patients and 500 controls. Three polymorphisms of Fyn gene (rs6916861, rs2182644 and rs12910) and three polymorphisms of Lyn gene (rs2668011, rs45587541 and rs45489500) were analyzed using Tetra-primer ARMS-PCR followed by DNA sequencing. SNP rs6916861 of Fyn gene mutant genotype (CC) showed statistically significant threefold increased risk of thyroid cancer (P < 0.0001). In case of rs2182644 of Fyn gene, mutant genotype (AA) indicated statistically significant 17-fold increased risk of thyroid cancer (P < 0.0001). Statistically significant threefold increased risk of thyroid cancer was observed in genotype AC (P < 0.0001) of Fyn gene polymorphism rs12910. In SNP rs2668011 of Lyn gene, TT genotype showed statistically significant threefold increased risk of thyroid cancer (P < 0.0001). In case of rs45587541 of Lyn gene, GA genotypes showed statistically significant 11-fold increased risk in thyroid cancer (P < 0.0001). Haplotype analysis revealed that AAATAG*, AGACAG*, AGCCAA*, AGCCAG*, CAATAG*, CGCCAG* and CGCCGA* haplotypes of Fyn and Lyn polymorphisms are associated with increased thyroid cancer risk. These results showed that genotypes and allele distribution of Fyn and Lyn are significantly linked with increased thyroid cancer risk and could be genetic adjuster for said disease.

DHF-7 Ameliorates Behavioral Disorders and White Matter Lesions by Regulating BDNF and Fyn in a Mouse Model of Schizophrenia Induced by Cuprizone and MK-801.

BACKGROUND: Schizophrenia is a psychiatric disorder including multiple clinical symptoms such as severe psychosis and cognitive dysfunction. DHF-7 is a novel dihydroflavanone derivative that was designed and synthesized to treat schizophrenia. This study aimed to investigate the effects and mechanisms of DHF-7 in a mouse model of schizophrenia induced by a combination of cuprizone and MK-801. METHODS: After intragastric administration of DHF-7 for 7 weeks, open field, Y-maze, and novel object recognition tests were performed to detect behavioral changes in the mouse model. White matter lesions and myelin loss were determined using transmission electron microscopy and oil red O staining. Western blotting and immunohistochemistry were used to detect the expression of the related proteins. RESULTS: The results showed that DHF-7 treatment significantly improved cognitive impairment and positive symptoms in the model mice. Moreover, DHF-7 alleviated white matter lesions and demyelination and promoted the differentiation and maturation of oligodendrocytes for remyelination in the corpus callosum of model mice. The mechanistic study showed that DHF-7 increased the expression of brain-derived neurotrophic factor and phosphorylated Fyn, thus activating the tyrosine kinase receptor B (Trk B)/Fyn/N-methyl-D-aspartate receptor subunit 2 B (NMDAR2B) and Raf/mitogen-activated protein kinase (MEK)/ extracellular signal-related kinase (ERK) signaling pathways. CONCLUSIONS: Our results provide an experimental basis for the development of DHF-7 as a novel therapeutic agent for schizophrenia.