Prognostic Significance of Phosphorylated Fyn in Patients with Lung Adenocarcinoma after Lung Resection.

PURPOSE: Src family tyrosine kinases, including Fyn, are non-receptor tyrosine kinases that drive malignancy in various kinds of cancers. Fyn has also been suggested to be an effector of epidermal growth factor receptor (EGFR) signaling, and is recognized as a potential therapeutic target. However, little is known about the clinical importance of phosphorylated Fyn (pFyn) in lung adenocarcinoma. The purpose of this study is to examine the prognostic significance of pFyn in this disease. METHODS: A total of 282 lung adenocarcinoma specimens were collected from patients who underwent surgery at our institute. A tissue microarray was assembled from paraffin-embedded tumor blocks. pFyn expression was analyzed through immunostaining of the tissue microarray and each case was classified as positive or negative. The association of clinical information with pFyn expression was analyzed statistically. RESULTS: pFyn was positive in 107 cases. A pFyn-positive status was significantly associated with male gender, p53 mutant, pathological stage, tumor size, plural invasion, lymphatic invasion, vascular invasion, and differentiation. pFyn positivity was associated with poor relapse-free survival (RFS; hazard ratio [HR]: 2.11, 95% confidence interval [CI]: 1.32-3.42, p <0.01) and poor overall survival (OS; HR: 1.95, 95% CI: 1.17-3.33, p = 0.01). CONCLUSION: pFyn expression may affect the prognosis of patients with lung adenocarcinoma after lung resection.

Sensitive FRET Biosensor Reveals Fyn Kinase Regulation by Submembrane Localization.

Fyn kinase plays crucial roles in hematology and T cell signaling; however, there are currently limited tools to visualize the dynamic Fyn activity in live cells. Here we developed and characterized a highly sensitive Fyn biosensor based on fluorescence resonance energy transfer (FRET) to monitor Fyn kinase activity in live cells. Our results show that Fyn kinase activity can be induced in both mouse embryonic fibroblasts (MEFs) and T cells by ligand engagement. Two different motifs were further introduced to target the biosensor at the cellular membrane microdomains in MEFs, revealing that the Fyn-tagged biosensor had 70% greater response to growth factor stimulation than the Lyn-tagged version. This suggests that the plasma membrane microdomains can be categorized into different functional subdomains. Further experiments show that while the membrane accessibility is necessary for Fyn activation, the localization of Fyn outside of its microdomains causes its hyperactivity, indicating that membrane microdomains provide a suppressive microenvironment for Fyn regulation in MEFs. Interestingly, a relatively high Fyn activity can be observed at perinuclear regions, further supporting the notion that the membrane microenvironment has a significant impact on the local molecular functions. Our work hence highlights a novel Fyn FRET biosensor for live cell imaging and its application in revealing an intricate submembrane regulation of Fyn in live MEFs.

Measurement of dissociation rate of biomolecular complexes using CE.

Fluorescence anisotropy (FA), non-equilibrium CE of equilibrium mixtures (NECEEM) and high-speed CE were evaluated for measuring dissociation kinetics of peptide-protein binding systems. Fyn-SH3-SH2, a protein construct consisting of the src homology 2 (SH2) and 3 (SH3) domain of the protein Fyn, and a fluorescein-labeled phosphopeptide were used as a model system. All three methods gave comparable half-life of approximately 53 s for Fyn-SH3-SH2:peptide complex. Achieving satisfactory results by NECEEM required columns over 30 cm long. When using Fyn-SH2-SH3 tagged with glutathione S-transferase (GST) as the binding protein, both FA and NECEEM assays gave evidence of two complexes forming with the peptide, yet neither method allowed accurate measurement of dissociation rates for both complexes because of a lack of resolution. High-speed CE, with a 7 s separation time, enabled separation of both complexes and allowed determination of dissociation rate of both complexes independently. The two complexes had half-lives of 22.0+/-2.7 and 58.8+/-6.1 s, respectively. Concentration studies revealed that the GST-Fyn-SH3-SH2 protein formed a dimer so that complexes had binding ratios of 2:1 (protein-to-peptide ratio) and 2:2. Our results demonstrate that although all methods are suitable for 1:1 binding systems, high-speed CE is unique in allowing multiple complexes to be resolved simultaneously. This property allows determination of binding kinetics of complicated systems and makes the technique useful for discovering novel affinity interactions.

Up-regulation of proproliferative genes and the ligand/receptor pair placental growth factor and vascular endothelial growth factor receptor 1 in hepatitis C cirrhosis.

BACKGROUND/AIMS: Cirrhosis can lead to hepatocellular carcinoma (HCC). Non-diseased liver and hepatitis C virus (HCV)-associated cirrhosis with or without HCC were compared. METHOD: Proliferation pathway genes, immune response genes and oncogenes were analysed by a quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunostaining. RESULTS: Real-time RT-PCR showed up-regulation of genes in HCV cirrhosis including the proliferation-associated genes bone morphogenetic protein 3 (BMP3), placental growth factor 3 (PGF3), vascular endothelial growth factor receptor 1 (VEGFR1) and soluble VEGFR1, the oncogene FYN, and the immune response-associated genes toll-like receptor 9 (TLR9) and natural killer cell transcript 4 (NK4). Expressions of TLR2 and the oncogenes B-cell CLL/lymphoma 9 (BCL9) and PIM2 were decreased in HCV cirrhosis. In addition, PIM2 and TLR2 were increased in HCV cirrhosis with HCC compared with HCV cirrhosis. The ligand/receptor pair PGF and VEGFR1 was intensely expressed by the portal tract vascular endothelium. VEGFR1 was expressed in reactive biliary epithelial structures in fibrotic septum and in some stellate cells and macrophages. CONCLUSION: PGF and VEGFR1 may have an important role in the pathogenesis of the neovascular response in cirrhosis.

Role of Src homology 2 domain-mediated PTK signaling in mouse zygotic development.

Fyn and other Src-family kinases play an essential role at several steps during egg activation following fertilization of externally fertilizing species, such as marine invertebrates, fish, and frogs. Recent studies demonstrate that the requirement for Src-family kinases in activation of the mammalian egg is different from lower species, and the objective of this study was to test the role of the Fyn kinase in the mouse egg activated by intracytoplasmic sperm injection (ICSI). An Src homology 2 (SH2) domain containing fusion protein was used to suppress Fyn function in the mouse zygote following ICSI. Eggs injected with the Fyn SH2 domain at an intracellular concentration of 4-8 microM exhibited reduced developmental potential with 100% of the zygotes being arrested following the first or the second cleavage. At higher concentrations, the protein blocked pronuclear congression and the zygotes remained at the pronuclear stage. The SH2 domain had no effect on sperm-induced calcium oscillations in distinct contrast to its effect on the eggs of lower species. The results indicate that the SH2 domain of Fyn kinase plays an important role in pronuclear congression as well as early cleavage events and that this effect appears not to involve disruption of calcium oscillations.