Eicosapentaenoic acid ameliorates pulmonary hypertension via inhibition of tyrosine kinase Fyn.

Pulmonary arterial hypertension (PAH) is a multifactorial disease characterized by pulmonary arterial vasoconstriction and remodeling. Src family tyrosine kinases, including Fyn, play critical roles in vascular remodeling via the inhibition of STAT3 signaling. EPA is known to inhibit Fyn kinase activity. This study investigated the therapeutic potential and underlying mechanisms of EPA and its metabolite, resolvin E1 (RvE1), to treat PAH using monocrotaline-induced PAH model rats (MCT-PAH), human pulmonary artery endothelial cells (HPAECs), and human pulmonary artery smooth muscle cells (HPASMCs). Administration of EPA 1 and 2 weeks after MCT injection both ameliorated right ventricular hypertrophy, remodeling and dysfunction, and medial wall thickening of the pulmonary arteries and prolonged survival in MCT-PAH rats. EPA attenuated the enhanced contractile response to 5-hydroxytryptamine in isolated pulmonary arteries of MCT-PAH rats. Mechanistically, the treatment with EPA and RvE1 or the introduction of dominant-negative Fyn prevented TGF-ß2-induced endothelial-to-mesenchymal transition and IL-6-induced phosphorylation of STAT3 in cultured HPAECs. EPA and RvE1 suppressed Src family kinases' activity as evaluated by their phosphorylation status in cultured HPAECs and HPASMCs. EPA and RvE1 suppressed vasocontraction of rat and human PA. Furthermore, EPA and RvE1 inhibited the enhanced proliferation and activity of Src family kinases in HPASMCs derived from patients with idiopathic PAH. EPA ameliorated PAH's pathophysiology by mitigating vascular remodeling and vasoconstriction, probably inhibiting Src family kinases, especially Fyn. Thus, EPA is considered a potent therapeutic agent for the treatment of PAH.

A Src-family-tyrosine kinase, Lyn, is required for efficient IFN-ß expression in pattern recognition receptor, RIG-I, signal pathway by interacting with IPS-1.

Retinoic acid-inducible gene I (RIG-I) plays an important role in antiviral immunity as a cytosolic receptor recognizing invading viruses. The activation of downstream signaling pathways led by IFN-ß promoter stimulator-1 (IPS-1), an adaptor, is known to culminate in the activation of IRFs and the expression of type I interferons. However, the role of Src-family-tyrosine kinases (STKs) in the RIG-I signaling pathway has not been fully evaluated. Through a combined approach of immunoprecipitation and micro reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) analysis, we established that Lyn, one of the STKs, is associated with RIG-I in macrophages. The association of Lyn and RIG-I was confirmed by co-immunoprecipitation study with 293T cells overexpressing Lyn and RIG-I. Suppression of Lyn by siRNA knockdown or a pharmacological inhibitor (PP2) resulted in the attenuation of IRF3 activation and IFN-ß expression induced by short poly I:C, a RIG-I agonist, in macrophages. Lyn activation, as determined by phosphorylation of Tyr396 residue, was observed upon short poly I:C stimulation in the mitochondria of macrophages. Short poly I:C induced the formation of speckle-like aggregates of Lyn, which are prominent in mitochondria. Lyn associated with IPS-1, an adaptor protein of RIG-I, which resides on mitochondria membrane. Helicase domain of RIG-I and CARD of IPS-1 are responsible for the interaction with Lyn while SH3 and SH2 domains in Lyn are required for the association with RIG-I and IPS-1. Collectively, our results indicate that Lyn plays a positive regulatory role in RIG-I-mediated interferon expression as a downstream component of IPS-1. They provide further information as to how tyrosine kinases such as STKs play a role in the regulation of antiviral immunity.

The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis.

The family of Abelson interactor (Abi) proteins is a component of WAVE regulatory complex (WRC) and a downstream target of Abelson (Abl) tyrosine kinase. The fact that Abi proteins also interact with diverse membrane proteins and intracellular signaling molecules places these proteins at a central position in the network that controls cytoskeletal functions and cancer cell metastasis. Here, we identified a motif in Abi proteins that conforms to consensus sequences found in a cohort of receptor and non-receptor tyrosine kinases that bind to Cbl-tyrosine kinase binding domain. The phosphorylation of tyrosine 213 in this motif is essential for Abi degradation. Double knockout of c-Cbl and Cbl B in Bcr-Abl-transformed leukemic cells abolishes Abi1, Abi2, and WAVE2 degradation. Moreover, knockout of Abi1 reduces Src family kinase Lyn activation in Bcr-Abl-positive leukemic cells and promotes EGF-induced EGF receptor downregulation in breast cancer cells. Importantly, Abi1 depletion impeded breast cancer cell invasion in vitro and metastasis in mouse xenografts. Together, these studies uncover a novel mechanism by which the WRC and receptor/non-receptor tyrosine kinases are regulated and identify Abi1 as a potential therapeutic target for metastatic breast cancer.

Purification of a Src family tyrosine protein kinase from bovine retinas.

Since tyrosine phosphorylation appears to play important functions in photoreceptor cells, we searched here for retinal nonreceptor tyrosine kinases of the Src family. We demonstrated that Src family tyrosine kinases were present in the cytosolic fraction of extracted bovine retinas. A Src family tyrosine kinase with an apparent molecular mass of about 62 kDa was purified to homogeneity from the soluble fraction of dark-adapted bovine retinas after three consecutive purification steps: ω-aminooctyl-agarose hydrophobic chromatography, Cibacron blue 3GA-agarose pseudo-affinity chromatography, and α-casein-agarose affinity chromatography. The purified protein was subjected to N-terminal amino acid sequencing and the sequence Gly-Ile-Ile-Lys-Ser-Glu-Glu was obtained, which displayed homology with the first seven residues of the Src family tyrosine kinase c-Yes from Bos taurus (Gly-Cys-Ile-Lys-Ser-Lys-Glu). Although the cytosolic fraction from dark-adapted retinas contained tyrosine kinases of the Src family capable of phosphorylating the α-subunit of transducin, which is the heterotrimeric G protein involved in phototransduction, the purified tyrosine kinase was not capable of using transducin as a substrate. The cellular role of this retinal Src family member remains to be found.

Src Family Kinases as Therapeutic Targets in Advanced Solid Tumors: What We Have Learned so Far.

Src is the prototypal member of Src Family tyrosine Kinases (SFKs), a large non-receptor kinase class that controls multiple signaling pathways in animal cells. SFKs activation is necessary for the mitogenic signal from many growth factors, but also for the acquisition of migratory and invasive phenotype. Indeed, oncogenic activation of SFKs has been demonstrated to play an important role in solid cancers; promoting tumor growth and formation of distant metastases. Several drugs targeting SFKs have been developed and tested in preclinical models and many of them have successfully reached clinical use in hematologic cancers. Although in solid tumors SFKs inhibitors have consistently confirmed their ability in blocking cancer cell progression in several experimental models; their utilization in clinical trials has unveiled unexpected complications against an effective utilization in patients. In this review, we summarize basic molecular mechanisms involving SFKs in cancer spreading and metastasization; and discuss preclinical and clinical data highlighting the main challenges for their future application as therapeutic targets in solid cancer progression.

Androgen receptor activation in castration-recurrent prostate cancer: the role of Src-family and Ack1 tyrosine kinases.

There is growing appreciation that castration-recurrent prostate cancer (CR-CaP) is driven by the continued expression of androgen receptor (AR). AR activation in CR-CaP through various mechanisms, including AR overexpression, expression of AR splice variants or mutants, increased expression of co-regulator proteins, and by post-translational modification, allows for the induction of AR-regulated genes in response to very low levels of tissue-expressed, so-called intracrine androgens, resulting in pathways that mediate CaP proliferation, anti-apoptosis and oncogenic aggressiveness. The current review focuses on the role played by Src-family (SFK) and Ack1 non-receptor tyrosine kinases in activating AR through direct phosphorylation, respectively, on tyrosines 534 or 267, and how these modifications facilitate progression to CR-CaP. The fact that SFK and Ack1 are central mediators for multiple growth factor receptor signaling pathways that become activated in CR-CaP, especially in the context of metastatic growth in the bone, has contributed to recent therapeutic trials using SFK/Ack1 inhibitors in monotherapy or in combination with antagonists of the AR activation axis.

Effects of activation of Src-family tyrosine kinases on human LECs apoptosis and EMT under the high glucose environment / 中华实验眼科杂志

Background Diabetic cataract is one of the major ocular complications in diabetes mellitus,including cortical cataract,nuclear cataract,subcapsular cataract and mixed cataract,and different cataractogenesis may be associated with lens epithelial cells (LECs).Subcapsular cataract is one of diabetic cataracts.Studying the biological behavior of LECs in subcapsular cataract is crucial for prevention and treatment.Objective This study was to investigate the effects of Src-family tyrosine kinases (SFKs) on apoptosis and epithelial-mesenchymal transition (EMT) of human LECs cultured by high glucose.Methods Human LECs (HLE-B3) were cultured for 24 hours with DMEM containing 5.5 mmol/L glucose (normal control group),DMEM containing 35.5 mmol/L glucose (high glucose group) and DMEM containing 35.5 mmol/L glucose + 10 μmol/L PP1,a specific inhibitor of SFKs (PP1 group).In 3,6,12 and 24 hours after culture,the apoptosis of human LECs was detected by flow cytometry assay;morphological change of human LECs was observed under the inverted microscope,and the expressions of the markers of EMT,E-cadherin and a-smooth muscle actin (α-SMA),in the cells were detected by immnofluorescence staining.In addition,the alternations of p-Src418 (active c-Src),bcl-xl,survivin,caspase-3,E-cadherin and α-SMA proteins were assayed by Western blot analysis.Results An elevated expression level of p-Src418 was found in LECs in the high glucose group and peaked 6 hours after cultured.The expressions of p-Src418(grey levels) were 0.125 ±0.036 in the high glucose group,and which was significantly higher than 0.042±0.011 in the normal control group and 0.035 ± 0.018 in the PP1 group,respectively (both at P＜0.01).No remarkable differences were seen in the apoptotic rates between the high glucose group and normal control group in 6,12 and 24 hours after culture (all at P＞0.05).The apoptotic rates of human LECs were(6.433±2.084)％,(10.333±2.610)％ and (8.033±2.967)％ in the PP1 group,which were higher than (3.233 ± 1.320) ％,(3.533 ± 1.159) ％,(5.733 ±0.230) ％ in the high glucose group and (3.133±1.170)％,(2.833±0.751)％,(3.333±1.201)％ in the normal control group (all at P＜0.05),however,there were significant differences in the apoptosis between the high glucose group and the normal control group (all at P＞0.05).In 6 hours and 12 hours after cell culture,the expression levels of bcl-xl and survivin (grey values) in human LECs were significantly declined,but the expression of caspase-3 was increased in the PP1 group compared with the the high glucose group and the normal control group (all at P＜0.05).The LECs showed slender in shape 24 hours after culture in the high glucose group,but the cell shape was close to the normal in the PP1 group.Western blot and immunofluorescence assay revealed that the expression of E-cadherin in human LECs was significantly reduced and that of α-SMA was significantly increased 6 hours after culture in the high glucose group compared with the PP1 group and the normal control group (all at P＜0.05).Conclusions High glucose activates c-Src kinase of LECs in high glucose environment and therefore induces EMT and inhibits apoptosis.However,PP1 impedes the biological process of EMT and apoptosis of LECs to maintain the epithelial characteristics even under the stress of high glucose.