Magic™ Anti-PhosphoY (Phospho Y) monoclonal antibody, clone 14G10

In response to viral infections, various pattern recognition receptors (PRRs) are activated for the production of type I interferon (IFN I). As a center of these receptor responses, TANK binding kinase-1 (TBK1) activates interferon regulatory factor 3 (IRF3). SRC is a member of Src family kinases (SFK) which participates in TBK1-mediated IFN I signaling pathway. In mammals, the immunological function of SRC is depended on its interaction with TBK1. To date, SRC has not been studied in fish. In this paper, we cloned the ORF of grass carp (Ctenopharyngodon idellus) SRC (CiSRC). CiSRC has a closer relationship with Sinocyclocheilus rhinocerous SRC (SrSRC). The expression level of CiSRC was significantly up-regulated following poly (I:C) stimulation in grass carp tissues and cells. Subcellular localization results showed that CiSRC is located both in the cytoplasm and nucleus, while CiTBK1 is only located in the cytoplasm of CIK cells. When GFP-CiSRC and FLAG-CiTBK1 were co-transfected into CIK cells, we found that they were co-localized in the cytoplasm. GST-pulldown and Co-immunoprecipitation analysis revealed that CiSRC and CiSRC tyrosine kinase domain deletion mutant (SRC-Delta Tyrkc) can interact with CiTBK1, respectively. CiSRC promotes the phosphorylation of CiTBK1. Furthermore, the phosphorylation of TBK1 is more strongly under poly (I:C) stimulation. We also demonstrated that SRC can up-regulate IFN I expression. These results above unraveled that CiSRC initiates innate immune response by binding to and then up-regulating the phosphorylation of TBK1.

EphA2 (EPH receptor A2) (erythropoietin-producing hepatocellular receptor tyrosine kinase subtype A2) plays a crucial role in human cancers, and is a promising target for the development of new anticancer drugs. In this study, we showed that the interaction of Annexin A1 (ANXA1) and EphA2 increased EphA2 stability by inhibiting its proteasome degradation in gastric cancer (GC) and colon cancer (CC) cells, and the amino acid residues 20-30 and 28-30 of ANXA1 N terminal were responsible for binding and stabilizing EphA2. Based on the amino acid residues of ANXA1 responsible for binding EphA2, we developed ANXA1-derived 3 amino acid-long (SKG) and 11 amino acid-long peptides (EYVQTVKSSKG) in fusion to cell-penetrating peptide, named as A1(28-30) and A1(20-30) respectively, and found that A1(28-30) and A1(20-30) blocked the binding of ANXA1 with EphA2, targeted EphA2 degradation, and suppressed the growth of GC and CC cellsin vitroand in mice. Our data demonstrated that ANXA1 was able to bind and stabilize EphA2 in GC and CC cells, and disruption of ANXA1-EphA2 interaction by the two ANXA1-derived peptides inhibited the growth of GC and CC cells by targeting EphA2 degradation, presenting a potential strategy for treating GC and CC with these peptides.