Abstract P47: Src Tyrosine Kinase Family is a Novel Upstream Modulator of Stat3 Activation in Urocortin-mediated Signal Transduction
Background: We previously identified in Src tyrosine kinase family a novel upstream modulator of MAP kinase subfamily, p42/p44, whose activation is required for urocortin (Ucn)-mediated cardioprotection. Src kinase was recently shown to reduce apoptosis in different cancer cell lines, enhancing phosphorylation and DNA binding affinity of Signal Transducer and Activator of Transcription (STAT)3. We also documented that modulation of the functional balance between STAT3 and STAT1, with preferential activation of pro-survival STAT3 over the pro-apoptotic STAT1, represents one mechanism of cardioprotection against ischemia.
Aim: To verify whether STAT3 plays a role in conveying survival signals downstream of Src tyrosine kinase in Ucn-mediated signal transduction.
Methods and Results: Western blot analysis using anti-STAT1, -STAT3, -phospho(p)STAT1Y701, -pSTAT1S727, -pSTAT3Y705, -pSTAT3S727, -Src and -pSrcT416 antibody was firstly performed. Incubation of 10 nM Ucn for 10 minutes with immortalized mouse atrial HL-1 cells was sufficient to induce phosphorylation, though not upregulation, of STAT3 in tyrosine 705, while expression level and activation of STAT1 were not affected. Ucn-induced STAT3 activation was followed by STAT3 nuclear translocation. This was substantiated by increased STAT3 DNA-binding, assessed by electromobility shift assay, and colocalization of active STAT3 with the nuclear staining TOPRO-3, assessed by immunocytochemistry and fluorescent microscopy evaluation. Importantly, a specific Src family kinase inhibitor, PP2, significantly reduced Ucn-induced phosphorylation of STAT3, as well as STAT3 DNA-binding and nuclear relocation, suggesting that Src is an upstream modulator of STAT3 activation. In line with this finding, cotransfection of HL-1 cells with a dominant negative cDNA for Src abolished Ucn-induced phosphorylarion of STAT3.
Conclusions: We report for the first time that short-term treatment with Ucn induces Src-mediated phosphorylation and subsequent nuclear translocation of STAT3. Further studies in animal models of ischemia/reperfusion injury are required to investigate the mechanisms by which Src modulates STAT3 activation in Ucn-mediated signal transduction.