MOLECULAR CHARACTERIZATION OF A SYK HOMOLOGUE FROM AYU, PLECOGLOSSUS ALTIVELIS : INSIGHTS INTO ITS CONSERVED ROLE IN MAPK SIGNALING ACTIVATION

Abstract

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase (NRTK). In mammals, SYK is an important adaptor that plays an essential role in the activation of immune cells, signal transduction of extracellular stimuli, and pathogen recognition etc. However, study regarding the function of this molecule in fish is limited. Therefore, the role of ayu Plecoglossus altivelis SYK ( Pa SYK) in pathogen infection and the activation of immune signaling pathways was studied. The cDNA sequence of Pa SYK was retrieved; Multiple sequence alignment and phylogenetic tree analysis were conducted to analyze the evolutionary status of Pa SYK. The expressions of SYK in healthy and Vibrio anguillarum infected tissues of ayu were revealed in real-time fluorescence quantitative PCR (RT-qPCR). Subcellular localization was conducted in HEK293T cells to detect the cellular distribution of Pa SYK. We overexpressed Pa SYK in HEK293T cells and ayu head kidney monocyte/macrophage (MO/MΦ), and its regulatory role on cytokine expression and the activation of MAPK signaling pathway were deciphered. Multiple sequence alignment indicated that the protein sequences and functional domains of SYK were highly conserved during evolution. Pa SYK was closely related to its homologue of river trout ( Salmo trutta ) as indicated in the phylogenetic tree. RT-qPCR results showed that the mRNA of SYK gene was expressed in all tissues examined, and the expression of Pa SYK was up-regulated in all tissues detected after V. anguillarum infection. Furthermore, Pa SYK significantly induced the expression of the pro-inflammatory factors TNF-α and IL-1β, and slightly inhibited the expression of anti-inflammatory factors TGF-β and IL-10. Western blot results demonstrated that, like its homologues in mammals, Pa SYK could activate the MAPK signaling pathway. Therefore, SYK is highly conserved during the evolution. Pa SYK and its mammalian homologues have conserved functions, and both could activate MAPK signaling pathway and play a role in inflammatory response.