Analysis and identification of tyrosine phosphorylated proteins in hemocytes from Fenneropenaeus chinensis (Decapoda: Penaeidae) infected with white spot syndrome virus

Abstract

The change in the level of protein tyrosine phosphorylation in hemocytes of Fenneropenaeus chinensis (Osbeck, 1765) (cf., Perez Farfante and Kensley, 1997) was detected by phospho-specific flow cytometry during white spot syndrome virus (WSSV) infection. Post WSSV infection, the hemocytes could be classified into two subsets with up- or down-regulated tyrosine phosphorylation level, whereas the geometric mean level of tyrosine phosphorylation in total hemocytes was significantly increased and exhibited two peaks at 1 and 12 hour post infection. Confocal microscopic observation revealed that the tyrosine phosphorylated proteins were localized in nucleus as well as cytoplasm, and five tyrosine phosphorylated proteins were detected in hemocytes by western blotting using phosphor-tyrosine-specific antibody, which were identified to be histone H2A, histone H3, histone H4, alpha-2-macroglobulin, and heat shock protein 70 by mass spectrometry. Moreover, the expression levels of h2a, h3, and h4 were examined to be significantly up-regulated post WSSV infection by quantitative real-time RT-PCR. These results indicated that protein tyrosine phosphorylation in shrimp hemocytes had a close correlation with WSSV infection.