Aggregation, tyrosine phosphorylation, and gene expression in hemocytes of the ascidian Halocynthia roretzi

Hemocyte aggregation is an early inflammatory response upon injury in the ascidian Halocynthia roretzi. We have already reported that the monoclonal antibody A74 strongly inhibits both hemocyte aggregation and phagocytosis of foreign substances in H. roretzi. The A74 antigen protein is a novel membrane glycoprotein with a molecular mass of 160 kDa, and it has two immunoreceptor tyrosine-based activation motifs (ITAMs) and several motifs that have been proposed to play a role in signal transduction. The A74 protein and several other proteins are tyrosine-phosphorylated during hemocyte aggregation. Immunoprecipitation analysis showed that tyrosine-phosphorylated A74 protein was associated with 90- and 75-kDa proteins, both of which were tyrosine-phosphorylated during hemocyte aggregation. To identify the proteins that function downstream of ITAMs in H. roretzi hemocytes, we carried out a differential display analysis to evaluate mRNAs differentially expressed before and after hemocyte aggregation. Four amplicons, the expression levels of which increased after induction of hemocyte aggregation, were isolated, and their expressions were found to be strongly inhibited by treatment with BAPTA-AM and Wortmannin. These results indicate that a signaling pathway mediated by ITAMs and tyrosine-phosphorylated proteins functions during hemocyte aggregation in H. roretzi, inducing the expression of several genes, and that calcium ions and a phosphatidylinositol 3-kinase-like enzyme are involved in their expressions in H. roretzi hemocytes.