ABSTRACT
In this study, we characterize the HIP-55 protein in the mosquito Aedes aegypti for the first time. HIP-55 is a 55-kDa HPK1-interacting protein that is also called SH3P7. HIP-55 constitutively binds HPK1 'via' an HPK1 proline-rich motif 2(PR2) through its C-terminal SH3 domain. HIP-55 critically interacts with ZAP-70, and this interaction was induced by TCR signalling. ZAP-70 phosphorylated HIP-55 at Tyr-334 and Tyr-344 in vitro and in vivo. In our previous findings, AaZAP gene expression strongly proved that AaZAP-70 was involved in immunity-like functions in mosquito. Northern blot analysis of HIP-55 mRNA expression confirmed that it is only expressed in the abdomen and haemocyte tissues; this prediction correlates 100% and a polyclonal antibody also confirmed its localization in haemocytes and the abdomen. We prepared extracts to show the cytoplasmic expression (CE) of this protein. Previous results had proven that this protein is secreted from the cytoplasm; thus, we confirmed here that the protein is a cytoplasmic adaptor protein in mosquitoes and mammalian systems. Furthermore, our polyclonal antibody against HIP-55 also demonstrated that this protein is found in haemocytes and abdomen tissues, which assumes that the protein may be involved in phagocytic-like functions. RNAi (siRNA) silencing studies were used to degrade mosquito HIP-55; however, silencing only slightly affected the HIP-55 sequence and the gene transcriptional level. To characterize this protein, we cloned 609 bp from the 1.6-kb full-length cDNA using a pET28 vector for polyclonal antibody production. Graphical abstract.
