RAF proto-oncogene serine/threonine-protein kinase (c-Raf) is a MAP kinase kinase kinase (MAPKKK) that
participates in the Erk1/2 pathway and plays an important role in lymphocyte activation. However, the study on
how c-Raf regulates adaptive immunity in non-mammal is still limited. In present study, based on analysis of
sequence characteristics of c-Raf from Oreochromis niloticus (On-c-Raf), we investigated its regulation roles on
teleost lymphocyte activation. The On-c-Raf was highly conserved during evolution, which was composed of a
Raf-like Ras-binding domain (RBD), a protein kinase C conserved region 1 (C1) domain and a serine/threonine
protein kinase catalytic (S_TKc) domain. Its mRNA showed a wide distribution in tissues of O. niloticus and with
the highest expression in gill. After Aeromonas hydrophila infection, during the adaptive immune stage transcription
level of On-c-Raf was significantly upregulated on day 8, but came back to original level on day 16 and
30, suggesting the potential involvement of On-c-Raf in primary response but not memory formation.
Furthermore, On-c-Raf mRNA in leukocytes of Nile tilapias was obviously induced by in vitro stimulation of T
cell mitogen PHA. More importantly, in vitro stimulation of lymphocytes agonist PMA augmented phosphorylation
level of On-c-Raf in leukocytes detected by western-blot and immunofluorescent. Thus, c-Raf regulated
lymphocyte activation of Nile tilapia on both mRNA and phosphorylation level. Together, our results revealed
that the c-Raf from teleost Nile tilapia engaged in adaptive immune response by regulating lymphocytes activation.
Since the regulatory mechanism of lymphocyte-mediated adaptive immunity is largely unknown in
teleost, our study provided important evidences to understand teleost adaptive immunity, and also shed a novel
perspective for the evolution of adaptive immune system.