Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 3 (MEKK3) is a serine/threonine protein kinase that acts as a key regulator and is widely involved in various innate and acquired immune signaling pathways. In this study, we first cloned the complete open reading frame (ORF) of the MEKK3 gene (named CcMEKK3) in a hybrid snakehead (Channa maculate ♀ × Channa argus ♂). The full-length ORF of CcMEKK3 is 1851 bp, and encodes a putative protein of 616 amino acids containing a serine/threonine kinase catalytic (S-TKc) domain and a Phox and Bem1p (PB1) domain. A sequence alignment and phylogenetic tree analysis showed that CcMEKK3 is highly conserved relative to the MEKK3 proteins of other teleost species. CcMEKK3 was constitutively expressed in all the healthy hybrid snakehead tissues tested, with greatest expression in the immune tissues, such as the head kidney and spleen. The expression of CcMEKK3 was usually upregulated in the head kidney, spleen, and liver at different time points after infection with Nocardia seriolae or Aeromonas schubertii. Similarly, the dynamic expression levels of CcMEKK3 in head kidney leukocytes after stimulation revealed that CcMEKK3 was induced by LTA, LPS, and poly(I:C). In the subcellular localization analysis, CcMEKK3 was evenly distributed in the cytoplasm of HEK293T cells, and its overexpression significantly promoted the activities of NF-κB and AP-1. These results suggest that CcMEKK3 is involved in the immune defense against these two pathogens, and plays a crucial role in activating the NF-κB and MAPK signaling pathways.

丝裂原活化蛋白激酶/细胞外信号调节激酶激酶 3 (MEKK3) 是一种丝氨酸/苏氨酸蛋白激酶，作为关键调节剂，广泛参与各种先天性和获得性免疫信号通路。在这项研究中，我们首先在杂交蛇头（Channa maculate ♀ ×  Channa argus ♂）中克隆了MEKK3基因（命名为CcMEKK3）的完整开放阅读框（ORF ）。CcMEKK3的全长 ORF是 1851 bp，并编码 616 个氨基酸的推定蛋白质，其中包含丝氨酸/苏氨酸激酶催化 (S-TKc) 域和 Phox 和 Bem1p (PB1) 域。序列比对和系统发育树分析表明 CcMEKK3 相对于其他硬骨鱼物种的 MEKK3 蛋白高度保守。CcMEKK3在所有测试的健康杂种蛇头组织中组成型表达，在免疫组织中表达最大，如头肾和脾脏。在感染诺卡氏菌或舒伯特气单胞菌后的不同时间点，CcMEKK3在头部肾脏、脾脏和肝脏中的表达通常上调。同样，CcMEKK3的动态表达水平刺激后头部肾脏白细胞中的细胞显示CcMEKK3是由 LTA、LPS 和 poly(I:C) 诱导的。在亚细胞定位分析中，CcMEKK3均匀分布于HEK293T细胞的细胞质中，其过表达显着促进了NF-κB和AP-1的活性。这些结果表明 CcMEKK3 参与了针对这两种病原体的免疫防御，并在激活 NF-κB 和 MAPK 信号通路中起关键作用。