Assessment of immune and pro-apoptotic functions of mitogen-activated protein kinase-interacting kinase 2 (MKNK2) in yellowtail clownfish (Amphiprion clarkii)

Mitogen-activated protein kinase-interacting serine/threonine-protein kinase 2 (MKNK2) regulates protein synthesis by phosphorylating eukaryotic initiation factor 4E (eIF4E). In this study, we characterized the yellowtail clownfish (Amphiprion clarkii) MKNK2 gene, AcMKNK2, by investigating its transcriptional responses and functional properties using different functional assays. The AcMKNK2 gene contains a 1425 bp open reading frame encoding 467 amino acids. The protein has a predicted molecular weight of 53.25 kDa and an isoelectric point value of 6.10. Two conserved active motifs (²¹⁰ENIL²¹³ and ²²⁹DLG²³¹) and a protein kinase ATP binding site were identified in the AcMKNK2 amino acid sequence. The highest constitutive expression of AcMKNK2 was found in muscle tissue under normal physiological conditions, while significant upregulation was observed under stimulations with polyinosinic:polycytidylic acid (poly(I:C)), lipopolysaccharide (LPS), and Vibrio harveyi in the blood, gill, and head kidney tissues. Subcellular localization analysis revealed that the AcMKNK2 protein is localized to the nucleus. Furthermore, AcMKNK2 overexpression increased the production of reactive oxygen species and the Bax/Bcl-2 mRNA expression ratio in fathead minnow cells exposed to H₂O₂, emphasizing the pro-apoptotic activities under oxidative stress conditions. Furthermore, RAW267.4 cells overexpressing AcMKNK2 demonstrated a significant increase in the expression of M1 marker genes and NO production after LPS stimulation. Overall, these findings suggest that AcMKNK2 mediates cellular stress responses and regulates host immunity in yellowtail clownfish by driving pro-apoptotic pathways and enhancing pro-inflammatory mechanisms.