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Molecular characterization, expression and function analysis of Epinephelus coioides MKK4 response to SGIV and Vibrio
Yu-Ling Su1, Yu-Feng Jiang2, He-Jia Chen1
1Joint Laboratory of Guangdong Province and Hong Kong Regions on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural University, Guangzhou 510642, Guangdong Province, PR China.
Abstract:
Mitogen-activated protein kinase 4 (MKK4), a member of the MAP kinase family, play important roles in response to many environmental and cellular stresses in mammals. In this study, three MKK4 subtypes, EcMKK4-1, EcMKK4-2 and EcMKK4-3, were obtained from grouper Epinephelus coioides. The open reading frame (ORF) of EcMKK4s are obtained and the EcMKK4s proteins contain highly conserved domains: a S_TKc domain, a canonical diphosphorylation group and two conserved MKKK ATP binding motifs, Asp-Phe-Gly (DFG) and Ala-Pro-Glu (APE). EcMKK4s could be found both in the cytoplasmic and nuclear. The EcMKK4s mRNA were detected in all E. coioides tissues examined with the different expression levels, and the expression were up-regulated during SGIV (Singapore grouper iridescent virus) or Vibrio alginolyticus infection. EcMKK4 could significantly reduce the activation of AP-1 reporter gene. The results suggested that EcMKK4s might play important roles in pathogen-caused inflammation.
Insights
Grouper Mitogen-activated protein kinase 4 (MKK4) subtypes were identified and found to be upregulated during pathogen infection. These MKK4s may play a role in regulating inflammation in fish.
Area of Science:
- Molecular Biology
- Immunology
- Aquaculture
Background:
- Mitogen-activated protein kinase 4 (MKK4) is crucial for cellular stress responses in mammals.
- Understanding fish immune responses is vital for aquaculture health and disease management.
Purpose of the Study:
- To identify and characterize MKK4 subtypes in grouper (Epinephelus coioides).
- To investigate the role of grouper MKK4 (EcMKK4) in response to viral and bacterial infections.
Main Methods:
- Cloning and sequencing of three EcMKK4 subtypes.
- Analysis of protein domains and subcellular localization.
- Quantitative analysis of EcMKK4 mRNA expression in various tissues and under infection conditions.
- Reporter gene assays to assess MKK4 activity.
Main Results:
- Three EcMKK4 subtypes (EcMKK4-1, -2, -3) were identified, possessing conserved kinase domains and motifs.
- EcMKK4 proteins were localized in both the cytoplasm and nucleus.
- EcMKK4 mRNA expression varied across tissues and was significantly upregulated during Singapore grouper iridescent virus (SGIV) and Vibrio alginolyticus infections.
- EcMKK4 significantly inhibited the activation of the AP-1 reporter gene.
Conclusions:
- EcMKK4s are conserved among fish and play a role in cellular defense mechanisms.
- Upregulation of EcMKK4 during SGIV and V. alginolyticus infections suggests involvement in the fish immune response.
- EcMKK4 likely contributes to the regulation of pathogen-induced inflammation by modulating signaling pathways like AP-1.
