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Updated: Jan 2, 2026
Author Spotlight: Integrating BRET-Based Assays and Rare Mutation Analysis to Decipher RAF Kinase Regulation in Live Cells
Published on: March 1, 2024
Identification and characterization of c-raf from orange-spotted grouper (Epinephelus coioides)
Ze-Quan Mo1, Xue-Li Lai2, Wan-Tao Wang3
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, China; College of Animal Science, South China Agricultural University, Guangzhou, 510642, Guangdong Province, China.
Abstract:
C-Raf proto-oncogene serine/threonine kinase is a mitogen-activated protein kinase (MAP) kinase kinase, which can initiate a mitogen-activated protein kinase (MAPK) cascade by phosphorylating the dual-specific MAP kinase kinases (MEK1/2), and in turn activate the extracellular signal-regulated kinases (ERK1/2). To study the function of c-Raf in teleost fish, a c-Raf cDNA sequence from orange-spotted grouper (Epinephelus coioides) was cloned. Ecc-Raf shared 81%-99% amino acid identity with other vertebrate c-Raf molecules, and shared the highest amino acid identity (99%) with Lates calcarifer c-Raf. Genomic structure analysis revealed that grouper c-Raf shared a conserved exon structure with other vertebrates. Tissue distribution showed that Ecc-Raf was mainly transcribed in systemic immune organs. Ecc-Raf was distributed throughout the cytoplasm of transfected GS cells and the overexpression of Ecc-Raf only slightly enhanced the activation of Activator protein 1. The phosphorylation levels of Ecc-Raf can be induced by PMA and H2O2 treatment, in contrast to DMSO or untreated HKLs. Moreover, the phosphorylation level of the Raf-MEK-ERK axis was downregulated after 24 h of SGIV infection. On the other hand, the total level and phosphorylation level of c-Raf significantly increased post C. irritans infection and showed an enhanced level post immunization. The results of this study suggested that the Raf-MEK-ERK cascade was involved in the response to viral or parasitic infections.
Insights
The study cloned grouper c-Raf (Ecc-Raf) and found its expression in immune organs. Ecc-Raf plays a role in the Raf-MEK-ERK pathway
Area of Science:
- Molecular Biology
- Immunology
- Fish Biology
Background:
- C-Raf proto-oncogene serine/threonine kinase (c-Raf) is a key component of the mitogen-activated protein kinase (MAPK) cascade, regulating cellular processes.
- Understanding c-Raf function in teleost fish is crucial for fish health and aquaculture.
- The Raf-MEK-ERK pathway is implicated in various cellular responses, including immune signaling.
Purpose of the Study:
- To clone and characterize the c-Raf cDNA from orange-spotted grouper (Epinephelus coioides).
- To investigate the tissue distribution and functional role of grouper c-Raf (Ecc-Raf).
- To elucidate the involvement of the Raf-MEK-ERK pathway in the immune response of teleost fish.
Main Methods:
- Cloning of Ecc-Raf cDNA from orange-spotted grouper.
- Sequence analysis and comparison with other vertebrate c-Raf molecules.
- Tissue distribution analysis using transcription assays.
- Cellular localization and functional assays in transfected cells.
- Analysis of Ecc-Raf phosphorylation levels under different treatments (PMA, H2O2).
- Investigation of the Raf-MEK-ERK axis in response to viral (SGIV) and parasitic (C. irritans) infections.
Main Results:
- Ecc-Raf shares high amino acid identity (81%-99%) with other vertebrate c-Raf, particularly Lates calcarifer c-Raf.
- Ecc-Raf is predominantly transcribed in systemic immune organs.
- Overexpression of Ecc-Raf showed a minor enhancement of Activator protein 1 activation.
- Ecc-Raf phosphorylation is inducible by PMA and H2O2.
- The Raf-MEK-ERK pathway was downregulated by SGIV infection but upregulated by C. irritans infection and immunization.
Conclusions:
- Grouper c-Raf (Ecc-Raf) is conserved among vertebrates and primarily expressed in immune tissues.
- The Raf-MEK-ERK signaling cascade is dynamically modulated during viral and parasitic infections in teleost fish.
- Ecc-Raf plays a role in the fish immune response, suggesting its involvement in pathogen defense mechanisms.
