| Background: MAPK/MAK/MRK Overlapping Kinase(MOK)belongs to MAP kinase superfamily,a class of serine/threonine protein kinases that are widely present in mammalian cells.The class of kinases are involved in synergistic signaling and shared by various cellular immune and different receptor-mediated signaling pathways.Caspase-3,as the final executor of apoptosis,plays an important role in caspase-mediated apoptotic signaling pathway.In caspases signalling pathway,caspases-1,4,5,11,12,13,14 are known as inflammatory caspases and are involved in the maturation of pro-inflammatory factors.Caspases-2,8,9,10 are located upstream of the cascade reaction and can initiate downstream caspases protein kinases.The caspases-3,6,and 7 are located downstream of the cascade reaction and play a role in performing the shearing of cellular structural proteins that directly cause apoptosis.Caspase-3 is a major effector of caspases and a key performer of apoptosis.In response to an apoptotic signal,inactivated caspase-3 is activated to trigger apoptosis,and therefore caspase-3 is becoming an important indicator for detecting apoptosis.In this paper,we investigated the regulation of apoptosis by the caspase signaling pathway mediated by MOK,a new member of MAP kinase superfamily.Methods: c DNA sequences of MOK and caspase-3(designated as CpMOK and CpCaspase-3)were cloned by RACE PCR.The expression of two genes was examined by real-time fluorescence quantitative PCR in seven tissues,including hepatopancreas,gill,kidney,mantle,adductor muscle,hemocytes and heart of healthy Cristaria plicata,and in hepatopancreas,gill and kidney after stimulation by lipopolysaccharide(LPS),peptidoglycan(PGN),poly I : C and Aeromonas hydrophila.The expression of CpMOK kinase at various time periods was detected during trauma repair by establishing a trauma repair model.The RNA interference of CpMOK was performed on the C.plicata,and its effects on the caspase signaling pathway cascade response and apoptosis were observed by q PCR,ELISA,Wester Blot and Tunel.The CpMOK and CpCaspase-3 genes were expressed using prokaryotic expression technology to obtain recombinant proteins of CpMOK and CpCaspase-3,and the in vitro binding of the proteins was analyzed.Subcellular localization and analysis of nucleoplasmic localization of recombinant CpMOK.Results: 1.The full-length c DNA sequence of CpMOK was 2290 bp,5’ UTR was 92 bp;3’ UTR was 853 bp;the open reading frame(ORF)had a base length of1413 bp and consisted of 470 amino acids.It contained an S＿TKC structural domain(23-304 aa).The theoretical molecular weight of the protein was 54.31 k Da with an isoelectric point of 9.51.The sequence of the cloned CpCaspase-3 gene was2425 bp in length and 969 bp in ORF,encoding a total of 322 amino acids.It contained a CASc structural domain and the deduced protein possessed a molecular weight of 36.44 k Da and an isoelectric point of 5.78.2.The m RNAs of CpMOK and CpCaspase-3 was expressed in hepatopancreas,gill,kidney,mantle,adductor muscle,hemocytes and heart.The expression of CpMOK was the highest in hepatopancreas and the least in hemocytes.CpCaspase-3 was the highest in hepatopancreas,followed by mantle.After stimulation by LPS,PGN,Poly I : C and A.hydrophila,the m RNA expression of CpMOK and CpCaspase-3 showed significant changes,with a general trend of increasing and then decreasing.A trauma model was constructed,where CpMOK was upregulated after the wound of the mantle,reaching the highest expression at 1 d and gradually recovered at 15 d.These suggested that CpMOK was involved in the immune response of C.plicata.3.The double-stranded RNA(ds RNA)of CpMOK was constructed for interference assay,and the m RNA expression of apoptotic gene signal caspase-1,caspase-3,caspase-7,caspase-8 and caspase-9 was up-regulated.Meanwhile,the proteins of caspase-1,-3,-7,-9 and cytochrome C(cyt-c)and tumor necrosis factor(TNF-α)was upregulated to some extent using ELISA assay.The detection of apoptotic cells using the Tunnel method revealed an increase in the number of apoptotic cells after interference.These results implied that knockdown of CpMOK might cause caspase-mediated apoptosis in C.plicata.4.Western-blot experiments showed that the phosphorylation levels of MOK kinase were significantly reduced in AH+ds GFP,AH+ds MOK,and AH groups compared to blank control,especially in the AH+ds MOK group.These results indicated that the knockdown of CpMOK might mediate the regulation of caspase signaling pathway by affecting the phosphorylation level of kinase.5.The p GEX 4T-1-CpMOK and p ET28a(+)-CpCaspase-3 expression plasmids were constructed to induce protein expression,and GST-CpMOK and HIS-CpCaspase-3 proteins were purified at 0.776 mg/m L and 0.453 mg/m L,respectively.In vitro binding result of CpMOK and CpCaspase-3 proteins was false positive.6.Cell localization experiments showed that CpMOK was distributed in the cytoplasm and also in the nucleus. |
