The Regulation Of RACK1 In JNK Signaling Pathway And The Molecular Mechanism In Human Hepatocellular Carcinoma

BackgroudJNK (c-Jun N-terminal protein kinase; also known as SAPK) is a member of the MAPK (mitogen-activated protein kinase) superfamily.?JNK is activated by a variety of extracellular stimuli, from the physical stresses, such as UV and osmotic shock, to proinflammatory cytokines like interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α). JNK is activated by a variety of extracellular stimuli,from stresses,Growth factor,proinflammatorycytokines.Extensive studies revealed an essential protumorigenic role of constitutively activated JNK in hepatocellular carcinoma (HCC). RACK1(the receptor of activated C kinase 1), is a 36-kDa protein containing seven internal Trp-Asp 40 (WD40) repeats.The WD repeats of RACK1 can be predicted to form a seven-bladed propeller structure,Therefore, RACK1 has multiple docking sites and could be implicated in the regulation of multiple signaling pathways.A recent study showed that RACK1 expression has been shown to be upregulated both in human HCC and the adjacent non-tumor liver as compared with control liver.It is of importance to investigate the possible reciprocal regulation between the JNK pathway and RACK1 in HCC.ObjectiveTo investigate the possible reciprocal regulation between the P-JNK and RACK1 in HCC; To confirm whether RACK1 and MKK7 could engage in a direct specific interaction;To map the interaction regions of RACK1 and MKK7; To investigate how the interaction between RACK1 and MKK7 affects hepatoma cell growth; To analyze the origin of the elevated expression of RACK1 protein. MethodsP-JNK and RACK1 expression was analyzed by immunohistochemical staining on a tissue microarray and by Western Blot in human hepatoma cell lines. Whether RACK1 and MKK7 could engage in a specific physical interaction in vivo was confirmed by indirect immunofluorescence microscopy. Whether RACK1 and MKK7 could interact in vivo, even in a more physiological context,was exzamined by immunoprecipitation. Further, the interaction between MKK7 and RACK1 was determined by in vitro GST pull-downand in vitro translation assays. Several RACK1 and MKK7 deletion mutant was constructed though molecular biology technologies. We further analyzed which domain mediates the interaction between RACK1 and MKK7 by coimmunoprecipitation. Molecular simulations of MKK7 and RACK1 were performed according to the reported 3D crystal structures of the proteins. The binding sites between MKK7 and RACK1 were predicted and the key residues were determined. Wether amino acids 269-272 and 275-280 are the specific determinants in RACK1 to bind with MKK7 was determined by coimmunoprecipitation. We investigate the biological effects of interaction between RACK1 and MKK7 by Soft agar assay and in vivo tumor growth in nude mice. The molecular mechanism by which RACK1 could enhance the phosphorylation of MKK7 was further explored. The origin of the elevated expression of RACK1 protein was analyzed by Western Blot.ResultsThe enhanced expression of RACK1 on human HCC tissue microarray positively correlated with the level of P-JNK. The pathological relationship between the activation of JNK and the protein level of RACK1 in human hepatoma cell lines is consistent with the microarray data. RACK1 overexpression led to enhanced MKK7 activation as well as JNK-c-Jun activation in various cell types including human hepatoma cells, whereas RACK1 knock down exhibited opposite effects. RACK1 and MKK7 interact with each other directly. Coimmunoprecipitation analysis revealed that MKK7 C-terminal interacts with RACK1. We further analyzed WD6,WD7 domain in WD5-7 mediates the interaction between RACK1 and MKK7. The binding sites between MKK7 and RACK1 were predicted and the key residues were determined. Amino acids 269-272 and 275-280 are the specific determinants in RACK1 to bind with MKK7. The data of Soft agar assay and in vivo tumor growth in nude mice suggest that the interaction of MKK7 with RACK1 is essential for hepatoma cell growth.ConclutionsThe enhanced expression of RACK1 in human primary HCC positively correlated with the level of P-JNK.The data in vivo and in vitro demonstrated that MKK7 and RACK1 could engage in a direct specific interaction. MKK7 and RACK1 interaction regions and determinants were mapped. Our data suggest that the interaction of MKK7 with RACK1 is essential for hepatoma cell growth. Exposure to inflammatory cytokine TNF-αleads to augmented RACK1 expression via the activation of the JNK pathway.