Molecular Mechanism Of ERK1/2S-nitrosylation In Cancer Cell Apoptosis

Apoptosis is of great significance in tumor cell development and tumor prevention. The design and screening of anti-cancer drugs is one of the major topics in pharmacology. During the past ten years, the delivery of exogenous NO is an attractive therapeutic option in the treatment of cancers. Nitric oxide (NO) is a short lived free radical and plays critical roles in the regulation of neuronal, immune, and cardiovascular systems. The multifaceted actions of nitric oxide can be classified into two categories:cGMP-dependent and protein nitrosylation. S-nitrosylation refers to the conversion of thiol groups to form S-nitrosothiols, which changes the protein conformation. S-nitrosylation regulates protein functions mainly by changing the protein activity, protein interactions and protein nucleocytoplasmic transport. This critical S-nitrosylation can regulate a plethora of biological processes such as cell proliferation, survival and especially apoptosis.Mitogen-activated protein kinase (MAPK) cascades are prominent pathways involved in cell survival, proliferation and apoptosis. In particular, extracellular signal-regulated kinase (ERK) has become an active area of intense research. ERK activity can be regulated by multiple mechanisms such as scaffolds, anchors, localization and inhibitors of ERK signaling. Aberrant upregulation and activation of ERK cascades may often lead to tumor cell development. However, the specific mechanism for the role that ERK plays in tumor cell is still unclear.Our work can be categorized into items as following: 1We tested the effect of nitric oxide donor sodium nitroprusside on MCF-7cell apoptosis and analyzed the impact of concentration of nitric oxide on cell apoptosis. These results showed that high concentration of nitric oxide could effectively induce MCF-7cell apoptosis, accompanied by the activation of caspase-9.2We next examined the effect of NO on the phosphorylation status of ERK. To further investigate the effect of NO on ERK phosphorylation and kinase activity, we transfected MCF-7cells with either wild type (WT) ERK or a constitutive active form (TEY-EED) harboring a point mutation. These results showed nitric oxide could induce cell apoptosis by inhibiting the phosphorylation of ERK.3We proved that ERK undergoes S-nitrosylation by endogenous nitric oxide (NO) by applying the biotin switch technique for the first time and found the potential cysteine residue for nitrosylation.4We constructed ERK1nitrosylation mutant plasmid by site-directed mutagenesis techniques. Next we focused on the relationship between ERK nitrosylation and phosphorylation and investigated the significance of ERK nitrosylation in nitric oxide induced cell apoptosis. Results showed nitric oxide inhibited ERK phosphorylation by ERK nitrosylation modification and thus induced MCF-7cell apoptosis. Further experiments showed that the mechanism of ERK nitrosylation regulation of its activity might be achieved partly by the way of regulation of ERK nucleocytoplasmic transport.5In order to prove the universality of ERK nitrosylation, we tested the level of ERK nitrosylation under other stress inducers such as TNF-a and hydrogen peroxide. The results showed that TNF-a. and hydrogen peroxide could effectively promote the nitrosylation of ERK.6In addition, we detected ERK nitrosylation in Hela cell apoptosis induced by nitric oxide. The results showed that EKR nitrosylation could regulate nitric oxide induced apoptosis in Hela cells.We described that ERK undergoes S-nitrosylation by endogenous nitric oxide (NO). ERK S-nitrosylation inhibits its phosphorylation and triggers apoptotic program as verified by massive apoptosis in fluorescence staining. The proapoptotic effect of NO induced S-nitrosylation is reversed by NO scavenger Haemoglobin (HB). Furthermore, an S-nitrosylation dead ERK mutant C183A also demolishes the proapoptotic potential of NO and favors cell survival. Therefore, Cys183might be a potential S-nitrosylation site in ERK. In addition, the S-nitrosylation effects are not restricted to NO donor SNP as exemplified by TNF-a and hydrogen peroxide treated cells suggesting that S-nitrosylation is a general phenomenon that regulate ERK activity. These findings identify a novel between NO-mediated S-nitrosylation and ERK regulation and provide critical insights into the control of apoptosis and tumor development.