Study Of The Molecular Mechanism For Phosphorylation-dependent Regulation Of Bcl-2Family Signaling Network By Small-molecule BH3Mimetics

The Bcl-2family proteins are the "switch" of endogenous apoptotic pathways and are the regulator of the balance between cell life and death. The members of this family form a multi-level, redundancy, connectivity and compensatory molecular networks by protein-protein interactions (PPI) through BH3domains. Several studies suggested that multiple Bcl-2family members can be phosphorylated by kinase pathways which is upstream of Bcl-2family PPI network. This kind of modification can change the characteristics of the Bcl-2family PPI network. Phosphorylation sites of Bcl-2family members have been elucidated. However, there is no effective research and persuasive conclusions to the influence of phosphorylation on Bcl-2family PPI network characteristics and the precise regulation of apoptosis.S is a small molecule BH3mimetic with independent intellectual property rights. It was developed by out group. Its specificity is higher than Gossypol, and is considerable with ABT-737which is undergoing clinical trial. S1induces apoptosis dependent on Bcl-2family network. Importantly, S1can simultaneously antagonize the two anti-apoptotic Bcl-2family network key nodes, Bcl-2and Mcl-1. But ABT-737can not antagonize Mcl-1. In this study, S1was used as a molecular tool to compare with Gossypol and ABT-737to study the anti-apoptotic function and phosphorylation signaling pathways of Bcl-2and Mcl-1phosphorylation in the Bcl-2family PPI network by using various methods including the induction of acquired resistance, Western blot, co-immunoprecipitations, exogenous transfections, gene silencing, quantitative PCR and isothermal titration calorimetry (ITC). The results revealed both the new features of the phosphorylated Bcl-2family of PPI networks and a new molecular mechanism of dynamic regulation.We showed novel mechanistic data demonstrating that BH3mimetics including S1and ABT-737induced endoplasmic reticulum (ER) stress in small cell lung cancer (SCLC) and then activated MAPK/ERK pathway, which resulted in Bcl-2transcriptional up-regulation and phosphorylation (S70). BH3mimetics stimulated ER stress then induced MAPK/ERK cascades with respect to Bcl-2transcriptional up-regulation and phosphorylation. Excess phosphorylated Bcl-2(S70)(pBcl-2(S70)) lead to cell survival by promoting ERK1/2activation and sequestrating more pro-apoptotic members, which BH3mimetics cannot release. These results provide the molecular evidence that ERK1/2mediate the crosstalk between Bcl-2family networks and ER stress-induced the activation of MEK/ERK pathway in SCLC cells. The dual essential function of Bcl-2phosphorylation (S70) in the regulatory of Bcl-2family network homeostasis is illustrated. The phosphorylation of Bcl-2(S70) resulted in enhanced primary and acquired resistance in SCLC not only by sequestrating more pro-apoptotic proteins and remodeling Bcl-2family PPI network, but also by positive feedback activating ERK1/2to further amplify the anti-apoptotic signaling.BH3mimetic S1was used as a tool for further confirming whether the anti-apoptotic function of Bcl-2can be enhanced by phosphorylation in both primary leukemia cells and cell lines. Bcl-2cannot interact to Bak. But in this study, pBcl-2(S70)/Bak dimers was found for the first time. Further studies showed that, pBcl-2(S70)/Bak was the key PPI by which Bcl-2phosphorylation (S70) maintain homeostasis of Bcl-2family network. pBcl-2(S70)/Bak reshaped the Bcl-2family PPI network features:by the stimulation of S1, Bak and other pro-apoptotic proteins can shuttle among the network nodes of Bcl-2, Mcl-1and pBcl-2(S70), while pBcl-2(S70) was the key node which S1can not antagonize. This new mode of action clarify the molecular mechanism of pBcl-2(S70)’s dynamic regulation of Bcl-2family network. Large data statistical analysis showed that pBcl-2(S70)/(Bcl-2+Mcl-1) ratio was the best predictive index for S1sensitivity in both primary and established cell lines. This reflects the anti-apoptotic function of Bcl-2family network can be enhanced by phosphorylation, and adjust the balance of the network to anti-apoptotic enhancement.S1was used to compare with ABT-737, Gossypol. We provided the evidence that Mcl-1phosphorylation at T163contributed to the resistance of melanoma cells to BH3mimetics. The results of our research showed that Mcl-1was phosphorylated by both ERK1/2and JNK pathways. A novel mechanism of the enhanced anti-apoptotic function of phosphorylated Mcl-1(T163)(pMcl-1(T163)) was revealed:pMcl-1(T163) antagonized the known BH3mimetics by sequestering pro-apoptotic Bim which was released from Bcl-2. pMcl-1(T163) was a "substituted" the network node, which displayed enhanced anti-apoptotic role in maintaining the homeostasis of the Bcl-2family network. These data reveal the promising therapeutic potential of targeting pMcl-1(T163) to treat melanoma. Furthermore, by using cytotoxicity test and ITC, a BH3mimetic Compound6, was identified to be the first small molecule to that induce endogenous apoptosis in melanoma cells by directly binding pMcl-1(T163) and disrupting the heterodimers of these proteins. Compound6can induce Mcl-1degradation and its induction of apoptosis is independent of Noxa.