8. International Drug Chemistry Conference, Antalya, Türkiye, 27 Şubat - 01 Mart 2020, ss.247
Bag-1 protein, which protects cells from apoptotic stimuli and is often over-expressed in human cancer cells, is an important member of the BAG family. The known interaction partners of Bag-1, C-Raf and B-Raf serine/threonine-protein kinases are located on the MAPK pathway, regulating proliferation and cell survival. The interaction of Bag-1 with C-Raf and B-Raf plays a critical role in long-term survival of cancer cells. However, the exact role of Bag-1 in the Raf activation cycle due to phosphorylation is unknown. In this study, the interactions between Raf kinases and pro-survival Bag-1 protein in breast cancer cells were investigated in the Raf activation cycle. In our study, to investigate the dependence of Bag-1/Raf kinase interactions on phosphorylation, the interactions of active or inactive Raf kinases with Bag-1S and Bag-1L isoforms were investigated by Hisprecipitation experiments. First, the His6x-labeled Bag-1S, Bag-1L, C-Raf and B-Raf cDNA sequences were cloned into the mammalian expression vectors and MCF-7 breast cancer cells were transiently transfected by the vectors. Bag-1/Raf interactions were tested by performing His-precipitation experiments with protein lysate from transfected cells. The amino acids Ser338 and Ser446, which served in the first step of the activation cycle of C-Raf and B-Raf, have been replaced by aspartate amino acid through site-directed mutagenesis that mimics phosphorylation. Inactive Raf kinase forms were created by converting the same serine amino acids into alanines to inhibit phosphorylation. His-precipitation experiments showed that Bag-1L interacts with the phosphomimetically active forms of Bag-1S predominantly while interacting with fully inactive C-Raf and B-Raf. Immunocytochemistry experiments with Bag-1S confirmed the dependence of Bag-1/Raf kinase interaction on phosphorylation. The active and inactive Raf kinase forms of Bag-1 and phosphorylation-dependent isoform-specific interaction suggest that Bag-1 protein stabilizes its active forms rather than activating Raf kinases. Clarifying the role of Bag-1 in the Raf activation cycle due to phosphorylation will contribute to target the MAPK pathway that is involved in long-term survival of cancer cells.