In vitro investigation of the interactions between Raf kinases and pro-survival BAG-1 in breast cancer cells


Türk M., Tatlı Ö. , Çebi E., Şahin Z., Dinler Doğanay G.

7th International Congress of the Molecular Biology Association of Turkey, İstanbul, Türkiye, 27 - 29 Eylül 2019, ss.51

  • Basıldığı Şehir: İstanbul
  • Basıldığı Ülke: Türkiye
  • Sayfa Sayısı: ss.51

Özet

Background/aim: Bcl-2-associated athanogene 1 (BAG-1) is a pro-survival molecule, which protect the cells from apoptotic stimuli and is frequently upregulated in human malignancies. C-Raf and B-Raf serine/threonine-protein kinases, known interaction partners of BAG-1, are involved in Ras/Raf/MEK/ERK pathway, which governs proliferation and cell survival. The interaction of BAG-1 with two Raf isoforms, C-Raf and B-Raf sets a critical role in the prolonged survival of cancer cells. However, the exact role of BAG-1 in phosphorylation-dependent Raf activation cycle remains unknown. Herein, we aim to probe phosphorylation-dependency of the interactions between Raf kinases and pro-survival BAG-1 in breast carcinoma.

 

Materials and methods: We cloned His6-tagged BAG-1S, BAG-1L, C-Raf and B-Raf genes into mammalian expression vector. To mimic phosphorylated and active state of Raf kinases, S446D and S338D substitutions were introduced into B-Raf and C-Raf, respectively. MCF-7 cells were transiently transfected with His6-tagged constructs and MTT assay was performed to screen the effect of overexpressed proteins in the viability of MCF-7 cells. After total protein was isolated from transfectants, cell lysates were used in His-pull down assays. Non-mutated Raf kinases were subjected to phosphatase treatment to fully inactivate kinases.

 

Results: Herein, to investigate phosphorylation-dependency of BAG-1/Raf kinase interactions, an aspartate substitution closely mimicking phosphorylated serine was introduced at residues S-338 and S-446 for C-Raf and B-Raf, respectively. The interactions of active/inactive Raf kinases with small and large isoforms of BAG-1 were investigated through His-pull down assays. His-pull down experiments showed that while only BAG-1L interacts with fully inactivated C-Raf and B-Raf, both BAG-1L and BAG-1S isoforms interact with phosphomimetic active forms of the kinases.

 

Conclusion: The isoform-specific interaction of BAG-1 with activated and inactivated forms of Raf kinases will contribute to understanding the role of BAG-1 in Ras/Raf/MEK/ERK pathway with critical prescription in the prolonged survival of cancer cells.

 

Key words: MAPK pathway, BAG-1, C-Raf, B-Raf

 

Acknowledgement and/or disclaimers, if any

This work is supported by TUBITAK research funds (Project No: 117Z848)