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Unveiling the Domain-Specific and RAS Isoform-Specific Details of BRAF Regulation
BRAF is a key member in the MAPK signaling pathway essential for cell growth, proliferation, and differentiation. Dysregulation or mutation of BRAF is often the underlying cause of various types of cancer. RAS, a small GTPase protein that acts upstream of BRAF, has been identified as a driver of up...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10168249/ https://www.ncbi.nlm.nih.gov/pubmed/37163002 http://dx.doi.org/10.1101/2023.04.24.538112 |
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author | Trebino, Tarah Markusic, Borna Nan, Haihan Banerjee, Shrhea Wang, Zhihong |
author_facet | Trebino, Tarah Markusic, Borna Nan, Haihan Banerjee, Shrhea Wang, Zhihong |
author_sort | Trebino, Tarah |
collection | PubMed |
description | BRAF is a key member in the MAPK signaling pathway essential for cell growth, proliferation, and differentiation. Dysregulation or mutation of BRAF is often the underlying cause of various types of cancer. RAS, a small GTPase protein that acts upstream of BRAF, has been identified as a driver of up to one-third of all cancers. When BRAF interacts with RAS via the RAS binding domain (RBD) and membrane recruitment, BRAF undergoes a conformational change from an inactive, autoinhibited monomer to an active dimer and subsequently phosphorylates MEK to propagate the signal. Despite the central role of BRAF in cellular signaling, the exact order and magnitude of its activation steps has yet to be confirmed experimentally. By studying the inter- and intramolecular interactions of BRAF, we unveil the domain-specific and isoform-specific details of BRAF regulation. We employed pulldown assays, open surface plasmon resonance (OpenSPR), and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the roles of the regulatory regions in BRAF activation and autoinhibition. Our results demonstrate that the BRAF specific region (BSR) and cysteine rich domain (CRD) play a crucial role in regulating the activity of BRAF. Moreover, we quantified the autoinhibitory binding affinities between the N-terminal domains and the kinase domain (KD) of BRAF and revealed the individual roles of the BRAF regulatory domains. Additionally, our findings provide evidence that the BSR negatively regulates BRAF activation in a RAS isoform-specific manner. Our findings also indicate that oncogenic BRAF-KD(D594G) mutant has a lower affinity for the regulatory domains, implicating that pathogenic BRAF acts through decreased propensity for autoinhibition. Collectively, our study provides valuable insights into the activation mechanism of BRAF kinase and may help to guide the development of new therapeutic strategies for cancer treatment. |
format | Online Article Text |
id | pubmed-10168249 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Cold Spring Harbor Laboratory |
record_format | MEDLINE/PubMed |
spelling | pubmed-101682492023-05-10 Unveiling the Domain-Specific and RAS Isoform-Specific Details of BRAF Regulation Trebino, Tarah Markusic, Borna Nan, Haihan Banerjee, Shrhea Wang, Zhihong bioRxiv Article BRAF is a key member in the MAPK signaling pathway essential for cell growth, proliferation, and differentiation. Dysregulation or mutation of BRAF is often the underlying cause of various types of cancer. RAS, a small GTPase protein that acts upstream of BRAF, has been identified as a driver of up to one-third of all cancers. When BRAF interacts with RAS via the RAS binding domain (RBD) and membrane recruitment, BRAF undergoes a conformational change from an inactive, autoinhibited monomer to an active dimer and subsequently phosphorylates MEK to propagate the signal. Despite the central role of BRAF in cellular signaling, the exact order and magnitude of its activation steps has yet to be confirmed experimentally. By studying the inter- and intramolecular interactions of BRAF, we unveil the domain-specific and isoform-specific details of BRAF regulation. We employed pulldown assays, open surface plasmon resonance (OpenSPR), and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the roles of the regulatory regions in BRAF activation and autoinhibition. Our results demonstrate that the BRAF specific region (BSR) and cysteine rich domain (CRD) play a crucial role in regulating the activity of BRAF. Moreover, we quantified the autoinhibitory binding affinities between the N-terminal domains and the kinase domain (KD) of BRAF and revealed the individual roles of the BRAF regulatory domains. Additionally, our findings provide evidence that the BSR negatively regulates BRAF activation in a RAS isoform-specific manner. Our findings also indicate that oncogenic BRAF-KD(D594G) mutant has a lower affinity for the regulatory domains, implicating that pathogenic BRAF acts through decreased propensity for autoinhibition. Collectively, our study provides valuable insights into the activation mechanism of BRAF kinase and may help to guide the development of new therapeutic strategies for cancer treatment. Cold Spring Harbor Laboratory 2023-09-27 /pmc/articles/PMC10168249/ /pubmed/37163002 http://dx.doi.org/10.1101/2023.04.24.538112 Text en https://creativecommons.org/licenses/by/4.0/This work is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) , which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. |
spellingShingle | Article Trebino, Tarah Markusic, Borna Nan, Haihan Banerjee, Shrhea Wang, Zhihong Unveiling the Domain-Specific and RAS Isoform-Specific Details of BRAF Regulation |
title | Unveiling the Domain-Specific and RAS Isoform-Specific Details of BRAF Regulation |
title_full | Unveiling the Domain-Specific and RAS Isoform-Specific Details of BRAF Regulation |
title_fullStr | Unveiling the Domain-Specific and RAS Isoform-Specific Details of BRAF Regulation |
title_full_unstemmed | Unveiling the Domain-Specific and RAS Isoform-Specific Details of BRAF Regulation |
title_short | Unveiling the Domain-Specific and RAS Isoform-Specific Details of BRAF Regulation |
title_sort | unveiling the domain-specific and ras isoform-specific details of braf regulation |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10168249/ https://www.ncbi.nlm.nih.gov/pubmed/37163002 http://dx.doi.org/10.1101/2023.04.24.538112 |
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