Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling
- 24 October 2013
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 110 (46), 18519-18524
- https://doi.org/10.1073/pnas.1318188110
Abstract
The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them. In the present work, we demonstrate that photoactivated localization microscopy (PALM), in combination with quantitative spatial analysis, provides sufficient resolution to directly visualize protein multimers in cells. Quantitative PALM imaging showed that CRAF exists predominantly as cytoplasmic monomers under resting conditions but forms dimers as well as trimers and tetramers at the cell membrane in the presence of active RAS. In contrast, N-terminal truncated CRAF (CatC) lacking autoinhibitory domains forms constitutive dimers and occasional tetramers in the cytoplasm, whereas a CatC mutant with a disrupted CRAF–CRAF dimer interface does not. Finally, artificially forcing CRAF to the membrane by fusion to a RAS CAAX motif induces multimer formation but activates RAF/MAPK only if the dimer interface is intact. Together, these quantitative results directly confirm the existence of RAF dimers and potentially higher-order multimers and their involvement in cell signaling, and showed that RAF multimer formation can result from multiple mechanisms and is a critical but not sufficient step for RAF activation.Keywords
This publication has 43 references indexed in Scilit:
- RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E)Nature, 2011
- Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysisNature Methods, 2011
- The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and functionNature Reviews Molecular Cell Biology, 2011
- Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulationNature, 2010
- Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanomaNature, 2010
- RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAFNature, 2010
- A dimerization-dependent mechanism drives RAF catalytic activationNature, 2009
- Photoactivatable mCherry for high-resolution two-color fluorescence microscopyNature Methods, 2009
- Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)Nature Methods, 2006
- The RAF proteins take centre stageNature Reviews Molecular Cell Biology, 2004