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@ARTICLE{Rohrer:276867,
      author       = {L. Rohrer and C. Spohr and C. Beha and R. Griffin and S.
                      Braun and S. Halbach$^*$ and T. Brummer$^*$},
      title        = {{A}nalysis of {RAS} and drug induced homo- and
                      heterodimerization of {RAF} and {KSR}1 proteins in living
                      cells using split {N}anoluc luciferase.},
      journal      = {Cell communication and signaling},
      volume       = {21},
      number       = {1},
      issn         = {1478-811X},
      address      = {London},
      publisher    = {Biomed Central},
      reportid     = {DKFZ-2023-01179},
      pages        = {136},
      year         = {2023},
      abstract     = {The dimerization of RAF kinases represents a key event in
                      their activation cycle and in RAS/ERK pathway activation.
                      Genetic, biochemical and structural approaches provided key
                      insights into this process defining RAF signaling output and
                      the clinical efficacy of RAF inhibitors (RAFi). However,
                      methods reporting the dynamics of RAF dimerization in living
                      cells and in real time are still in their infancy. Recently,
                      split luciferase systems have been developed for the
                      detection of protein-protein-interactions (PPIs), incl.
                      proof-of-concept studies demonstrating the
                      heterodimerization of the BRAF and RAF1 isoforms. Due to
                      their small size, the Nanoluc luciferase moieties LgBiT and
                      SmBiT, which reconstitute a light emitting holoenzyme upon
                      fusion partner promoted interaction, appear as well-suited
                      to study RAF dimerization. Here, we provide an extensive
                      analysis of the suitability of the Nanoluc system to study
                      the homo- and heterodimerization of BRAF, RAF1 and the
                      related KSR1 pseudokinase. We show that KRASG12V promotes
                      the homo- and heterodimerization of BRAF, while considerable
                      KSR1 homo- and KSR1/BRAF heterodimerization already occurs
                      in the absence of this active GTPase and requires a salt
                      bridge between the CC-SAM domain of KSR1 and the
                      BRAF-specific region. We demonstrate that loss-of-function
                      mutations impairing key steps of the RAF activation cycle
                      can be used as calibrators to gauge the dynamics of
                      heterodimerization. This approach identified the RAS-binding
                      domains and the C-terminal 14-3-3 binding motifs as
                      particularly critical for the reconstitution of RAF mediated
                      LgBiT/SmBiT reconstitution, while the dimer interface was
                      less important for dimerization but essential for downstream
                      signaling. We show for the first time that BRAFV600E, the
                      most common BRAF oncoprotein whose dimerization status is
                      controversially portrayed in the literature, forms
                      homodimers in living cells more efficiently than its
                      wildtype counterpart. Of note, Nanoluc activity
                      reconstituted by BRAFV600E homodimers is highly sensitive to
                      the paradox-breaking RAFi PLX8394, indicating a dynamic and
                      specific PPI. We report the effects of eleven ERK pathway
                      inhibitors on RAF dimerization, incl. third-generation
                      compounds that are less-defined in terms of their dimer
                      promoting abilities. We identify Naporafenib as a potent and
                      long-lasting dimerizer and show that the split Nanoluc
                      approach discriminates between type I, I1/2 and II RAFi.
                      Video Abstract.},
      keywords     = {BRAF (Other) / Belvarafenib (Other) / KRAS (Other) / KSR1
                      (Other) / LgBiT (Other) / MAPK pathway (Other) / NanoBit
                      Oplophorus luciferase (Other) / RAF1 (Other) / SmBiT (Other)
                      / Sorafenib (Other)},
      cin          = {FR01},
      ddc          = {570},
      cid          = {I:(DE-He78)FR01-20160331},
      pnm          = {899 - ohne Topic (POF4-899)},
      pid          = {G:(DE-HGF)POF4-899},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:37316874},
      doi          = {10.1186/s12964-023-01146-9},
      url          = {https://inrepo02.dkfz.de/record/276867},
}