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Effects of Raf dimerization and its inhibition on normal and disease-associated Raf signaling.

ABSTRACT: Raf kinases are essential for normal Ras-Raf-MEK-ERK pathway signaling, and activating mutations in components of this pathway are associated with a variety of human cancers, as well as the related developmental disorders Noonan, LEOPARD, and cardiofaciocutaneous syndromes. Although the Raf kinases are known to dimerize during normal and disease-associated Raf signaling, the functional significance of Raf dimerization has not been fully elucidated. Here, using mutational analysis and a peptide inhibitor, we show that dimerization is required for normal Ras-dependent Raf activation and for the biological function of disease-associated Raf mutants with moderate, low, or impaired kinase activity. However, dimerization is not needed for the function of B-Raf mutants with high catalytic activity, such as V600E-B-Raf. Importantly, we find that a dimer interface peptide can effectively block Raf dimerization and inhibit Raf signaling when dimerization is required for Raf function, thus identifying the Raf dimer interface as a therapeutic target.

SUBMITTER: Freeman AK

PROVIDER: S-EPMC3582845 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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The mechanism of Raf activation through dimerization.

Project description:Raf, a threonine/serine kinase in the Raf/MEK/ERK pathway, regulates cell proliferation. Raf's full activation requires dimerization. Aberrant activation through dimerization is an important therapeutic target. Despite its clinical importance, fundamental questions, such as how the side-to-side dimerization promotes the OFF-to-ON transition of Raf's kinase domain and how the fully activated ON-state kinase domain is stabilized in the dimer for Raf signaling, remain unanswered. Herein, we decipher an atomic-level mechanism of Raf activation through dimerization, clarifying this enigma. The mechanism reveals that the replacement of intramolecular π-π stacking by intermolecular π-π stacking at the dimer interface releases the structural constraint of the αC-helix, promoting the OFF-to-ON transition. During the transition, the inhibitory hydrophobic interactions were disrupted, making the phosphorylation sites in A-loop approach the HRD motif for cis-autophosphorylation. Once fully activated, the ON-state kinase domain can be stabilized by a newly identified functional N-terminal basic (NtB) motif in the dimer for Raf signaling. This work provides atomic level insight into critical steps in Raf activation and outlines a new venue for drug discovery against Raf dimerization.

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SHOC2 phosphatase-dependent RAF dimerization mediates resistance to MEK inhibition in RAS-mutant cancers.

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Wildtype C-Raf gene dosage and dimerization drive prostate cancer metastasis

Project description:To investigate the function of C-Raf overexpression in driving metastasis, we established C-Raf overexpression lines and C-Raf metastasis derived lines from mice in RWPE-1 and BPH-1 cells.

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(V600E)BRAF is associated with disabled feedback inhibition of RAF-MEK signaling and elevated transcriptional output of the pathway.

Project description:Tumors with mutant BRAF and those with receptor tyrosine kinase (RTK) activation have similar levels of phosphorylated ERK, but only the former depend on ERK signaling for proliferation. The mitogen-activated protein kinase, extracellular signal-regulated kinase kinase (MEK)/ERK-dependent transcriptional output was defined as the genes whose expression changes significantly 8 h after MEK inhibition. In (V600E)BRAF cells, this output is comprised of 52 genes, including transcription factors that regulate transformation and members of the dual specificity phosphatase and Sprouty gene families, feedback inhibitors of ERK signaling. No such genes were identified in RTK tumor cells, suggesting that ERK pathway signaling output is selectively activated in BRAF mutant tumors. We find that RAF signaling is feedback down-regulated in RTK cells, but is insensitive to this feedback in BRAF mutant tumors. Physiologic feedback inhibition of RAF/MEK signaling down-regulates ERK output in RTK cells; evasion of this feedback in mutant BRAF cells is associated with increased transcriptional output and MEK/ERK-dependent transformation.

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Development of Highly Sensitive Biosensors of RAF Dimerization in Cells.

Project description:The BRAF inhibitors dabrafenib and vemurafenib induce remarkable clinical responses in patients with BRAF-mutated melanomas. However, adverse events, including the emergence of secondary tumors and drug resistance, have been reported. Studies have revealed that undesirable RAF dimerization induced by inhibitors promotes these adverse effects. Here, we developed highly sensitive biosensors of RAF dimerization in cells utilizing the split enhanced click beetle luciferase (Emerald Luc, ELuc) complementation technique. We demonstrated that our biosensor system works effectively for high-throughput screens in the microplate format. A comprehensive analysis of commercially available RAF inhibitors performed using this assay system revealed that the inhibitors exhibit various potencies in inducing the dimerization of RAF isoforms, and their dimerization potencies do not always correlate with the RAF enzyme inhibition. This sensitive assay system will become a powerful tool to discover next-generation BRAF inhibitors with safer profiles.

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Inhibition of mutant BRAF splice variant signaling by next-generation, selective RAF inhibitors.

Project description:Vemurafenib and dabrafenib block MEK-ERK1/2 signaling and cause tumor regression in the majority of advanced-stage BRAF(V600E) melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next-generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAF(V600E) melanoma cells without paradoxical effects in wild-type cells. Furthermore, PLX7904 blocks the growth of vemurafenib-resistant BRAF(V600E) cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAF(V600E) splice variant-mediated vemurafenib-resistant cells. We show that paradox-breaker RAF inhibitors potently block MEK-ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720-resistant cells harboring distinct BRAF(V600E) splice variants. These data support the further investigation of paradox-breaker RAF inhibitors as a second-line treatment option for patients failing on vemurafenib or dabrafenib.

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Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization.

Project description:RAF kinases have a prominent role in cancer. Their mode of activation is complex but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and, as a result, undesirably stimulate RAS/ERK pathway-mediated cell growth. The mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe bioluminescence resonance energy transfer-based biosensors for the extended RAF family that enable the detection of RAF dimerization in living cells. Notably, we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization and for probing structural determinants of RAF dimerization in vivo. Our findings, which seem generalizable to other kinase families allosterically regulated by kinase domain dimerization, suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain.

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