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Regulation of RAF protein kinases in ERK signalling

Key Points

  • The three cellular RAF family kinases (ARAF, BRAF and CRAF) were identified 30 years ago on the basis of their homology to the viral oncoprotein v-raf. They were then found to be core members of the RAS–ERK pathway.

  • Kinase suppressor of RAS (KSR) pseudokinases were identified in 1995 as modulators of the RAS–ERK pathway. They are closely related to RAF proteins and were originally defined as scaffolding proteins.

  • In quiescent cells, inactive RAF is maintained in the cytosol in an auto-inhibited state stabilized by 14-3-3 proteins. Upon cell stimulation, RAF is recruited to the plasma membrane by activated RAS. This relieves RAF auto-inhibition, which is concomitant with its phosphorylation on various activating residues.

  • The catalytic activation of RAF also relies on an allosteric mechanism implemented by the formation of homotypic or heterotypic kinase domain dimers. KSR proteins also dimerize with RAF proteins and promote their catalytic activation.

  • BRAF oncogenic mutations are found at high frequency in diverse tumour types. BRAF and CRAF mutant alleles have also been detected in developmental syndromes termed RASopathies.

  • RAF inhibitors have been developed and exhibit significant clinical efficacy against metastatic melanoma driven by the Val600Glu BRAF mutation. However, relapse invariably occurs within a year. Moreover, these inhibitors paradoxically induce ERK signalling in activated RAS mutant cells by their ability to promote RAF dimerization. Paradox-breaking RAF inhibitors are currently being developed.

Abstract

RAF family kinases were among the first oncoproteins to be described more than 30 years ago. They primarily act as signalling relays downstream of RAS, and their close ties to cancer have fuelled a large number of studies. However, we still lack a systems-level understanding of their regulation and mode of action. The recent discovery that the catalytic activity of RAF depends on an allosteric mechanism driven by kinase domain dimerization is providing a vital new piece of information towards a comprehensive model of RAF function. The fact that current RAF inhibitors unexpectedly induce ERK signalling by stimulating RAF dimerization also calls for a deeper structural characterization of this family of kinases.

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Figure 1: Three decades of research on RAF family proteins.
Figure 2: RAF and KSR proteins within the RAS–ERK pathway.
Figure 3: The RAF activation cycle.
Figure 4: Regulation of the RAF kinase domain by dimerization.
Figure 5: Regulation of RAF and KSR proteins by phosphorylation.
Figure 6: Prevalence of RAF mutations in human diseases.

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Acknowledgements

The authors apologize to their colleagues whose work could not be cited owing to space restrictions. They thank their colleagues from M.T.'s laboratory and from the laboratory of F. Sicheri at the Lunenfeld–Tanenbaum Research Institute for discussions. H.L. was the recipient of a Canadian Institutes of Health Research (CIHR) Banting postdoctoral fellowship. Relevant research in the laboratory of M.T. is sponsored by an Impact Grant from the Canadian Cancer Society Research Institute (702319) and by operating funds from the CIHR (MOP119443).

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Supplementary information

Supplementary information S1 (figure)

RAF (A) and KSR (B) proteins undergo multiple phosphorylation events. (PDF 254 kb)

Supplementary information S2 (table)

Knockout and transgenic mouse models available for RAF family proteins. (PDF 171 kb)

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Glossary

RAS GTP-loading

The process of loading GTP onto RAS to make it active, which is mediated by guanine nucleotide exchange factors (GEFs). Active GTP-bound RAS reverts to the basal, inactive GDP-bound state by the action of GTPase-activating proteins (GAPs).

RASopathies

A group of developmental diseases — including neurofibromatosis type 1, Legius syndrome, Costello syndrome, cardio-facio-cutaneous syndrome, LEOPARD syndrome and Noonan syndrome — that share a common set of clinical manifestations. They are characterized by germline gain-of-function mutations in various genes encoding components of the RAS–ERK pathway. So far, only cardio-facio-cutaneous, LEOPARD and Noonan syndromes have been associated with mutations in CRAF or BRAF.

Prenylation

Post-translational addition of a lipidic polyisoprenyl group (either a 15-carbon long farnesyl or a 20-carbon long geranylgeranyl moiety) on the Cys residue of a CAAX box. Prenylation of proteins facilitates their association with cellular membranes.

CAAX box

Sequence motif found at the carboxyl terminal end of most small GTPases and also in other types of proteins. C stands for Cys, and A for an aliphatic residue; X represents any amino acid. A Leu residue at the X position dictates geranylgeranylation, whereas other residues specify farnesylation.

Noonan syndrome

A genetic disorder that prevents normal development in various parts of the body; clinical presentation includes unusual facial characteristics, short stature and heart defects. Mutations in Tyr protein phosphatase non-receptor type 11 (PTPN11), CBL, SOS1, RASA2, SHOC2, KRAS, NRAS, RIT1, CRAF and MEK1 have been linked to this condition.

LEOPARD syndrome

A genetic disorder, the mnemonic name of which stands for lentigines (skin lesions), electrocardiographic conduction abnormalities (heart malfunction), ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retarded growth and deafness. It is also sometimes referred to as Noonan syndrome with multiple lentigines. Mutations in PTPN11, BRAF, CRAF and MEK1 have been associated with this disease.

Cardio-facio-cutaneous syndrome

A genetic disorder, the clinical presentation of which includes distinctive facial appearance, unusually sparse, brittle, curly scalp hair, a range of skin abnormalities, various heart malformations, delayed growth and foot abnormalities. Mutations in KRAS, BRAF, MEK1 and MEK2 have been associated with this disease.

COSMIC database

(Catalogue of somatic mutations in cancer database). A publicly available sequence repository and associated information that reports somatically acquired mutations found in diverse human cancers.

Indels

Insertion or deletion of nucleotides within genes.

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Lavoie, H., Therrien, M. Regulation of RAF protein kinases in ERK signalling. Nat Rev Mol Cell Biol 16, 281–298 (2015). https://doi.org/10.1038/nrm3979

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