RESUMO
Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved "S259" inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Quinases raf/química , Quinases raf/metabolismo , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Edição de Genes , Técnicas de Inativação de Genes , Humanos , Fosforilação , Multimerização Proteica , Proteínas ras/metabolismoRESUMO
Dephosphorylation of the inhibitory "S259" site on RAF kinases (S259 on CRAF, S365 on BRAF) plays a key role in RAF activation. The MRAS GTPase, a close relative of RAS oncoproteins, interacts with SHOC2 and protein phosphatase 1 (PP1) to form a heterotrimeric holoenzyme that dephosphorylates this S259 RAF site. MRAS and SHOC2 function as PP1 regulatory subunits providing the complex with striking specificity against RAF. MRAS also functions as a targeting subunit as membrane localization is required for efficient RAF dephosphorylation and ERK pathway regulation in cells. SHOC2's predicted structure shows remarkable similarities to the A subunit of PP2A, suggesting a case of convergent structural evolution with the PP2A heterotrimer. We have identified multiple regions in SHOC2 involved in complex formation as well as residues in MRAS switch I and the interswitch region that help account for MRAS's unique effector specificity for SHOC2-PP1. MRAS, SHOC2, and PPP1CB are mutated in Noonan syndrome, and we show that syndromic mutations invariably promote complex formation with each other, but not necessarily with other interactors. Thus, Noonan syndrome in individuals with SHOC2, MRAS, or PPPC1B mutations is likely driven at the biochemical level by enhanced ternary complex formation and highlights the crucial role of this phosphatase holoenzyme in RAF S259 dephosphorylation, ERK pathway dynamics, and normal human development.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Síndrome de Noonan/metabolismo , Proteína Fosfatase 1/metabolismo , Quinases raf/metabolismo , Proteínas ras/metabolismo , Proteínas de Transporte , Linhagem Celular , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases , Modelos Moleculares , Mutação , Síndrome de Noonan/genética , Fosforilação , Proteína Fosfatase 1/genética , Alinhamento de Sequência , Proteínas ras/genéticaRESUMO
The MAP kinase-interacting kinases (Mnk1 and Mnk2) are activated by ERK and are best known for phosphorylating the translation initiation factor eIF4E. Genetic knockout of the Mnks impaired the migration of embryonic fibroblasts both in two-dimensional wound-healing experiments and in three-dimensional migration assays. Furthermore, a novel and selective Mnk inhibitor, Mnk-I1, which potently blocks eIF4E phosphorylation, blocked the migration of fibroblasts and cancer cells, without exerting 'off-target' effects on other signalling pathways such as Erk. Mnk-I1 or genetic knockout of the Mnks decreased the expression of vimentin, a marker of mesenchymal cells, without affecting vimentin mRNA levels. Vimentin protein levels were much lower in Mnk1/2-knockout cells than in controls, although mRNA levels were similar. Our data suggest that the Mnks regulate the translation of the vimentin mRNA and the stability of the vimentin protein. Inhibition or genetic knockout of the Mnks increased the binding of eIF4E to the cytoplasmic FMRP-interacting protein 1 (CYFIP1), which binds the fragile-X mental retardation protein, FMRP, a translational repressor. Since FMRP binds mRNAs for proteins involved in metastasis, the Mnk-dependent release of CYFIP1 from eIF4E is expected to release the repression of translation of FMRP-bound mRNAs, potentially providing a molecular mechanism for the control of cell migration by the Mnks. As Mnk1/2 are not essential for viability, inhibition of the Mnks may be a useful approach to tackling cancer metastasis, a key process contributing to mortality in cancer patients.
Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Vimentina/metabolismo , Cicatrização , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos/citologia , Fator de Iniciação 4E em Eucariotos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Proteínas Mutantes/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/genética , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Estabilidade Proteica/efeitos dos fármacos , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vimentina/química , Vimentina/genética , Cicatrização/efeitos dos fármacosRESUMO
Targeted inhibition of the ERK-MAPK pathway, upregulated in a majority of human cancers, has been hindered in the clinic by drug resistance and toxicity. The MRAS-SHOC2-PP1 (SHOC2 phosphatase) complex plays a key role in RAF-ERK pathway activation by dephosphorylating a critical inhibitory site on RAF kinases. Here we show that genetic inhibition of SHOC2 suppresses tumorigenic growth in a subset of KRAS-mutant NSCLC cell lines and prominently inhibits tumour development in autochthonous murine KRAS-driven lung cancer models. On the other hand, systemic SHOC2 ablation in adult mice is relatively well tolerated. Furthermore, we show that SHOC2 deletion selectively sensitizes KRAS- and EGFR-mutant NSCLC cells to MEK inhibitors. Mechanistically, SHOC2 deletion prevents MEKi-induced RAF dimerization, leading to more potent and durable ERK pathway suppression that promotes BIM-dependent apoptosis. These results present a rationale for the generation of SHOC2 phosphatase targeted therapies, both as a monotherapy and to widen the therapeutic index of MEK inhibitors.