RESUMO
BACKGROUND: RHEB is a unique member of the RAS superfamily of small GTPases expressed in all tissues and conserved from yeast to humans. Early studies on RHEB indicated a possible RHEB-RAF interaction, but this has not been fully explored. Recent work on cancer genome databases has revealed a reoccurring mutation in RHEB at the Tyr35 position, and a recent study points to the oncogenic potential of this mutant that involves activation of RAF/MEK/ERK signaling. These developments prompted us to reassess the significance of RHEB effect on RAF, and to compare mutant and wild type RHEB. METHODS: To study RHEB-RAF interaction, and the effect of the Y35N mutation on this interaction, we used transfection, immunoprecipitation, and Western blotting techniques. We generated cell lines stably expressing RHEB WT, RHEB Y35N, and KRAS G12V, and monitored cellular transforming properties through cell proliferation, anchorage independent growth, cell cycle analysis, and foci formation assays. RESULTS: We observe a strong interaction between RHEB and BRAF, but not with CRAF. This interaction is dependent on an intact RHEB effector domain and RHEB-GTP loading status. RHEB overexpression decreases RAF activation of the RAF/MEK/ERK pathway and RHEB knockdown results in an increase in RAF/MEK/ERK activation. RHEB Y35N mutation has decreased interaction with BRAF, and RHEB Y35N cells exhibit greater BRAF/CRAF heterodimerization resulting in increased RAF/MEK/ERK signaling. This leads to cancer transformation of RHEB Y35N stably expressing cell lines, similar to KRAS G12 V expressing cell lines. CONCLUSIONS: RHEB interaction with BRAF is crucial for inhibiting RAF/MEK/ERK signaling. The RHEB Y35N mutant sustains RAF/MEK/ERK signaling due to a decreased interaction with BRAF, leading to increased BRAF/CRAF heterodimerization. RHEB Y35N expressing cells undergo cancer transformation due to decreased interaction between RHEB and BRAF resulting in overactive RAF/MEK/ERK signaling. Taken together with the previously established function of RHEB to activate mTORC1 signaling, it appears that RHEB performs a dual function; one is to suppress the RAF/MEK/ERK signaling and the other is to activate mTORC1 signaling.
Assuntos
Carcinogênese/genética , Neoplasias/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Mutação , Neoplasias/patologia , Fosforilação , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis.
Assuntos
Aspartato Carbamoiltransferase/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Di-Hidro-Orotase/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neuropeptídeos/metabolismo , Nucleosídeos de Pirimidina/biossíntese , Proteínas ras/metabolismo , Animais , Proliferação de Células/genética , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Complexos Multiproteicos/metabolismo , Neuropeptídeos/genética , Ligação Proteica , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Serina-Treonina Quinases TOR/metabolismo , Proteínas ras/genéticaRESUMO
In this review we highlight recent progress in the study of Rheb family GTPases. Structural studies using X-ray crystallography and NMR have given us insight into unique features of this GTPase. Combined with mutagenesis studies, these works have expanded our understanding of residues that affect Rheb GTP/GDP bound ratios, effector protein interactions, and stimulation of mTORC1 signaling. Analysis of cancer genome databases has revealed that several human carcinomas contain activating mutations of the protein. Rheb's role in activating mTORC1 signaling at the lysosome in response to stimuli has been further elucidated. Rheb has also been suggested to play roles in other cellular pathways including mitophagy and peroxisomal ROS response. A number of studies in mice have demonstrated the importance of Rheb in development, as well as in a variety of functions including cardiac protection and myelination. We conclude with a discussion of future prospects in the study of Rheb family GTPases.
