RESUMO
A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts.
Assuntos
GTP Fosfo-Hidrolases , Transdução de Sinais , Animais , Cristalografia por Raios X , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Mamíferos/metabolismo , Nucleotídeos , ProteínasRESUMO
The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.
Assuntos
Proteínas Argonautas/fisiologia , Células-Tronco Embrionárias/metabolismo , Glioma/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Feminino , Regulação da Expressão Gênica , Glioma/genética , Glioma/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Hidrolases/genética , Camundongos , Camundongos Knockout , MicroRNAs/genética , Ligação Proteica , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
Targeted mass spectrometry-based assays typically rely on previously acquired large data sets for peptide target selection. Such repositories are widely available for unlabeled peptides. However, they are less common for isobaric tagged peptides. Here we have assembled two series of six data sets originating from a mouse embryonic fibroblast cell line (NIH/3T3). One series is of peptides derived from a tryptic digest of a whole cell proteome and a second from enriched phosphopeptides. These data sets encompass three labeling approaches (unlabeled, TMT11-labeled, and TMTpro16-labeled) and two data acquisition strategies (ion trap MS2 with and without FAIMS-based gas phase separation). We identified a total of 1â¯509â¯526 peptide-spectrum matches which covered 11â¯482 proteins from the whole cell proteome tryptic digest, and 188â¯849 phosphopeptides from the phosphopeptide enrichment. The data sets were of similar depth, and while overlap across data sets was modest, protein overlap was high, thus reinforcing the comprehensiveness of these data sets. The data also supported FAIMS as a means to increase data set depth. These data sets provide a rich resource of peptides that may be used as starting points for targeted assays. Future data sets may be compiled for any genome-sequenced organism using the technologies and strategies highlighted herein. The data have been deposited in the ProteomeXchange Consortium with data set identifier PXD024298.
Assuntos
Fibroblastos , Proteômica , Animais , Proteínas Reguladoras de Apoptose , Espectrometria de Massas , Camundongos , Fosfopeptídeos , ProteomaRESUMO
Current treatments for KRAS-mutant colorectal cancers (CRCs) are often limited by cellular plasticity and rewiring responses. Here we describe a promising therapeutic strategy that simultaneously targets epigenetic and oncogenic signals. Specifically, we show that inhibitors of the histone methyltransferase, EZH2, synergize with various RAS pathway inhibitors and promote dramatic tumor regression in vivo. Together these agents cooperatively suppress WNT-driven transcription and drive CRCs into a more differentiated cell state by inducing the Groucho/TLE corepressor, TLE4, along with a network of WNT pathway inhibitors and intestinal differentiation proteins. However, these agents also induce the pro-apoptotic protein BMF, which subsequently kills these more differentiated cells. Accordingly, cell death can be prevented by activating ß-catenin, blocking differentiation, or by ablating BMF expression. Collectively, these studies reveal a new therapeutic approach for treating KRAS-mutant CRCs and illustrate a critical convergence of EZH2 and RAS on oncogenic WNT signals, intestinal differentiation, and apoptosis.
RESUMO
The highest frequencies of KRAS mutations occur in colorectal carcinoma (CRC) and pancreatic ductal adenocarcinoma (PDAC). The ability to target downstream pathways mediating KRAS oncogenicity is limited by an incomplete understanding of the contextual cues modulating the signaling output of activated K-RAS. We performed mass spectrometry on mouse tissues expressing wild-type or mutant Kras to determine how tissue context and genetic background modulate oncogenic signaling. Mutant Kras dramatically altered the proteomes and phosphoproteomes of preneoplastic and neoplastic colons and pancreases in a context-specific manner. We developed an approach to statistically humanize the mouse networks with data from human cancer and identified genes within the humanized CRC and PDAC networks synthetically lethal with mutant KRAS. Our studies demonstrate the context-dependent plasticity of oncogenic signaling, identify non-canonical mediators of KRAS oncogenicity within the KRAS-regulated signaling network, and demonstrate how statistical integration of mouse and human datasets can reveal cross-species therapeutic insights.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Neoplasias Colorretais/metabolismo , Redes Reguladoras de Genes , Redes e Vias Metabólicas , Proteogenômica/métodos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Carcinogênese , Carcinoma Ductal Pancreático/genética , Microambiente Celular , Neoplasias Colorretais/genética , Biologia Computacional , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Humanos , Camundongos , Mutação/genética , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais , Microambiente TumoralRESUMO
Adenomatous Polyposis Coli (APC) is the most frequently mutated gene in colorectal cancer. APC negatively regulates the Wnt signaling pathway by promoting the degradation of ß-catenin, but the extent to which APC exerts Wnt/ß-catenin-independent tumor-suppressive activity is unclear. To identify interaction partners and ß-catenin-independent targets of endogenous, full-length APC, we applied label-free and multiplexed tandem mass tag-based mass spectrometry. Affinity enrichment-mass spectrometry identified more than 150 previously unidentified APC interaction partners. Moreover, our global proteomic analysis revealed that roughly half of the protein expression changes that occur in response to APC loss are independent of ß-catenin. Combining these two analyses, we identified Misshapen-like kinase 1 (MINK1) as a putative substrate of an APC-containing destruction complex. We validated the interaction between endogenous MINK1 and APC and further confirmed the negative, and ß-catenin-independent, regulation of MINK1 by APC. Increased Mink1/Msn levels were also observed in mouse intestinal tissue and Drosophila follicular cells expressing mutant Apc/APC when compared with wild-type tissue/cells. Collectively, our results highlight the extent and importance of Wnt-independent APC functions in epithelial biology and disease. IMPLICATIONS: The tumor-suppressive function of APC, the most frequently mutated gene in colorectal cancer, is mainly attributed to its role in ß-catenin/Wnt signaling. Our study substantially expands the list of APC interaction partners and reveals that approximately half of the changes in the cellular proteome induced by loss of APC function are mediated by ß-catenin-independent mechanisms.