RESUMO
ARHGAP36 is an atypical Rho GTPase-activating protein (GAP) family member that drives both spinal cord development and tumorigenesis, acting in part through an N-terminal motif that suppresses protein kinase A and activates Gli transcription factors. ARHGAP36 also contains isoform-specific N-terminal sequences, a central GAP-like module, and a unique C-terminal domain, and the functions of these regions remain unknown. Here we have mapped the ARHGAP36 structure-activity landscape using a deep sequencing-based mutagenesis screen and truncation mutant analyses. Using this approach, we have discovered several residues in the GAP homology domain that are essential for Gli activation and a role for the C-terminal domain in counteracting an N-terminal autoinhibitory motif that is present in certain ARHGAP36 isoforms. In addition, each of these sites modulates ARHGAP36 recruitment to the plasma membrane or primary cilium. Through comparative proteomics, we also have identified proteins that preferentially interact with active ARHGAP36, and we demonstrate that one binding partner, prolyl oligopeptidase-like protein, is a novel ARHGAP36 antagonist. Our work reveals multiple modes of ARHGAP36 regulation and establishes an experimental framework that can be applied towards other signaling proteins.
Assuntos
Cílios , Proteínas Ativadoras de GTPase , Transdução de Sinais , Animais , Cílios/química , Cílios/genética , Cílios/metabolismo , Proteínas Ativadoras de GTPase/biossíntese , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Domínios Proteicos , Isoformas de Proteínas , Relação Estrutura-AtividadeRESUMO
Activating mutations in RAS GTPases drive many cancers, but limited understanding of less-studied RAS interactors, and of the specific roles of different RAS interactor paralogs, continues to limit target discovery. We developed a multistage discovery and screening process to systematically identify genes conferring RAS-related susceptibilities in lung adenocarcinoma. Using affinity purification mass spectrometry, we generated a protein-protein interaction map of RAS interactors and pathway components containing hundreds of interactions. From this network, we constructed a CRISPR dual knockout library targeting 119 RAS-related genes that we screened for KRAS-dependent genetic interactions (GI). This approach identified new RAS effectors, including the adhesion controller RADIL and the endocytosis regulator RIN1, and >250 synthetic lethal GIs, including a potent KRAS-dependent interaction between RAP1GDS1 and RHOA. Many GIs link specific paralogs within and between gene families. These findings illustrate the power of multiomic approaches to uncover synthetic lethal combinations specific for hitherto untreatable cancer genotypes. SIGNIFICANCE: We establish a deep network of protein-protein and genetic interactions in the RAS pathway. Many interactions validated here demonstrate important specificities and redundancies among paralogous RAS regulators and effectors. By comparing synthetic lethal interactions across KRAS-dependent and KRAS-independent cell lines, we identify several new combination therapy targets for RAS-driven cancers.This article is highlighted in the In This Issue feature, p. 1775.
Assuntos
Neoplasias Pulmonares/genética , Proteômica/métodos , Proteínas ras/genética , HumanosRESUMO
Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.
Assuntos
Cromograninas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Neoplasias Pulmonares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteína Fosfatase 2/metabolismo , Proteômica/métodos , Carcinoma de Pequenas Células do Pulmão/metabolismo , Células A549 , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cromograninas/genética , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteína Fosfatase 2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
E2F transcription factors are central regulators of cell division and cell fate decisions. E2F4 often represents the predominant E2F activity in cells. E2F4 is a transcriptional repressor implicated in cell cycle arrest and whose repressive activity depends on its interaction with members of the RB family. Here we show that E2F4 is important for the proliferation and the survival of mouse embryonic stem cells. In these cells, E2F4 acts in part as a transcriptional activator that promotes the expression of cell cycle genes. This role for E2F4 is independent of the RB family. Furthermore, E2F4 functionally interacts with chromatin regulators associated with gene activation and we observed decreased histone acetylation at the promoters of cell cycle genes and E2F targets upon loss of E2F4 in RB family-mutant cells. Taken together, our findings uncover a non-canonical role for E2F4 that provide insights into the biology of rapidly dividing cells.
Assuntos
Fator de Transcrição E2F4/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteína do Retinoblastoma/metabolismo , Ativação Transcricional , Animais , Ciclo Celular , Divisão Celular , Fator de Transcrição E2F4/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Família Multigênica , Proteína do Retinoblastoma/genéticaRESUMO
During tissue repair, fibronectin is converted from a soluble, inactive form into biologically active extracellular matrix (ECM) fibrils through a cell-dependent process. ECM fibronectin promotes numerous cell processes that are critical to tissue repair and regulates the assembly of other proteins into the matrix. Nonhealing wounds show reduced levels of ECM fibronectin. To functionally mimic ECM fibronectin, a series of fibronectin matrix mimetics was developed by directly coupling the matricryptic, heparin-binding fragment of the first type III repeat of fibronectin (FNIII1H) to various sequences from the integrin-binding domain (FNIII8-10). The recombinant proteins were produced as glutathione-S-transferase (GST)-tagged fusion proteins for ease of production and purification. Full-thickness, excisional wounds were produced in genetically diabetic mice, and fibronectin matrix mimetics were applied directly to the wounds. A significant enhancement of wound closure was observed by day 9 in response to GST/III1H,8-10 versus GST-treated controls (73.9%±4.1% vs. 58.1%±4.7% closure, respectively). Two weeks after injury, fibronectin matrix mimetic-treated wounds had developed a multi-layered epithelium that completely covered the wound space. Furthermore, significant increases in granulation tissue thickness were observed in response to treatment with GST/III1H,8-10 (4.05±0.93-fold), GST/III1H,8,10 (2.91±0.49-fold), or GST/III1H,8(RGD) (3.55±0.59-fold) compared with GST controls, and was accompanied by dense collagen deposition, the presence of myofibroblasts, and functional vasculature. Thus, the recombinant fibronectin matrix analogs normalized the impairment in repair observed in this chronic wound model and may provide a new approach to accelerate the healing of diabetic wounds.