RESUMO
Mutations in SPOP, the gene most frequently point-mutated in primary prostate cancer, are associated with a high degree of genomic instability and deficiency in homologous recombination repair of DNA but the underlying mechanisms behind this defect are currently unknown. Here we demonstrate that SPOP knockdown leads to spontaneous replication stress and impaired recovery from replication fork stalling. We show that this is associated with reduced expression of several key DNA repair and replication factors including BRCA2, ATR, CHK1 and RAD51. Consequently, SPOP knockdown impairs RAD51 foci formation and activation of CHK1 in response to replication stress and compromises recovery from replication fork stalling. An SPOP interactome analysis shows that wild type (WT) SPOP but not mutant SPOP associates with multiple proteins involved in transcription, mRNA splicing and export. Consistent with the association of SPOP with transcription, splicing and RNA export complexes, the decreased expression of BRCA2, ATR, CHK1 and RAD51 occurs at the level of transcription.
Assuntos
Replicação do DNA/genética , Instabilidade Genômica/genética , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA/genética , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Masculino , Mutação , Neoplasias da Próstata/patologia , Splicing de RNA/genética , RNA Mensageiro/genética , Rad51 Recombinase/genéticaRESUMO
A fascinating conundrum in cell signaling is how stimulation of the same receptor tyrosine kinase with distinct ligands generates specific outcomes. To decipher the functional selectivity of EGF and TGF-α, which induce epidermal growth factor receptor (EGFR) degradation and recycling, respectively, we devised an integrated multilayered proteomics approach (IMPA). We analyzed dynamic changes in the receptor interactome, ubiquitinome, phosphoproteome, and late proteome in response to both ligands in human cells by quantitative MS and identified 67 proteins regulated at multiple levels. We identified RAB7 phosphorylation and RCP recruitment to EGFR as switches for EGF and TGF-α outputs, controlling receptor trafficking, signaling duration, proliferation, and migration. By manipulating RCP levels or phosphorylation of RAB7 in EGFR-positive cancer cells, we were able to switch a TGF-α-mediated response to an EGF-like response or vice versa as EGFR trafficking was rerouted. We propose IMPA as an approach to uncover fine-tuned regulatory mechanisms in cell signaling.