RESUMO
Splicing abnormalities have profound impact in human cancer. Several splicing factors, including SAM68, have pro-oncogenic functions, and their increased expression often correlates with human cancer development and progression. Herein, we have identified using mass spectrometry proteins that interact with endogenous SAM68 in prostate cancer (PCa) cells. Among other interesting proteins, we have characterized the interaction of SAM68 with SND1, a transcriptional co-activator that binds spliceosome components, thus coupling transcription and splicing. We found that both SAM68 and SND1 are upregulated in PCa cells with respect to benign prostate cells. Upregulation of SND1 exerts a synergic effect with SAM68 on exon v5 inclusion in the CD44 mRNA. The effect of SND1 on CD44 splicing required SAM68, as it was compromised after knockdown of this protein or mutation of the SAM68-binding sites in the CD44 pre-mRNA. More generally, we found that SND1 promotes the inclusion of CD44 variable exons by recruiting SAM68 and spliceosomal components on CD44 pre-mRNA. Inclusion of the variable exons in CD44 correlates with increased proliferation, motility and invasiveness of cancer cells. Strikingly, we found that knockdown of SND1, or SAM68, reduced proliferation and migration of PCa cells. Thus, our findings strongly suggest that SND1 is a novel regulator of alternative splicing that promotes PCa cell growth and survival.
Assuntos
Processamento Alternativo , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Ativação Transcricional , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases , Éxons , Técnicas de Silenciamento de Genes , Humanos , Receptores de Hialuronatos/genética , Masculino , Ligação Proteica , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
E2A, HEB and E2-2 genes encode a group of basic helix-loop-helix (bHLH) transcription factors that are structurally and functionally similar. Deletion of the genes encoding either of these proteins leads to early lethality and a block in B lymphocyte development. Evidence for a function in T lymphocyte development has, however, only been reported for E2A and HEB. To further elucidate the role of E2-2 at developmental stages that have proven difficult to study due to the early lethality phenotype of mice defective in E2-2, we generated and analyzed mice conditionally mutated in the E2-2 gene. These mice are mosaic with respect to E2-2 expression, consisting of cells with either one functional and one null mutated E2-2 allele or two null mutated alleles. Using this experimental model, we find that cells with a homozygous null mutated E2-2 gene are under-represented in B lymphocyte as well as T lymphocyte cell lineages as compared to other hematopoietic or non-hematopoietic cell lineages. Our data suggests that E2-2 deficiency leads to a partial block in both B and T lymphocyte development. The block in T cell development appears to occur at an early stage in differentiation, since skewing in the mosaicism is observed already in CD4+8+ double-positive thymocytes.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Sequências Hélice-Alça-Hélice , Hematopoese/genética , Síndromes de Imunodeficiência/genética , Proteínas do Tecido Nervoso , Linfócitos T/patologia , Transativadores/fisiologia , Fatores de Transcrição , Alelos , Animais , Linfócitos B/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Diferenciação Celular , Linhagem da Célula , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Marcação de Genes , Genótipo , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/patologia , Contagem de Linfócitos , Subpopulações de Linfócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mosaicismo , Baço/patologia , Fatores de Transcrição TCF , Timo/patologia , Transativadores/deficiência , Transativadores/genética , Fator de Transcrição 4RESUMO
Among the members of the fibroblast growth factor receptor family the FGFR4 has demonstrated strong dependence on heparin-like material for its activation by fibroblast growth factors. We have produced and characterized a recombinant human FGFR4 extracellular domain (FGFR4ed), in order to study its biochemical properties in isolated conditions. The FGFR4ed was expressed in an insect cell system and purified from the culture medium by Ni(2+)-affinity and gel filtration chromatography. Pure FGFR4ed was tested for FGF- and heparin-binding by covalent crosslinking experiments and by biosensor analysis. In solution, FGFR4ed formed complexes with acidic FGF (FGF-1) and basic FGF (FGF-2), both in the presence and absence of heparin. Immobilized FGFR4 also bound FGF-8 besides FGF-1 and FGF-2. Furthermore, heparin alone induced receptor oligomerization on the surface of the receptor coupled chip. Thus, the recombinant FGFR4ed revealed properties described for the cellular form of this receptor and can be used for interaction studies.
Assuntos
Histidina , Engenharia de Proteínas/métodos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Técnicas Biossensoriais , Células Cultivadas , Cromatografia de Afinidade , Dimerização , Fator 1 de Crescimento de Fibroblastos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 8 de Crescimento de Fibroblasto , Fatores de Crescimento de Fibroblastos/metabolismo , Heparina/metabolismo , Humanos , Insetos/citologia , Peptídeos/genética , Peptídeos/metabolismo , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The basic helix-loop-helix (bHLH) family of transcription factors is essential for numerous developmental and growth control processes. The regulation of bHLH proteins occurs at many levels, including tissue specific expression, differential oligomerization and DNA binding specificities, interaction with negatively acting HLH proteins and post-translational modifications. This review focuses on what is emerging as another level of bHLH protein regulation, calcium regulation through interaction with Ca2+ loaded calmodulin and S-100 proteins. The mechanism and implications of these Ca2+ regulated interactions are discussed.
