RESUMO
Microspherule protein Msp58 (or MCRS1) plays a role in numerous cellular processes including transcriptional regulation and cell proliferation. It is not well understood either how Msp58 mediates its myriad functions or how it is itself regulated. Here, by immunoprecipitation, we identify EDD (E3 identified by differential display) as a novel Msp58-interacting protein. EDD, also called UBR5, is a HECT-domain (homologous to E6-AP carboxy-terminus) containing ubiquitin ligase that plays a role in cell proliferation, differentiation and DNA damage response. Both in vitro and in vivo binding assays show that Msp58 directly interacts with EDD. Microscopy studies reveal that these two proteins co-localize in the nucleus. We have also found that depletion of EDD leads to an increase of Msp58 protein level and extends the half-life of Msp58, demonstrating that EDD negatively regulates Msp58's protein stability. Furthermore, we show that Msp58 is upregulated in multiple different cell lines upon the treatment with proteasome inhibitor MG132 and exogenously expressed Msp58 is ubiquitinated, suggesting that Msp58 is degraded by the ubiquitin-proteasome pathway. Finally, knockdown of either Msp58 or EDD in human lung fibroblast WI-38 cells affects the levels of cyclins B, D and E, as well as cell cycle progression. Together, these results suggest a role for the Msp58/EDD interaction in controlling cell cycle progression. Given that both Msp58 and EDD are often aberrantly expressed in various human cancers, our findings open a new direction to elucidate Msp58 and EDD's roles in cell proliferation and tumorigenesis.
Assuntos
Ciclo Celular , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Sequência de Aminoácidos , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Ciclo Celular/fisiologia , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Leupeptinas/farmacologia , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Inibidores de Proteassoma/farmacologia , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/fisiologia , Mapeamento de Interação de Proteínas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
Specific chromatin marks keep master regulators of differentiation silent yet poised for activation by extracellular signals. We report that nodal TGF-ß signals use the poised histone mark H3K9me3 to trigger differentiation of mammalian embryonic stem cells. Nodal receptors induce the formation of companion Smad4-Smad2/3 and TRIM33-Smad2/3 complexes. The PHD-Bromo cassette of TRIM33 facilitates binding of TRIM33-Smad2/3 to H3K9me3 and H3K18ac on the promoters of mesendoderm regulators Gsc and Mixl1. The crystal structure of this cassette, bound to histone H3 peptides, illustrates that PHD recognizes K9me3, and Bromo binds an adjacent K18ac. The interaction between TRIM33-Smad2/3 and H3K9me3 displaces the chromatin-compacting factor HP1γ, making nodal response elements accessible to Smad4-Smad2/3 for Pol II recruitment. In turn, Smad4 increases K18 acetylation to augment TRIM33-Smad2/3 binding. Thus, nodal effectors use the H3K9me3 mark as a platform to switch master regulators of stem cell differentiation from the poised to the active state.
