RESUMO
In response to DNA double-strand breaks (DSBs), H2AX is rapidly phosphorylated at Ser139 to promote DSB repair. Here we show that H2AX is rapidly stabilized in response to DSBs to efficiently generate γH2AX foci. This mechanism operated even in quiescent cells that barely expressed H2AX. H2AX stabilization resulted from the inhibition of proteasome-mediated degradation. Synthesized H2AX ordinarily underwent degradation through poly-ubiquitination mediated by the E3 ligase HUWE1; however, H2AX ubiquitination was transiently halted upon DSB formation. Such rapid H2AX stabilization by DSBs was associated with chromatin incorporation of H2AX and halting of its poly-ubiquitination mediated by the ATM kinase, the sirtuin protein SIRT6, and the chromatin remodeler SNF2H. H2AX Ser139, the ATM phosphorylation site, was essential for H2AX stabilization upon DSB formation. Our results reveal a pathway controlled by ATM, SIRT6, and SNF2H to block HUWE1, which stabilizes H2AX and induces its incorporation into chromatin only when cells are damaged.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Sirtuínas/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Quebras de DNA de Cadeia Dupla , Células HeLa , Histonas/genética , Humanos , Camundongos , Fosforilação , Sirtuínas/genética , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases/metabolismoRESUMO
In order to clarify the molecular mechanism involved in renal carcinogenesis, and to identify molecular targets for development of novel treatments of renal cell carcinoma (RCC), we previously analyzed genome-wide gene expression profiles of clear-cell types of RCC by cDNA microarray. Among the transcativated genes, we herein focused on functional significance of TMEM22 (transmembrane protein 22), a transmembrane protein, in cell growth of RCC. Northern blot and semi-quantitative RT-PCR analyses confirmed up-regulation of TMEM22 in a great majority of RCC clinical samples and cell lines examined. Immunocytochemical analysis validated its localization at the plasma membrane. We found an interaction between TMEM22 and RAB37 (Ras-related protein Rab-37), which was also up-regulated in RCC cells. Interestingly, knockdown of either of TMEM22 or RAB37 expression by specific siRNA caused significant reduction of cancer cell growth. Our results imply that the TMEM22/RAB37 complex is likely to play a crucial role in growth of RCC and that inhibition of the TMEM22/RAB37 expression or their interaction should be novel therapeutic targets for RCC.
