[Show abstract][Hide abstract] ABSTRACT: Ran-binding protein M (RanBPM) is a nucleocytoplasmic protein of yet unknown function. We have previously shown that RanBPM inhibits expression of the anti-apoptotic factor Bcl-2 and promotes apoptosis induced by DNA damage. Here we show that the effects of RanBPM on Bcl-2 expression occur through a regulation of the ERK signaling pathway. Transient and stable down-regulation of RanBPM stimulated ERK phosphorylation, leading to Bcl-2 up-regulation, while re-expression of RanBPM reversed these effects. RanBPM was found to inhibit MEK and ERK activation induced by ectopic expression of active RasV12. Activation of ERK by active c-Raf was also prevented by RanBPM. Expression of RanBPM correlated with a marked decrease in the protein levels of ectopically expressed active c-Raf and also affected the expression of endogenous c-Raf. RanBPM formed a complex with both active c-Raf, consisting of the C-terminal kinase domain, and endogenous c-Raf in mammalian cells. In addition, RanBPM was found to decrease the binding of Hsp90 to c-Raf. Finally, we show that loss of RanBPM expression confers increased cell proliferation and cell migration properties to HEK293 cells. Altogether, these findings establish RanBPM as a novel inhibitor of the ERK pathway through an interaction with the c-Raf complex and a regulation of c-Raf stability, and provide evidence that RanBPM loss of expression results in constitutive activation of the ERK pathway and promotes cellular events leading to cellular transformation and tumorigenesis.
[Show abstract][Hide abstract] ABSTRACT: RanBPM is a ubiquitous protein that has been reported to regulate several cellular processes through interactions with various proteins. However, it is not known whether RanBPM may regulate gene expression patterns. As it has been shown that RanBPM interacts with a number of transcription factors, we hypothesized that it may have wide ranging effects on gene expression that may explain its function. To test this hypothesis, we generated stable RanBPM shRNA cell lines to analyze the effect of RanBPM on global gene expression. Microarray analyses were conducted comparing the gene expression profile of Hela and HCT116 RanBPM shRNA cells versus control shRNA cells. We identified 167 annotated genes significantly up- or down-regulated in the two cell lines. Analysis of the gene set revealed that down-regulation of RanBPM led to gene expression changes that affect regulation of cell, tissue, and organ development and morphology, as well as biological processes implicated in tumorigenesis. Analysis of Transcription Factor Binding Sites (TFBS) present in the gene set identified several significantly over-represented transcription factors of the Forkhead, HMG, and Homeodomain families of transcription factors, which have previously been demonstrated as having important roles in development and tumorigenesis. In addition, the combined results of these analyses suggested that several signaling pathways were affected by RanBPM down-regulation, including ERK1/2, Wnt, Notch, and PI3K/Akt pathways. Lastly, analysis of selected target genes by quantitative RT-qPCR confirmed the changes revealed by microarray. Several of the genes up-regulated in RanBPM shRNA cells encode proteins with known oncogenic functions, such as the RON tyrosine kinase, the adhesion molecule L1CAM, and transcription factor ELF3/ESE-1, suggesting that RanBPM functions as a tumor suppressor to prevent deregulated expression of these genes. Altogether, these results suggest that RanBPM does indeed function to regulate many genomic events that regulate embryonic, tissue, and cellular development as well as those involved in cancer development and progression.
Full-text · Article · Sep 2012 · American Journal of Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Ran-binding protein M (RanBPM) is a nucleocytoplasmic protein previously implicated in various signaling pathways, but whose function remains enigmatic. Here, we provide evidence that RanBPM functions as an activator of apoptotic pathways induced by DNA damage. First, transient expression of RanBPM in HeLa cells induced cell death through caspase activation, and in the long-term, forced expression of RanBPM impaired cell viability. RanBPM COOH-terminal domain stimulated the ability of RanBPM to induce caspase activation, whereas this activity was negatively regulated by the central SPRY domain. Second, small interfering RNA-directed knockdown of RanBPM prevented DNA damage-induced apoptosis, as evidenced by the marked reduction in caspase-3 and caspase-2 activation. This correlated with a magnitude fold increase in the survival of RanBPM-depleted cells. Following ionizing radiation treatment, we observed a progressive relocalization of RanBPM from the nucleus to the cytoplasm, suggesting that the activation of apoptotic pathways by RanBPM in response to ionizing radiation may be regulated by nucleocytoplasmic trafficking. Finally, RanBPM downregulation was associated with a marked decrease of mitochondria-associated Bax, whereas Bcl-2 overall levels were dramatically upregulated. Overall, our results reveal a novel proapoptotic function for RanBPM in DNA damage-induced apoptosis through the regulation of factors involved in the mitochondrial apoptotic pathway.
Full-text · Article · Dec 2009 · Molecular Cancer Research