Src kinase phosphorylates RUNX3 at tyrosine residues and localizes the protein in the cytoplasm.

RUNX3 is a transcription factor that functions as a tumor suppressor. In some cancers, RUNX3 expression is down-regulated, usually due to promoter hypermethylation. Recently, it was found that RUNX3 can also be inactivated by the mislocalization of the protein in the cytoplasm. The molecular mechanisms controlling this mislocalization are poorly understood. In this study, we found that the overexpression of Src results in the tyrosine phosphorylation and cytoplasmic localization of RUNX3. We also found that the tyrosine residues of endogenous RUNX3 are phosphorylated and that the protein is localized in the cytoplasm in Src-activated cancer cell lines. We further showed that the knockdown of Src by small interfering RNA, or the inhibition of Src kinase activity by a chemical inhibitor, causes the re-localization of RUNX3 to the nucleus. Collectively, our results demonstrate that the tyrosine phosphorylation of RUNX3 by activated Src is associated with the cytoplasmic localization of RUNX3 in gastric and breast cancers.

Runt-related transcription factor RUNX3 is a target of MDM2-mediated ubiquitination.

The p14(ARF)-MDM2-p53 pathway constitutes an effective mechanism for protecting cells from oncogenic stimuli such as activated Ras and Myc. Importantly, Ras activation induces p14(ARF) and often occurs earlier than p53 inactivation during cancer development. Here, we show that RUNX3, a tumor suppressor in various tumors including stomach, bladder, colon, and lung, is stabilized by Ras activation through the p14(ARF)-MDM2 signaling pathway. RUNX3 directly binds MDM2 through its Runt-related DNA-binding domain. MDM2 blocks RUNX3 transcriptional activity by interacting with RUNX3 through an acidic domain adjacent to the p53-binding domain of MDM2 and ubiquitinates RUNX3 on key lysine residues to mediate nuclear export and proteasomal degradation. Our data indicate that the lineage-specific tumor suppressor RUNX3 and the ubiquitous p53 protein are both principal responders of the p14(ARF)-MDM2 cell surveillance pathway that prevents pathologic consequences of abnormal oncogene activation.

Jab1/CSN5 induces the cytoplasmic localization and degradation of RUNX3.

Runt-related (RUNX) transcription factors play pivotal roles in neoplastic development and have tissue-specific developmental roles in hematopoiesis (RUNX1), osteogenesis (RUNX2), as well as neurogenesis and thymopoiesis (RUNX3). RUNX3 is a tumor suppressor in gastric carcinoma, and its expression is frequently inactivated by DNA methylation or its protein mislocalized in many cancer types, including gastric and breast cancer. Jun-activation domain-binding protein 1 (Jab1/CSN5), a component of the COP9 signalosome (CSN), is critical for nuclear export and the degradation of several tumor suppressor proteins, including p53, p27(Kip1), and Smad4. Here, we find that Jab1 facilitates nuclear export of RUNX3 that is controlled by CSN-associated kinases. RUNX3 sequestered in the cytoplasm is rapidly degraded through a proteasome-mediated pathway. Our results identify a novel mechanism of regulating nuclear export and protein stability of RUNX3 by the CSN complex.

E1A physically interacts with RUNX3 and inhibits its transactivation activity.

The adenoviral gene, termed early region 1A (E1A), is crucial for transformation and has been used very effectively as a tool to determine the molecular mechanisms that underlie the basis of cellular transformation. pRb, p107, p130, p300/CBP, p400, TRRAP, and CtBP were identified to be E1A-binding proteins and their roles in cellular transformation have been established. Although the major function of E1A is considered to be the regulation of gene expression that is critical for differentiation and cell cycle exit, one of the most significant questions relating to E1A transformation is how E1A mediates this regulation. RUNX3 is a transcription factor that was first described as a gastric cancer tumor suppressor but is now known to be involved in many different cancers. Exogenous expression of RUNX3 strongly inhibits the growth of cells. Here, we show that the adenovirus oncoprotein E1A interacts with RUNX3 in vitro and in vivo. RUNX3 interacts with the N-terminus (amino acids 2-29) of E1A, which is known to interact with p300/CBP, p400, and TRRAP. E1A interacts directly with the Runt domain of RUNX3 but does not interfere with CBFbeta-RUNX3 interactions. In addition, E1A inhibits the transactivation activity of RUNX3 on the p21(WAF1/CIP1) promoter. Consistent with these observations, the growth inhibition induced by RUNX3 is reduced by E1A. These results demonstrate that E1A specifically binds to RUNX3 and inactivates its transactivation activity. We propose that one of the mechanisms for the oncogenic activity of E1A is the inhibition of RUNX3, similar to that of RB and p300/CBP.

Bone morphogenetic protein-2 stimulates Runx2 acetylation.

Runx2/Cbfa1/Pebp2aA is a global regulator of osteogenesis and is crucial for regulating the expression of bone-specific genes. Runx2 is a major target of the bone morphogenetic protein (BMP) pathway. Genetic analysis has revealed that Runx2 is degraded through a Smurf-mediated ubiquitination pathway, and its activity is inhibited by HDAC4. Here, we demonstrate the molecular link between Smurf, HDACs and Runx2, in BMP signaling. BMP-2 signaling stimulates p300-mediated Runx2 acetylation, increasing transactivation activity and inhibiting Smurf1-mediated degradation of Runx2. HDAC4 and HDAC5 dea-cetylate Runx2, allowing the protein to undergo Smurf-mediated degradation. Inhibition of HDAC increases Runx2 acetylation, and potentiates BMP-2-stimulated osteoblast differentiation and increases bone formation. These results demonstrate that the level of Runx2 is controlled by a dynamic equilibrium of acetylation, deacetylation, and ubiquitination. These findings have important medical implications because BMPs and Runx2 are of tremendous interest with regard to the development of therapeutic agents against bone diseases.

Epistatic interactions between modifier genes confer strain-specific redundancy for Tgfb1 in developmental angiogenesis.

Tgfbm1 (chromosome 5, P = 8 x 10(-5)) and Tgfbm3 (chromosome 12, P = 6 x 10(-11)) were identified as loci that modify developmental angiogenesis of Tgfb1 -/- mice. Congenic mice validated these loci and demonstrated epistatic interaction between them. The novel locus, Tgfbm3, encompasses approximately 22 genes, colocalizes with both tumor susceptibility and atherosclerosis susceptibility loci, and is enriched in genes regulating cell growth and morphogenesis. The use of gene knockout and/or transgenic mice that predispose to a complex trait, such as vascular development/angiogenesis, facilitates the identification of modifiers by simplifying genetic analysis. Identification of genes that modify response to lack of transforming growth factor beta1 (TGFbeta1) will enhance the understanding of TGFbeta1 action in vivo and may help predict which patients would respond well to anti-TGFbeta therapy. Identification of angiogenesis-modifying genes may provide new targets for angiogenesis therapies and analysis of polymorphisms therein may contribute to assessment of risk for diseases involving angiogenesis.

RNT-1, the C. elegans homologue of mammalian RUNX transcription factors, regulates body size and male tail development.

The rnt-1 gene is the only Caenorhabditis elegans homologue of the mammalian RUNX genes. Several lines of molecular biological evidence have demonstrated that the RUNX proteins interact and cooperate with Smads, which are transforming growth factor-beta (TGF-beta) signal mediators. However, the involvement of RUNX in TGF-beta signaling has not yet been supported by any genetic evidence. The Sma/Mab TGF-beta signaling pathway in C. elegans is known to regulate body length and male tail development. The rnt-1(ok351) mutants show the characteristic phenotypes observed in mutants of the Sma/Mab pathway, namely, they have a small body size and ray defects. Moreover, RNT-1 can physically interact with SMA-4 which is one of the Smads in C. elegans, and double mutant animals containing both the rnt-1(ok351) mutation and a mutation in a known Sma/Mab pathway gene displayed synergism in the aberrant phenotypes. In addition, lon-1(e185) mutants was epistatic to rnt-1(ok351) mutants in terms of long phenotype, suggesting that lon-1 is indeed downstream target of rnt-1. Our data reveal that RNT-1 functionally cooperates with the SMA-4 proteins to regulate body size and male tail development in C. elegans.

Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein.

The Runx family of transcription factors plays pivotal roles during normal development and in neoplasias. In mammals, Runx family genes are composed of Runx1 (Pebp2alphaB/Cbfa2/Aml1), Runx2 (Pebp2alphaA/Cbfa1/Aml3) and Runx3 (Pebp2alphaC/Cbfa3/Aml2). Runx1 and Runx3 are known to be involved in leukemogenesis and gastric carcinogenesis, respectively. Runx2, on the other hand, is a common target of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) and plays an essential role in osteoblast differentiation. Runx2 is induced by the receptor-activated Smad; Runx2 mediates the blockage of myogenic differentiation and induces osteoblast differentiation in C2C12 pluripotent mesenchymal precursor cells. However, Smad does not directly induce Runx2 expression; an additional step of de novo protein synthesis is required. Here we report that Smad-induced junB functions as an upstream activator of Runx2 expression. Furthermore, not only the Smad pathway but also the mitogen-activated protein kinase (MAPK) cascades are involved in the induction of Runx2 by TGF-beta1 and BMP-2. Our results demonstrate that following TGF-beta and BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation.