The transcription corepressor CtBP is often recruited to the target promoter via interaction with a conserved PxDLS motif in the interacting repressor. In this study, we demonstrate that CtBP1 was SUMOylated and that its SUMOylation profoundly affected its subcellular localization. SUMOylation occurred at a single Lys residue, Lys428, of CtBP1. CtBP2, a close homolog of CtBP1, lacked the SUMOylation site and was not modified by SUMO-1. Mutation of Lys428 into Arg (K428R) shifted CtBP1 from the nucleus to the cytoplasm, while it had little effect on its interaction with the PxDLS motif. Consistent with a change in localization, the K428R mutation abolished the ability of CtBP1 to repress the E-cadherin promoter activity. Notably, SUMOylation of CtBP1 was inhibited by the PDZ domain of nNOS, correlating with the known inhibitory effect of nNOS on the nuclear accumulation of CtBP1. This study identifies SUMOylation as a regulatory mechanism underlying CtBP1-dependent transcriptional repression.

Tumor suppressor Smad4/DPC4 is a central intracellular signal transducer for transforming growth factor-␤ (TGF-␤) signaling. We recently reported that transcriptional potential of Smad4 was regulated by SUMOylation in transfected HeLa cells (1), but the precise mechanism and function of Smad4 SUMOylation in TGF-␤ signaling remain to be elucidated. Here, we describe the regulation of TGF-␤ signaling by SUMOylation through the control of Smad4 metabolic stability and subcellular localization. We found that SUMO-1 overexpression strongly increases Smad4 levels, while inhibition of SUMOylation by small interfering RNA (siRNA)-mediated knockdown of the E2 enzyme Ubc9 reduces endogenous Smad4 levels. Concomitantly, SUMO-1 overexpression enhances and Ubc9 knockdown reduces levels of intranuclear Smad4, growth inhibitory response, as well as transcriptional responses to TGF-␤. Comparison of wild type and mutant forms of Smad4 for SUMOylation, ubiquitination, and half-life allows the conclusion that SUMO-1 modification serves to protect Smad4 from ubiquitin-dependent degradation and consequently enhances the growth inhibitory and transcriptional responses of Smad4.

To assess whether Smad signaling affects skin development, we generated transgenic mice in which a Smad antagonist, Smad7, was induced in keratinocytes, including epidermal stem cells. Smad7 transgene induction perturbed hair follicle morphogenesis and differentiation, but accelerated sebaceous gland morphogenesis. Further analysis revealed that independent of its role in anti-Smad signaling, Smad7 bound beta-catenin and induced beta-catenin degradation by recruiting an E3 ligase, Smurf2, to the Smad7/beta-catenin complex. Consequently, Wnt/beta-catenin signaling was suppressed in Smad7 transgenic hair follicles. Coexpression of the Smurf2 and Smad7 transgenes exacerbated Smad7-induced abnormalities in hair follicles and sebaceous glands. Conversely, when endogenous Smad7 was knocked down, keratinocytes exhibited increased beta-catenin protein and enhanced Wnt signaling. Our data reveal a mechanism for Smad7 in antagonizing Wnt/beta-catenin signaling, thereby shifting the skin differentiation program from forming hair follicles to sebaceous glands.

Smads are important intracellular effectors in signaling pathways of the transforming growth factor-␤ (TGF-␤) superfamily. Upon activation by TGF-␤, receptor-phosphorylated Smads form a complex with tumor suppressor Smad4/DPC4, and the Smad complexes then are imported into the nucleus. Although diverse pathways regulate the activity and expression of receptorphosphorylated and inhibitory Smads, cellular factors modulating the activity of the common Smad4 remain unidentified. Here we describe the involvement of the small ubiquitin-like modifier-1 (SUMO-1) conjugation pathway in regulating the growth inhibitory and transcriptional responses of Smad4. The MH1 domain of Smad4 was shown to associate physically with Ubc9, the ubiquitin carrier protein (E2) conjugating enzyme in sumoylation. In cultured cells, Smad4 is modified by SUMO-1 at the endogenous level. The sumoylation sites were identified as two evolutionarily conserved lysine residues, Lys-113 and Lys-159, in the MH1 domain. We found that the mutations at Lys-113 and Lys-159 did not alter the ability of Smad4 to form a complex with Smad2 and FAST on the Mix.2 promoter. Importantly, SUMO-1 overexpression enhanced TGF-␤-induced transcriptional responses. These findings identify sumoylation as a unique mechanism to modulate Smad4-dependent cellular responses.Transforming growth factor-␤ (TGF-␤) 1 is a secreted multifunctional protein that exhibits a diverse set of cellular responses including cell proliferation and differentiation. TGF-␤ functions as a potent growth inhibitor and induces the expression of a variety of genes during growth and development (1-4).TGF-␤ signals are transduced by transmembrane serine/threonine kinase receptors and intracellular effectors called Smads (2,5,6). Upon TGF-␤ stimulation, Smad2 and/or Smad3 are phosphorylated by the activated type I receptor and then form complexes with Smad4. The heteromeric complexes of R-Smads and Smad4 then are translocated into the nucleus, where they exert ligand-induced changes in the transcription of a variety of genes (2, 6, 7). The heteromeric Smad complex activates transcription through its ability to cooperate functionally with several promoter-specific transcription factors and/or to bind specific DNA sequences (7,8).Proper TGF-␤ signaling requires precise control of Smad functions. Recent studies have shown that R-Smads are modified post-translationally by ubiquitin and can be removed irreversibly by the proteasome-mediated degradation system (9 -13). A number of ubiquitin-related proteins also are present in eukaryotic cells. These proteins, including the small ubiquitinlike modifier-1 (SUMO-1), utilize a conjugation system that is similar to ubiquitination (14 -16). SUMO-1 uses Aos1/Uba2 as E1 activating enzymes (17, 18) and Ubc9 as an E2 conjugating enzyme (19,20). Unlike ubiquitination, SUMO-1 modifications of target proteins do not promote their degradation. In contrast, SUMO-1 modifications of I B preclude its ubiquitination (21). Functional consequences of SUMO-1 modification vary dep...

SummaryMicroRNAs are known to play regulatory roles in gene expression associated with cancer development. We analyzed levels of the microRNA miR-24 in patients with breast carcinoma and found that miR-24 was higher in breast carcinoma samples than in benign breast tissues. We generated constructs expressing miR-24 and studied its functions using both in vitro and in vivo techniques. We found that the ectopic expression of miR-24 promoted breast cancer cell invasion and migration. In vivo experiments in mice indicated that the expression of miR-24 enhanced tumor growth, invasion into local tissues, metastasis to lung tissues and decreased overall mouse survival. In the miR-24-expressing cells and tumors, EGFR was highly phosphorylated, whereas expression of the phosphatases tyrosine-protein phosphatase non-receptor type 9 (PTPN9) and receptor-type tyrosine-protein phosphatase F (PTPRF) were repressed. We confirmed that miR-24 could directly target both PTPN9 and PTPRF. Consistent with this, we found that the levels of phosphorylated epidermal growth factor receptor (pEGFR) were higher whereas the levels of PTPN9 and PTPRF were lower in the patients with metastatic breast carcinoma. Ectopic expression of PTPN9 and PTPRF decreased pEGFR levels, cell invasion, migration and tumor metastasis. Furthermore, we found that MMP2, MMP11, pErk, and ADAM15 were upregulated, whereas TIMP2 was downregulated; all of which supported the roles of miR-24 in tumor invasion and metastasis. Our results suggest that miR-24 plays a key role in breast cancer invasion and metastasis. miR-24 could potentially be a target for cancer intervention.

Bone morphogenetic proteins (BMPs) are secreted polypeptides belonging to the transforming growth factor-␤ (TGF-␤) superfamily that activates a broad range of biological responses in the metazoan organism. The BMP-initiated signaling pathway is under tight control by processes including regulation of the ligands, the receptors, and the key downstream intracellular effector Smads. A critical point of control in BMP signaling is the phosphorylation of Smad1, Smad5, and Smad8 in their C-terminal SXS motif. Although such phosphorylation, which is mediated by the type I BMP receptor kinases in response to BMP stimulation, is well characterized, biochemical mechanisms underlying Smad dephosphorylation remain to be elucidated. In this study, we have found that PPM1A, a metal ion-dependent protein serine/threonine phosphatase, physically interacts with and dephosphorylates Smad1 both in vitro and in vivo. Functionally, overexpression of PPM1A abolishes BMP-induced transcriptional responses, whereas RNA interference-mediated knockdown of PPM1A enhances BMP signaling. Collectively, our study suggests that PPM1A plays an important role in controlling BMP signaling through catalyzing Smad dephosphorylation.Bone morphogenetic proteins (BMPs), 3 originally identified by their ability to cause bone differentiation (1), are signaling molecules that belong to the transforming growth factor-␤ (TGF-␤) superfamily. Presently, the biological functions of BMPs have been greatly expanded. BMPs regulate skeletal development as well as many non-osteogenic developmental processes, such as mesoderm patterning, left-right symmetry, neuronal patterning, and hematopoiesis (2-5). Accumulating evidence indicates that BMPs play an important role in the regulation of stem cell properties (3, 6 -8). Signals from BMP ligands are transduced through binding to type I and II receptors on the cell surface, where type II receptors activate type I receptors, which in turn phosphorylate the downstream Smad1, Smad5, and Smad8. The phospho-R-Smads (P-Smads) form a complex with Smad4 and translocate into the nucleus, where they bind to the Smad binding sites and cooperate with other transcription factors to regulate BMP-induced gene expression (5, 9, 10).Despite substantial effort devoted to understanding the actions of BMP/TGF-␤ and Smads, the precise regulation of Smads remains enigmatic. Regulation of Smads can be accomplished via various post-translational mechanisms, including phosphorylation and ubiquitin-dependent modifications (11,12). Among these, BMP-induced phosphorylation of Smad1/ 5/8, which is carried out by the BMP type I receptor (BMPRIA or BMPRIB) and occurs at the C-terminal SXS motif of Smad1/ 5/8, represents the most critical step in Smad signaling. The SXS phosphorylation triggers a cascade of intracellular events from Smad complex assembly in the cytoplasm to transcriptional control in the nucleus.The reversible phosphorylation and dephosphorylation represents a fundamental strategy used by eukaryotic organisms to regulate a battery of...

Smad6 and Smad7 are inhibitory Smads induced by transforming growth factor ␤-Smad signal transduction pathways in a negative-feedback mechanism. Previously it has been thought that inhibitory Smads bind to the type I receptor and block the phosphorylation of receptor-activated Smads, thereby inhibiting the initiation of Smad signaling. Conversely, few studies have suggested the possible nuclear functions of inhibitory Smads. Here, we present compelling evidence demonstrating that Smad6 repressed bone morphogenetic proteininduced Id1 transcription through recruiting transcriptional corepressor C-terminal binding protein (CtBP). A consensus CtBP-binding motif, PLDLS, was identified in the linker region of Smad6. Our findings show that mutation in the motif abolished the Smad6 binding to CtBP and subsequently its repressor activity of transcription. We conclude that the nuclear functions and physical interaction of Smad6 and CtBP provide a novel mechanism for the transcriptional regulation by inhibitory Smads.

The c-Myc oncogene has been implicated in the genesis of diverse human tumors. Ectopic expression of the c-Myc gene in cultured epithelial cells causes resistance to the antiproliferative effects of TGF-beta. However, little is known about the precise mechanisms of c-Myc-mediated TGF-beta resistance. In this study, we reveal that c-Myc physically interacts with Smad2 and Smad3, two specific signal transducers involved in TGF-beta signaling. Through its direct interaction with Smads, c-Myc binds to the Sp1-Smad complex on the promoter of the p15(Ink4B) gene, thereby inhibiting the TGF-beta-induced transcriptional activity of Sp1 and Smad/Sp1-dependent transcription of the p15(Ink4B) gene. These results suggest that oncogenic c-Myc promotes cell growth and cancer development partly by inhibiting the growth inhibitory functions of Smads.