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EVOLUTIONARY HOMOLOGS

DAF-5 is a Ski oncoprotein homolog that functions in a TGFβ pathway to regulate C. elegans dauer development

An unconventional TGFß superfamily pathway plays a crucial role in the decision between dauer diapause and reproductive growth. The daf-5 gene, along with the daf-3 Smad gene (homolog of Drosophila Medea), is antagonized by upstream receptors and receptor-regulated Smads. DAF-5 is a novel member of the Sno/Ski superfamily that binds to DAF-3 Smad, suggesting that DAF-5, like Sno/Ski, is a regulator of transcription in a TGFß superfamily signaling pathway. However, evidence is presented that DAF-5 is an unconventional Sno/Ski protein, because DAF-5 acts as a co-factor, rather than an antagonist, of a Smad protein. Expressing DAF-5 in the nervous system rescues a daf-5 mutant, whereas muscle or hypodermal expression does not. Previous work suggested that DAF-5 and DAF-3 function in pharyngeal muscle to regulate gene expression, but analysis of regulation of a pharynx specific promoter suggests otherwise. A model is presented in which DAF-5 and DAF-3 control the production or release of a hormone from the nervous system by either regulating the expression of biosynthetic genes or by altering the connectivity or the differentiated state of neurons (da Graca, 2004).

Sno and Ski are found in humans and all major groups of vertebrates, but in insects have only one ortholog of these two proteins. Sno and Ski are more similar to each other than either is to the single Drosophila or mosquito ortholog; therefore Sno and Ski are probably paralogs that were duplicated after the divergence of the protostome and deuterostome lineages. The insect genes have been named Snowski (Snk) to reflect the orthology to both Sno and Ski (da Graca, 2004).

A new family of proteins closely related to the Snowski group is described. Humans have two genes in this group. These genes have been named Skate (for Ski-related gene) and Icy (for Ski sequence family). Drosophila and mosquito each have a gene that is much more similar to human Icy and Skate than to Drosophila Snk. The single Drosophila and mosquito genes have been named iceskate (isk) to reflect their orthology to both Icy and Skate (da Graca, 2004).

It is suggested that DAF-5 is an ortholog of Snowski or Iceskate. (1)DAF-5 clearly has an SDS box, which is not found in any other protein in C. elegans or C. briggsae. This SDS box is more similar to the Snowski group than to the Iceskate group, including amino acids that are important for the ability of Ski to bind Smad proteins. (2) DAF-5 binds the DAF-3 Smad. This binding is mediated by the SDS box in Ski, and may thus be a conserved function of the SDS box. (3) The rate of divergence in the Snowski/Iceskate family is so high that relatively modest sequence conservation is not surprising. This rapid change can be seen when examining the SDS box of B. malayi and potato cyst nematode Snowski. These two nematode proteins are more different from each other than insect Snowski is from Human Sno and Ski. The DAF-5 gene is even more rapidly diverging in the Caenorhabditis genus. C. briggsae and C. elegans proteins average more than 70% amino acid identity. The DAF-5 sequence is only 40% identical overall between C. briggsae and C. elegans. In fact, in the Dachbox and SDS box, the difference between C. elegans and C. briggsae DAF-5 is greater than the difference between insect and human Snowski (da Graca, 2004).

Tissue-specific expression of daf-5 was used to identify cells in which it functions. daf-5cDNA::GFP was expressed with various tissue-specific promoters; these constructs had GFP inserted at the same site as a genomic construct that rescued a daf-5 mutant. Rescue was assayed in daf-7; daf-5 double mutants. Rescued animals would be expected to have the Daf-c phenotype of a daf-7 mutant. pF25B3.3 strongly expressed daf-5::GFP exclusively in nervous system and rescued daf-5 mutants as well as two positive controls. Similarly, punc-14, which expresses daf-5::GFP in nervous system at high level in addition to some non-neuronal expression, also shows strong rescue. Weak ubiquitous expression of daf-5::GFP from the pdpy-30 promoter gives partial rescue. unc-119::daf-5::GFP is weakly expressed in the nervous system but does not rescue, perhaps owing to the low level of expression. Strong expression of daf-5::GFP from the muscle promoter pmyo-3 do not rescue at all. Initial tests of two strains of pdpy-7::daf-5::GFP gave very weak expression and no rescue. Therefore, additional lines were isolated that had strong hypodermal expression of daf-5::GFP, and these did not rescue either. Overall, the results show neuronal expression of daf-5 is sufficient to rescue daf-5 dauer formation defect, while muscle or hypodermal expression is not (da Graca, 2004).

Ski/Sno proteins antagonizes TGF-β and BMP signaling

Smad proteins mediate transforming growth factor-β signaling to regulate cell growth and differentiation. The SnoN oncoprotein interacts with Smad2 and Smad4 and represses their abilities to activate transcription through recruitment of the transcriptional corepressor N-CoR. Immediately after TGF-β stimulation, SnoN is rapidly degraded by the nuclear accumulation of Smad3, allowing the activation of TGF-β target genes. By 2 hours, TGF-β induces a marked increase in SnoN expression, resulting in termination of Smad-mediated transactivation. Thus, SnoN maintains the repressed state of TGF-β-responsive genes in the absence of ligand and participates in negative feedback regulation of TGF-β signaling (Stroschein, 1999).

The Ski family of nuclear oncoproteins represses transforming growth factor-β signaling through inhibition of transcriptional activity of Smad proteins. A novel gene, fussel-15 (functional smad suppressing element on chromosome 15), has been identified with high homology to the recently discovered Fussel-18 protein. Both, Fussel-15 and Fussel-18, share important structural features, significant homology and similar genomic organization with the homolog Ski family members, Ski and SnoN. Unlike Ski and SnoN, which are ubiquitously expressed in human tissues, Fussel-15 expression, like Fussel-18, is much more restricted in its expression and is principally found in the nervous system of mouse and humans. Interestingly, Fussel-15 expression is even more restricted in adulthood to Purkinje cells of human cerebellum. In contrast to Fussel-18 that interacts with Smad 2, Smad3 and Smad4 and has an inhibitory activity on TGF-β signaling, Fussel-15 interacts with Smad1, Smad2 and Smad3 molecules and suppresses mainly BMP signaling pathway but has only minor effects on TGF-β signaling. This new protein expands the family of Ski/Sno proto-oncoproteins and represents a novel molecular regulator of BMP signaling (Arndt, 2007).

Cytoplasmic SnoN in normal tissues and nonmalignant cells antagonizes TGF-β signaling by sequestration of the Smad proteins

Transforming growth factor-β signaling requires the action of Smad proteins in association with other DNA-binding factors and coactivator and corepressor proteins to modulate target gene transcription. Smad2 and Smad3 both associate with the c-Ski and Sno oncoproteins to repress transcription of Smad target genes via recruitment of a nuclear corepressor complex. Ski-interacting protein (SKIP), a nuclear hormone receptor coactivator, was examined as a possible modulator of transcriptional regulation of the TGF-β-responsive promoter from the plasminogen activator inhibitor gene-1. SKIP augmented TGF-β-dependent transactivation in contrast to Ski/Sno-dependent repression of this reporter. SKIP interacts with Smad2 and Smad3 proteins in vivo in yeast and in mammalian cells through a region of SKIP between amino acids 201-333. In vitro, deletion of the Mad homology domain 2 (MH2) domain of Smad3 abrogated SKIP binding, like Ski/Sno, but the MH2 domain of Smad3 alone is not sufficient for protein-protein interaction. Overexpression of SKIP partially overcomes Ski/Sno-dependent repression, whereas Ski/Sno overexpression attenuates SKIP augmentation of TGF-β-dependent transcription. These results suggest a potential mechanism for transcriptional control of TGF-β signaling that involves the opposing and competitive actions of SKIP and Smad MH2-interacting factors, such as Ski and/or Sno. Thus, SKIP appears to modulate both TGF-β and nuclear hormone receptor signaling pathways (Leong, 2001).

The Ski family of nuclear oncoproteins represses TGF-beta signaling through interactions with the Smad proteins. The crystal structure of the Smad4 binding domain of human c-Ski in complex with the MH2 domain of Smad4 reveals specific recognition of the Smad4 L3 loop region by a highly conserved interaction loop (I loop) from Ski. The Ski binding surface on Smad4 significantly overlaps with that required for binding of the R-Smads. Indeed, Ski disrupts the formation of a functional complex between the Co- and R-Smads, explaining how it could lead to repression of TGF-beta, activin, and BMP responses. Intriguingly, the structure of the Ski fragment, stabilized by a bound zinc atom, resembles the SAND domain, in which the corresponding I loop is responsible for DNA binding (Wu, 2002).

c-Ski is a transcriptional corepressor that interacts strongly with Smad2, Smad3, and Smad4 but only weakly with Smad1 and Smad5. Through binding to Smad proteins, c-Ski suppresses signaling of transforming growth factor-β as well as bone morphogenetic proteins (BMPs). A mutant of c-Ski, termed c-Ski (ARPG) inhibits TGF-β/activin signaling but not BMP signaling. Selectivity was confirmed in luciferase reporter assays and by determination of cellular responses in mammalian cells (BMP-induced osteoblastic differentiation of C2C12 cells and TGF-β-induced epithelial-to-mesenchymal transdifferentiation of NMuMG cells) and Xenopus embryos. The ARPG mutant recruited histone deacetylases 1 (HDAC1) to the Smad3-Smad4 complex but not to the Smad1/5-Smad4 complex. c-Ski (ARPG) was unable to interact with Smad4, and the selective loss of suppression of BMP signaling by c-Ski (ARPG) was attributed to the lack of Smad4 binding. It was also found that c-Ski interacts with Smad3 or Smad4 without disrupting Smad3-Smad4 heteromer formation. c-Ski (ARPG) would be useful for selectively suppressing TGF-β/activin signaling (Takeda, 2004; full text of article).

c-Ski inhibits transforming growth factor-β signaling through interaction with Smad proteins. c-Ski represses Smad-mediated transcriptional activation, probably through its action as a transcriptional co-repressor. c-Ski also inhibits TGF-β-induced downregulation of genes such as c-myc. However, mechanisms for transcriptional regulation of target genes by c-Ski have not been fully determined. This study examines how c-Ski inhibits both TGF-β-induced transcriptional activation and repression. DNA-affinity precipitation analysis revealed that c-Ski enhances the binding of Smad2 and 4, and to a lesser extent Smad3, to both CAGA and TGF-β1 inhibitory element probes. A c-Ski mutant, which is unable to interact with Smad4, failed to enhance the binding of Smad complex on these probes and to inhibit the Smad-responsive promoter. These results suggest that stabilization of inactive Smad complexes on DNA is a critical event in c-Ski-mediated inhibition of TGF-β signaling (Suzuki, 2004).

TGF-β is a ubiquitously expressed cytokine that signals through the Smad proteins to regulate many diverse cellular processes. SnoN is an important negative regulator of Smad signaling. It has been described as a nuclear protein, based on studies of ectopically expressed SnoN and endogenous SnoN in cancer cell lines. In the nucleus, SnoN binds to Smad2, Smad3, and Smad4 and represses their ability to activate transcription of TGF-β target genes through multiple mechanisms. Whereas SnoN is localized exclusively in the nucleus in cancer tissues or cells, in normal tissues and nontumorigenic or primary epithelial cells, SnoN is predominantly cytoplasmic. Upon morphological differentiation or cell-cycle arrest, SnoN translocates into the nucleus. In contrast to nuclear SnoN that represses the transcriptional activity of the Smad complexes, cytoplasmic SnoN antagonizes TGF-β signaling by sequestering the Smad proteins in the cytoplasm. Interestingly, cytoplasmic SnoN is resistant to TGF-β-induced degradation and therefore is more potent than nuclear SnoN in repressing TGF-β signaling. Thus, this study has identified a mechanism for regulation of TGF-β signaling via differential subcellular localization of SnoN that is likely to produce different patterns of downstream TGF-β responses and may influence the proliferation or differentiation states of epithelial cells (Krakowski, 2005; full text of article).

Ski/Sno as a transcriptional co-repressor acting through interaction with smads

Ski was first identified as a viral oncogene (v-ski) from the avian Sloan-Kettering retrovirus (SKV) that transforms chicken embryo fibroblasts. The human cellular homolog c-ski was later cloned based on its homology with v-ski and was found to encode a nuclear protein of 728 amino acids. Compared with c-Ski, v-Ski is truncated mostly at the carboxyl terminus. However, this truncation is not responsible for the activation of ski as an oncogene. Overexpression of wild-type c-Ski also results in oncogenic transformation of chicken and quail embryo fibroblasts. The transforming activity of Ski is likely attributable to overexpression, not truncation, of the c-Ski protein. Consistent with this notion, an elevated level of c-Ski has been detected in several human tumor cell lines derived from neuroblastoma, melanoma, and prostate cancer. c-ski is a unique oncogene; in addition to affecting cell growth, it is also involved in regulation of muscle differentiation. Overexpression of Ski results in muscle differentiation of quail embryo cells and hypertrophy of skeletal muscle in mice. Furthermore, mice lacking c-ski display defective muscle and neuronal differentiation. At the molecular level, Ski can function either as a transcriptional activator or as a repressor depending on the specific promoters involved. It has been shown to bind to DNA, but only in conjunction with other cellular proteins. Ski is a component of the histone deacetylase (HDAC1) complex through binding to the nuclear hormone receptor corepressor (N-CoR) and mSin3A, and mediated transcriptional repression of the thyroid hormone receptor, Mad and pRb. The interaction between Ski and N-CoR is mediated by the amino-terminal part of Ski. This region is also essential for the transforming activity of c-Ski and is conserved among ski family members, including v-Ski and c-SnoN. This raises an interesting possibility that the transforming activity of Ski may be linked to its function as a transcriptional corepressor (Luo, 1999 and references therein).

Ski can interact directly with Smad2, Smad3, and Smad4 on a TGF beta-responsive promoter element and repress their abilities to activate transcription through recruitment of the nuclear transcriptional corepressor N-CoR, and possibly its associated histone deacetylase complex. Thus Ski is a transcriptional corepressor of Smads. Overexpression of Ski in a TGF beta-responsive cell line renders it resistant to TGF beta-induced growth inhibition and defective in activation of JunB expression. This ability to overcome TGF beta-induced growth arrest may be responsible for the transforming activity of Ski in human and avian cancer cells. These studies suggest a new paradigm for inactivation of the Smad proteins by an oncoprotein through transcriptional repression (Luo, 1999).

Using a nuclear extract from c-ski-transformed cells, a specific DNA-binding site for Ski and its associated proteins was identified (GTCTAGAC) by cyclic amplification and selection of targets (CASTing). The Ski binding site was found to mediate transcriptional repression by Ski, suggesting that Ski may bind to DNA through interaction with the Smads. Ski/Smad3 and Ski/Smad4 complexes can bind to SBE and repress Smad-mediated transcriptional activation. Thus, Smad3 and Smad4 are the DNA-binding partners of Ski in these c-ski-transformed cells. In addition to SBE, Ski has also been found to interact with the nuclear factor I (NFI) binding site through interaction with the NFI protein. However, in this context, Ski functions to potentiate, not repress, NFI-stimulated transcriptional activation. Thus, Ski may interact with different DNA-binding factors and regulate transcription both positively and negatively depending on the proper cellular context or interacting partners (Luo, 1999 and references).

Ski also binds directly to Rb and retinoic acid receptor and to repress transactivation induced by these proteins, probably through similar mechanisms. N-CoR was originally identified as a corepressor that mediates transcriptional repression by the thyroid hormone receptor and Mad. It is a protein of 270 kD and contains three repressor domains in its amino-terminal region. It shows a striking homology to another corepressor, SMRT, and represses transcription by forming complexes with mSin3 and HDAC. Although no specific interactions between the Smads and endogenous mSin3A or HDAC could be detected because of technical difficulties, the recruitment of an N-CoR complex to the Smads suggests that repression of Smad-mediated transcription by Ski may involve deacetylation of nucleosomal histones. Recently, Smad2 has been shown to interact with TGIF, another transcriptional corepressor that recruits HDAC to the Smads. Thus, repression of Smad-mediated transactivation may involve multiple corepressors. Future studies will allow for a determination of whether Ski, Smads, N-CoR, and TGIF are in the same complex or whether Smads interact with different corepressors depending on the expression level of these corepressors in different cell types or at different developmental stages (Luo, 1999 and references therein).

Smads are intracellular signaling mediators of the transforming growth factor-β superfamily that regulates a wide variety of biological processes. Among them, Smads 2 and 3 are activated specifically by TGF-β. c-Ski has been identified as a Smad2 interacting protein. c-Ski is the cellular homologue of the v-ski oncogene product and has been shown to repress transcription by recruiting histone deacetylase (HDAC). Smad2/3 interacts with c-Ski through its C-terminal MH2 domain in a TGF-β-dependent manner. c-Ski contains two distinct Smad-binding sites with different binding properties. c-Ski strongly inhibits transactivation of various reporter genes by TGF-β. c-Ski is incorporated in the Smad DNA binding complex, interferes with the interaction of Smad3 with a transcriptional co-activator, p300, and in turn recruits HDAC. c-Ski is thus a transcriptional co-repressor that links Smads to HDAC in TGF-β signaling (Akiyoshi, 1999).

The N-CoR/SMRT complex containing mSin3 and histone deacetylase (HDAC) mediates transcriptional repression by nuclear hormone receptors and Mad. The proteins encoded by the ski proto-oncogene family directly bind to N-CoR/SMRT and mSin3A, and forms a complex with HDAC. c-Ski and its related gene product Sno are required for transcriptional repression by Mad and thyroid hormone receptor (TRbeta). The oncogenic form, v-Ski, which lacks the mSin3A-binding domain, acts in a dominant-negative fashion, and abrogates transcriptional repression by Mad and TRbeta. In ski-deficient mouse embryos, the ornithine decarboxylase gene, whose expression is normally repressed by Mad-Max, is expressed ectopically. These results show that Ski is a component of the HDAC complex and that Ski is required for the transcriptional repression mediated by this complex. The involvement of c-Ski in the HDAC complex indicates that the function of the HDAC complex is important for oncogenesis (Nomura, 1999).

The Ski and Sno oncoproteins are components of a macromolecular complex containing the co-repressor N-CoR/SMRT, mSin3 and histone deacetylase. This complex has been implicated in the transcriptional repression exerted by a number of repressors including nuclear hormone receptors and Mad. Further more, Ski and Sno negatively regulate TGF-beta signaling by recruiting this complex to Smads. Loss of one copy of sno increases susceptibility to tumorigenesis in mice. Mice lacking sno die at an early stage of embryogenesis, and sno is required for blastocyst formation. Heterozygous (sno^+/-) mice develop spontaneous lymphomas at a low frequency and show an increased level of tumor formation relative to wild-type mice when challenged with a chemical carcinogen. sno^+/- embryonic fibroblasts have an increased proliferative capacity and the introduction of activated Ki-ras into these cells resulted in neoplastic transformation. The B cells, T cells and embryonic fibroblasts of sno^+/- mice have a decreased sensitivity to apoptosis or cell cycle arrest. These findings demonstrate that sno acts as a tumor suppressor at least in some types of cells (Shinagawa, 2000).

SnoN and Ski oncoproteins are co-repressors for Smad proteins and repress TGF-β-responsive gene expression. The smad7 gene is a TGF-β target induced by Smad signaling, and its promoter contains the Smad-binding element (SBE) required for a positive regulation by the TGF-β/Smad pathway. SnoN and Ski co-repressors also bind SBE but regulate negatively smad7 gene. Ski along with Smad4 binds and represses the smad7 promoter, whereas the repression mechanism by SnoN is not clear. Ski and SnoN overexpression inhibits smad7 reporter expression induced through TGF-β signaling. Using chromatin immunoprecipitation assays, it was found that SnoN binds smad7 promoter at the basal condition, whereas after a short TGF-β treatment for 15-30 min SnoN is downregulated and no longer bound smad7 promoter. Interestingly, after a prolonged TGF-β treatment SnoN is upregulated and returns to its position on the smad7 promoter, functioning probably as a negative feedback control. Thus, SnoN also seems to regulate negatively the TGF-β-responsive smad7 gene by binding and repressing its promoter in a similar way to Ski (Briones-Orta, 2006).

c-Ski activates MyoD in the nucleus of myoblastic cells through suppression of histone deacetylases

c-Ski, originally identified as an oncogene product, induces myogenic differentiation in nonmyogenic fibroblasts through transcriptional activation of muscle regulatory factors. Although c-Ski does not bind to DNA directly, it binds to DNA through interaction with Smad proteins and regulates signaling activities of transforming growth factor-β. c-Ski has been shown to activate the myogenin promoter independently of regulation of endogenous TGF-β signaling. Expression of myogenin is regulated by a transcription factor complex containing proteins of the MyoD family and the myocyte enhancer factor 2 (MEF2) family. c-Ski acts on the MyoD-MEF2 complex and modulates the activity of MyoD in myogenin promoter regulation. Interestingly, histone deacetylase (HDAC) inhibitors up-regulated basal activity of transcription from a MyoD-responsive reporter, although c-Ski failed to further augment this transcription in the presence of HDAC inhibitors. c-Ski is observed both in the cytoplasm and in the nucleus, but its nuclear localization is required for myogenic differentiation. It is concluded that c-Ski induces myogenic differentiation through acting on MyoD and inhibiting HDAC activity in the nucleus of myogenic cells (Kobayashi, 2007).

Competition between Ski and CBP for binding to Smads in TGF-β signaling

The family of Smad proteins mediates transforming growth factor-β signaling in cell growth and differentiation. Smad proteins repress or activate TGF-β signaling by interacting with corepressors (e.g., Ski) or coactivators (e.g., CREB binding protein [CBP]), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads, and whether they can interact with Smad3 at the same binding surface on Smad3. This study investigated the interactions among purified constructs of Smad, Ski and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Ski (aa 16-192) interacts directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g., Smad2 or Smad3, to form a hexamer; Ski (aa 16-192) interacts with an R-Smad/Smad4 heterotrimer to form a pentamer. CBP (aa 1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer, and with an R-Smad/Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP compete with each other for binding to Smad3. Mutational studies reveal that domains of Ski and CBP interact with Smad3 at a portion of the Smad anchor for receptor activation (SARA)-binding surface. These results suggest that Ski negatively regulates TGF-β signaling by replacing CBP in R-Smad complexes. A working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-β pathway (Chen, 2007),

Ski activates Wnt/β-catenin signalling in human melanoma

Overexpression of the oncoprotein SKI correlates with the progression of human melanoma in vivo. SKI is known to curtail the growth inhibitory activity of tumor growth factor β through the formation of repressive transcriptional complexes with Smad2 and Smad3 at the p21(Waf-1) promoter. This study shows that SKI also stimulates growth by activating the Wnt signaling pathway. From a yeast two-hybrid screen and immunoprecipitation studies, the protein FHL2/DRAL was identified as a novel SKI binding partner. FHL2, a LIM-only protein, binds β-catenin and can function as either a transcriptional repressor or activator of the Wnt signaling pathway. SKI enhances the activation of FHL2 and/or β-catenin- regulated gene promoters in melanoma cells. Among the SKI targets were microphthalmia-associated transcription factor and Nr-CAM, two proteins associated with melanoma cell survival, growth, motility, and transformation. Transient overexpression of SKI and FHL2 in ski-/- melanocytes synergistically enhanced cell growth, and stable overexpression of SKI in a poorly clonogenic human melanoma cell line was sufficient to stimulate rapid proliferation, decreasing the number of cells in the G₁ phase of the cell cycle, and dramatically increasing clonogenicity, colony size and motility. Taken together, these results suggest that by targeting members of the tumor growth factor β and β-catenin pathways, SKI regulates crucial events required for melanoma growth, survival, and invasion (Chen, 2003).

c-Jun associates with the oncoprotein Ski and suppresses Smad2 transcriptional activity

The Smad proteins are key intracellular effectors of transforming growth factor-β cytokines. The ability of Smads to modulate transcription results from a functional cooperativity with the coactivators p300/cAMP-response element-binding protein-binding protein (CBP), or the corepressors TGIF and Ski. The c-Jun N-terminal kinase (JNK) pathway, another downstream target activated by TGF-β receptors, has also been suggested to inhibit TGF-β signaling through interaction of c-Jun with Smad2 and Smad3. c-Jun directly interacts with Ski to enhance the association of Ski with Smad2 in the basal state. Interestingly, TGF-β signaling induces dissociation of c-Jun from Ski, thereby relieving active repression by c-Jun. Moreover, activation of JNK pathway suppresses the ability of TGF-β to induce dissociation of c-Jun from ski. Thus, the formation of a c-Jun/Ski complex maintains the repressed state of Smad2-responsive genes in the absence of ligand and participates in negative feedback regulation of TGF-β signaling by the JNK cascade (Pessah, 2002).

Ski is involved in transcriptional regulation by the repressor and full-length forms of Gli3

Transcription factor Glioblastoma-3 (Gli3) is cleaved in the anterior region of the limb bud to generate its repressor form. In contrast, Sonic hedgehog (Shh) signaling from the posterior zone of polarizing activity blocks Gli3 processing and then induces the expression of Gli3 target genes, including Gli1. This study reports that the Ski corepressor binds to Gli3 and recruits the histone deacetylase complex. The Gli3-mediated repression was impaired by anti-Ski antibody and in Ski-deficient fibroblasts, and Shh-induced Gli1 gene transcription mediated by full-length Gli3 was inhibited by Ski. Furthermore, a Ski mutation enhanced the digit abnormalities caused by the Gli3 gene mutation. Thus, Ski plays an important role in pattern formation (Dai, 2003).

Ski interaction with Myb

The c-myb proto-oncogene product (c-Myb) regulates proliferation of hematopoietic cells by inducing the transcription of a group of target genes. Removal or mutations of the negative regulatory domain (NRD) in the C-terminal half of c-Myb leads to increased transactivating capacity and oncogenic activation. TIF1β directly binds to the NRD and negatively regulates the c-Myb-dependent trans-activation. In addition, three corepressors (Ski, N-CoR, and mSin3A) bind to the DNA-binding domain of c-Myb together with TIF1β and recruit the histone deacetylase complex to c-Myb. Furthermore, the Drosophila TIF1β homolog, Bonus, negatively regulates Drosophila Myb activity. The Ski corepressor competes with the coactivator CBP for binding to c-Myb, indicating that the selection of coactivators and corepressors is a key event for c-Myb-dependent transcription. Mutations or deletion of the NRD of c-Myb and the mutations found in the DNA-binding domain of v-Myb decrease the interaction with these corepressors and weaken the corepressor-induced negative regulation of Myb activity. These observations have conceptual implications for understanding how the nuclear oncogene is activated (Nomura, 2004).

Ski negatively regulates erythroid differentiation through its interaction with GATA1

The Ski oncoprotein dramatically affects cell growth, differentiation, and/or survival. Recently, Ski was shown to act in distinct signaling pathways including those involving nuclear receptors, transforming growth factor β, and tumor suppressors. These divergent roles of Ski are probably dependent on Ski's capacity to bind multiple partners with disparate functions. In particular, Ski alters the growth and differentiation program of erythroid progenitor cells, leading to malignant leukemia. However, the mechanism underlying this important effect has remained elusive. This study shows that Ski interacts with GATA1, a transcription factor essential in erythropoiesis. Using a Ski mutant deficient in GATA1 binding, it was shown that this Ski-GATA1 interaction is critical for Ski's ability to repress GATA1-mediated transcription and block erythroid differentiation. Furthermore, the repression of GATA1-mediated transcription involves Ski's ability to block DNA binding of GATA1. This finding is in marked contrast to those in previous reports on the mechanism of repression by Ski, which have described a model involving the recruitment of corepressors into DNA-bound transcription complexes. It is proposed that Ski cooperates in the process of transformation in erythroid cells by interfering with GATA1 function, thereby contributing to erythroleukemia (Ueki, 2004).

Sumoylatation of SnoN

The transcriptional modulator SnoN controls a diverse set of biological processes, including cell proliferation and differentiation. The mechanisms by which SnoN regulates these processes remain incompletely understood. Recent studies have shown that SnoN exerts positive or negative regulatory effects on transcription. Because post-translational modification of proteins by small ubiquitin-like modifier (SUMO; see Drosophila SUMO) represents an important mechanism in the control of the activity of transcriptional regulators, it was asked if this modification regulates SnoN function. This study shows that SnoN is sumoylated. The data demonstrate that the SUMO-conjugating E2 enzyme Ubc9 is critical for SnoN sumoylation and that the SUMO E3 ligase PIAS1 selectively interacts with and enhances the sumoylation of SnoN. Lysine residues 50 and 383 have been as the SUMO acceptor sites in SnoN. Analyses of SUMO 'loss-of-function' and 'gain-of-function' SnoN mutants in transcriptional reporter assays reveal that sumoylation of SnoN contributes to the ability of SnoN to repress gene expression in a promoter-specific manner. Although this modification has little effect on SnoN repression of the plasminogen activator inhibitor-1 promoter and only modestly potentiates SnoN repression of the p21 promoter, SnoN sumoylation robustly augments the ability of SnoN to suppress transcription of the myogenesis master regulatory gene myogenin. In addition, the SnoN SUMO E3 ligase, PIAS1, at its endogenous levels, suppresses myogenin transcription. Collectively, these findings suggest that SnoN is directly regulated by sumoylation leading to the enhancement of the ability of SnoN to repress transcription in a promoter-specific manner. This study also points to a physiological role for SnoN sumoylation in the control of myogenin expression in differentiating muscle cells (Hsu, 2006).

Recent progress has been made on the role of oncoproteins c-Ski and related SnoN in the control of cellular transformation. c-Ski/SnoN potently repress TGF-β antiproliferative signaling through physical interaction with signal transducers called Smads. Overexpression of c-Ski/SnoN also induces skeletal muscle differentiation, but how c-Ski/SnoN function in myogenesis is largely unknown. During an investigation on the role of sumoylation in TGF-β signaling, it was inadvertently found that SnoN is modified by small ubiquitin-like modifier-1 (SUMO-1). SnoN sumoylation was characterized in detail and the physiological function of the modification is reported. Sumoylation occurs primarily at lysine 50 (Lys-50). PIAS1 and PIASx proteins physically interact with SnoN to stimulate its sumoylation, thus serving as SUMO-protein isopeptide ligases (E3) for SnoN sumoylation. SnoN sumoylation does not alter its metabolic stability or its ability to repress TGF-β signaling. Notably, loss of sumoylation in the Lys-50 site (via a Lys-to-Arg point mutation) potently activates muscle-specific gene expression and enhances myotube formation. This study suggests a novel role for SUMO modification in the regulation of myogenic differentiation (Wrighton, 2007).

Nuclear and cytoplasmic c-Ski differently modulate cellular functions

c-Ski is a proto-oncogene product that induces morphologic transformation, anchorage independence, and myogenic differentiation when it is over-expressed in mesenchymal cells. c-Ski also inhibits signaling of transforming growth factor-β superfamily members through interaction with Smad proteins. Although c-Ski is predominantly localized in the nucleus, aberrant cytoplasmic localization of it has also been reported in some tumor tissues and cell lines. In the present study, the nuclear localization signal (NLS) in c-Ski was identified. By introducing a mutation to abolish NLS activity, the function of cytoplasmic c-Ski was examined. Although cytoplasmic c-Ski suppresses TGF-β superfamily-induced Smad signaling through sequestration of activated Smad complex to the cytoplasm, it fails to exhibit some of the activities that require nuclear localization of c-Ski, including suppression of basal transcription of the Smad7 gene. These findings indicate that subcellular localization of c-Ski affects its biologic activities. c-Ski accumulates in the cytoplasm when proteasome activity is inhibited. Mapping of the regions required for cytoplasmic accumulation by proteasome inhibitors suggests that subcellular localization of c-Ski may be regulated by proteasome-sensitive processes through amino acid residues 94-210 and 491-548 (Nagata, 2006).

Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN

Smad proteins mediate transforming growth factor-β signaling to regulate cell growth and differentiation. SnoN is an important negative regulator of TGF-β signaling that functions to maintain the repressed state of TGF-β target genes in the absence of ligand. On TGF-β stimulation, Smad3 and Smad2 translocate into the nucleus and induce a rapid degradation of SnoN, allowing activation of TGF-β target genes. Smad2- or Smad3-induced degradation of SnoN requires the ubiquitin-dependent proteasome and can be mediated by the anaphase-promoting complex (APC) and the UbcH5 family of ubiquitin-conjugating enzymes. Smad3 and to a lesser extent, Smad2, interact with both the APC and SnoN, resulting in the recruitment of the APC to SnoN and subsequent ubiquitination of SnoN in a destruction box (D box)-dependent manner. In addition to the D box, efficient ubiquitination and degradation of SnoN also requires the Smad3 binding site in SnoN as well as key lysine residues necessary for ubiquitin attachment. Mutation of either the Smad3 binding site or lysine residues results in stabilization of SnoN and in enhanced antagonism of TGF-β signaling. These studies elucidate an important mechanism and pathway for the degradation of SnoN and more importantly, reveal a novel role of the APC in the regulation of TGF-β signaling (Stroschein, 2001).

TAK1 MAPKKK mediates TGF-β signaling by targeting SnoN oncoprotein for degradation

Transforming growth factor-β regulates a variety of physiologic processes through essential intracellular mediators Smads. The SnoN oncoprotein is an inhibitor of TGF-β signaling. SnoN recruits transcriptional repressor complex to block Smad-dependent transcriptional activation of TGF-β-responsive genes. Following TGF-β stimulation, SnoN is rapidly degraded, thereby allowing the activation of TGF-β target genes. This study reports the role of TAK1 as a SnoN protein kinase. TAK1 interacts with and phosphorylates SnoN, and this phosphorylation regulates the stability of SnoN. Inactivation of TAK1 prevents TGF-β-induced SnoN degradation, and impairs induction of the TGF-β-responsive genes. These data suggest that TAK1 modulates TGF-β dependent cellular responses by targeting SnoN for degradation (Kajino, 2007).

Mutation and knock-down of Sno

The proto-oncogene Sno has been shown to be a negative regulator of transforming growth factor beta (TGF-beta) signaling in vitro, using overexpression and artificial reporter systems. To examine Sno function in vivo, two targeted deletions at the Sno locus were made: a 5' deletion, with reduced Sno protein (hypomorph), and an exon 1 deletion removing half the protein coding sequence, in which Sno protein is undetectable in homozygotes (null). Homozygous Sno hypomorph and null mutant mice are viable without gross developmental defects. Sno mRNA was found constitutively expressed in normal thymocytes and splenic T cells, with increased expression 1 h following T-cell receptor ligation. Although thymocyte and splenic T-cell populations appeared normal in mutant mice, T-cell proliferation in response to activating stimuli was defective in both mutant strains. This defect could be reversed by incubation with either anti-TGF-beta antibodies or exogenous interleukin-2 (IL-2). Together, these findings suggest that Sno-dependent suppression of TGF-beta signaling is required for upregulation of growth factor production and normal T-cell proliferation following receptor ligation. Indeed, both IL-2 and IL-4 levels are reduced in response to anti-CD3 epsilon stimulation of mutant T cells, and transfected Sno activated an IL-2 reporter system in non-T cells. Mutant mouse embryo fibroblasts also exhibited a reduced cell proliferation rate that could be reversed by administration of anti-TGF-beta. These data provide strong evidence that Sno is a significant negative regulator of antiproliferative TGF-beta signaling in both T cells and other cell types in vivo (Pearson-White, 2003).

The transforming growth factor-β family of secreted proteins have pleiotropic functions that are critical to normal development and homeostasis. However, the intracellular mechanisms by which the TGF-β proteins elicit cellular responses remain incompletely understood. The Smad proteins provide a major means for the propagation of the TGF-β signal from the cell surface to the nucleus, where the Smad proteins regulate gene expression leading to TGF-β-dependent cellular responses including the inhibition of cell proliferation. Recent studies have suggested that a nuclear Smad-interacting protein termed SnoN, when overexpressed in cells, suppresses TGF-β-induced Smad signaling and TGF-β inhibition of cell proliferation. However, the physiologic function of endogenous SnoN in TGF-β-mediated biological responses remains to be elucidated. This study determined the effect of genetic knock-down of SnoN by RNA interference on TGF-β responses in mammalian cells. Unexpectedly, SnoN knock-down was found to specifically inhibit TGF-β-induced transcription in the lung epithelial cell line Mv1Lu but not in HeLa or HaCaT cells. SnoN knock-down was also found to block TGF-β-dependent cell cycle arrest in Mv1Lu cells. Collectively, these data indicate that rather than suppressing TGF-β-induced responses, endogenous SnoN acts as a positive mediator of TGF-β-induced transcription and cell cycle arrest in lung epithelial cells. This study also shows that SnoN couples the TGF-β signal to gene expression in a cell-specific manner (Sarker, 2005).

DACH1 inhibits TGF-beta signaling through binding Smad4

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, it is hypothesized that DACH1 might play a similar cellular function. DACH1 has been found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-ß-induced apoptosis. DACH1 represses TGF-ß induction of AP-1 and Smad signaling in gene reporter assays and represses endogenous TGF-ß-responsive genes by microarray analyses. DACH1 binds to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localizes with NCoR in nuclear dotlike structures. NCoR enhances DACH1 repression, and the repression of TGF-ß-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH is sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibits the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-ß signaling by interacting with NCoR and Smad4 (Wu, 2003).

DACH1 functions as a transcriptional repressor of TGF-ß signaling. DACH1 represses TGF-ß-induced activity of both Smad/FAST1 Binding Element (SBE) and AP-1 activity and inhibits TGF-ß-induced apoptosis in MDA-MB-231 cells. NCoR enhances repression of TGF-ß signaling by DACH1. Repression by DACH1 requires Smad4, being abrogated in Smad4-deficient cells and restored by Smad4 coexpression. Repression by DACH1 requires a conserved DS domain that binds the transcriptional co-repressor NCoR. DACH1 and NCoR co-localize in a substantial proportion of subnuclear dotlike structures by confocal microscopy. Together, these findings suggest NCoR may participate in DACH1-mediated repression of gene expression (Wu, 2003).

DACH1 is detectable in MDA-MB-231 cells by Western blotting, and genome-wide analysis of DACH1-responsive genes in these cells indicates that 422 genes of 17,000 are regulated >2-fold by DACH1 expression. Consistent with the reporter gene analysis demonstrating DACH1 inhibition of AP-1 activity, several AP-1-responsive genes are repressed by DACH1 expression, including c-fos, Egr1, cyclin E2, neuregulin, tumor necrosis factor alpha-induced protein 3, cdc25A, FGF5, GRO3, MEF2C, ETR101, and BMP4. A comparison between genes regulated significantly by DACH1 with recent studies of TGF-ß signaling using a similar approach has demonstrated that genes induced by TGF-ß in other cell types are repressed by DACH1 (ATF3, interleukin-11, P2RY2) and several genes repressed by TGF-ß are induced by DACH1 (ID1 and interleukin-1-ß). Comparison between genome wide analysis 'fingerprints' must be considered with caution; however, it is of interest that of 70 genes regulated by TGF-ß, 22 of those genes are also significantly regulated by DACH1 expression; similarly, there is overlap with TGF-ß response genes in recent publications. The functions of these genes are diverse and include cell division, transcriptional regulation, cellular adhesion, extracellular matrix remodeling, and signal transduction. The use of genome-wide expression studies to identify clusters of genes representing a molecular signature of DACH1-regulated activity suggests a normal function for DACH1 in the inhibition of AP-1-regulated genes. The current studies suggest DACH1 may function to regulate aberrant TGF-ß signals that play important roles in human breast cancer progression. TGF-ß itself plays an important role in cancer progression by functioning both as an antiproliferative factor and as a tumor promoter. The numerous components of the signal conduction pathway are tumor suppressors that are functionally mutated in cancer (Wu, 2003).

DACH1 was found within a complex bound to a FAST1/SBE DNA binding site with Smad4. Immunopurified DACH1, however, does not bind DNA directly, suggesting that Smad4 serves as a DNA-bound platform to recruit DACH1. The DACH1 DS domain alone is insufficient for Smad4 binding, which requires the EYAD domain and is defective in SBE and AP-1 repression. DACH1 co-immunoprecipitates with Smad4 from cultured cells, and the association of DACH1 with Smad4 was observed in reciprocal immunoprecipitation. DACH1 associates with Smad4 in vitro using GST pull-down experiments, and, like Ski, multiple domains in DACH1 are required, including both the DS and EYA domains. Using saturating immunoprecipitation, the relative amount of co-precipitated Smad4 was greater for Ski than DACH1. In contrast, the relative abundance of NCoR coprecipitating with DACH1 is relatively greater than that associated with Ski. The finding that the DACH1DeltaDS domain mutant abrogates Ski-mediated repression of SBE activity suggests that DACH1 and Ski may function in a similar pathway (Wu, 2003).

DACH1, like Ski, represses Smad3-regulated transactivation of either SBE or AP-1 activity. These findings with Ski are similar to previous findings but contrast with the effect of Sno-N, which has little effect on Smad3 transactivation. Sno-N is degraded rapidly in response to Smad3 or TGF-ß, whereas Ski expression and DACH1 expression are not affected greatly by TGF-ß. These findings suggest distinct roles for Sno-N versus Ski-N and DACH1 in TGF-ß signaling (Wu, 2003).

DACH1 inhibits TGF-ß- and Smad-induced AP-1 activity. Inhibition of TGF-ß and Smad-induced AP-1 activity requires the DACH1 DS domain. TGF-ß induction of several genes, including PAI-1, clusterin, monocyte chemoattractant protein-1 (JE/MCP-1), type I collagen, and TGF-ß itself depends on AP-1 DNA-binding sites in the promoter region of these genes. Induction of AP-1 activity by TGF-ß involves interactions between Smads and AP-1 transcription factors. Smads bind directly to the Jun family, and both Smad3 and Smad4 can bind JunB, c-Jun, and JunD. Since the regions of DACH1 that bind Smads are required for repression of TGF-ß-induced AP-1 activity, it is likely that DACH1 mediates AP-1 repression through Smad4 association (Wu, 2003).

The identification of DACH1 as a new co-repressor of TGF-ß signaling extends understanding of this key pathway. The role of TGF-ß in cancer includes a complex function as both an antiproliferative activity and as a tumor promoter. DACH1, like Sno-N and v-Ski oncogenes, bind directly to NCoR/SMRT and mSin3. TGF-ß controls a plethora of cellular functions and regulates development and homeostasis. Since DACH1 and SKI have only partially overlapping expression patterns, with DACH1 expressed in neuroblastomas and in cell lines derived from pancreas and breast cancer cell lines, it is possible that DACH1 contributes in a cell type-specific manner to regulate TGF-ß signaling (Wu, 2003).

Requirement for the SnoN oncoprotein in transforming growth factor β-induced oncogenic transformation of fibroblast cells

Transforming growth factor β was originally identified by virtue of its ability to induce transformation of the AKR-2B and NRK fibroblasts but was later found to be a potent inhibitor of the growth of epithelial, endothelial, and lymphoid cells. Although the growth-inhibitory pathway of TGF-β mediated by the Smad proteins is well studied, the signaling pathway leading to the transforming activity of TGF-β in fibroblasts is not well understood. This study shows that SnoN, a member of the Ski family of oncoproteins, is required for TGF-β-induced proliferation and transformation of AKR-2B and NRK fibroblasts. TGF-β induces upregulation of snoN expression in both epithelial cells and fibroblasts through a common Smad-dependent mechanism. However, a strong and prolonged activation of snoN transcription that lasts for 8 to 24 h is detected only in these two fibroblast lines. This prolonged induction is mediated by Smad2 and appears to play an important role in the transformation of both AKR-2B and NRK cells. Reduction of snoN expression by small interfering RNA or shortening of the duration of snoN induction by a pharmacological inhibitor impaired TGF-β-induced anchorage-independent growth of AKR-2B cells. Interestingly, Smad2 and Smad3 play opposite roles in regulating snoN expression in both fibroblasts and epithelial cells. The Smad2/Smad4 complex activates snoN transcription by direct binding to the TGF-β-responsive element in the snoN promoter, while the Smad3/Smad4 complex inhibits it through a novel Smad inhibitory site. Mutations of Smad4 that render it defective in heterodimerization with Smad3, which are found in many human cancers, convert the activity of Smad3 on the snoN promoter from inhibitory to stimulatory, resulting in increased snoN expression in cancer cells. Thus, this study demonstrates a novel role of SnoN in the transforming activity of TGF-β in fibroblasts and also uncovered a mechanism for the elevated SnoN expression in some human cancer cells (Zhu, 2005; full text of article).

Dual role of SnoN in mammalian tumorigenesis

SnoN is an important negative regulator of transforming growth factor β signaling through its ability to interact with and repress the activity of Smad proteins. It was originally identified as an oncoprotein based on its ability to induce anchorage-independent growth in chicken embryo fibroblasts. However, the roles of SnoN in mammalian epithelial carcinogenesis have not been well defined. This study shows that SnoN plays an important but complex role in human cancer. SnoN expression is highly elevated in many human cancer cell lines, and this high level of SnoN promotes mitogenic transformation of breast and lung cancer cell lines in vitro and tumor growth in vivo, consistent with its proposed pro-oncogenic role. However, this high level of SnoN expression also inhibits epithelial-to-mesenchymal transdifferentiation. Breast and lung cancer cells expressing the shRNA for SnoN exhibited an increase in cell motility, actin stress fiber formation, metalloprotease activity, and extracellular matrix production as well as a reduction in adherens junction proteins. Supporting this observation, in an in vivo breast cancer metastasis model, reducing SnoN expression was found to moderately enhance metastasis of human breast cancer cells to bone and lung. Thus, SnoN plays both pro-tumorigenic and antitumorigenic roles at different stages of mammalian malignant progression. The growth-promoting activity of SnoN appears to require its ability to bind to and repress the Smad proteins, while the antitumorigenic activity can be mediated by both Smad-dependent and Smad-independent pathways and requires the activity of small GTPase RhoA. This study has established the importance of SnoN in mammalian epithelial carcinogenesis and revealed a novel aspect of SnoN function in malignant progression (Zhu, 2007).

Cell-intrinsic regulation of axonal morphogenesis by the Cdh1-APC target SnoN

Axonal growth is fundamental to the establishment of neuronal connectivity in the brain. However, the cell-intrinsic mechanisms that govern axonal morphogenesis remain to be elucidated. The ubiquitin ligase Cdh1-anaphase-promoting complex (Cdh1-APC) suppresses the growth of axons in postmitotic neurons. Cdh1-APC operates in the nucleus to inhibit axonal growth. The transcriptional corepressor SnoN is a key target of neuronal Cdh1-APC that promotes axonal growth. Cdh1 forms a physical complex with SnoN and stimulates the ubiquitin-dependent proteasomal degradation of SnoN in neurons. Knockdown of SnoN in neurons significantly reduces axonal growth and suppresses Cdh1 RNAi enhancement of axonal growth. In addition, SnoN knockdown in vivo suggests an essential function for SnoN in the development of granule neuron parallel fibers in the cerebellar cortex. These findings define Cdh1-APC and SnoN as components of a cell-intrinsic pathway that orchestrates axonal morphogenesis in a transcription-dependent manner in the mammalian brain (Stegmuller, 2006).

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