SIAH1介导TRB3的泛素化和降解

目的利用酵母回转实验和免疫共沉淀实验验证SIAHI和TRB3之间的相互作用并探讨其功能相关性。方法将全长形式的TRB3基因和SIAH1基因分别克隆入酵母表达载体pDBLeu和pPC86中,共转化至MaV203酵母感受态细胞,验证其相互作用,然后分别构建至真核表达载体pCMV—Myc和pFLAG—CMV-2中,采用免疫共沉淀实验进行进一步验证。通过体内泛素化实验检测SIAH1对TRB3蛋白稳定性及泛素化修饰的影响。结果通过在酵母细胞中的回转实验和HEK293rr细胞中的免疫共沉淀实验证实了TRB3与SIAH1之间的相互作用。通过体内泛素化实验证实了S1AH1介导了TRB3的泛素化修饰和降解。结论证实了TRB3与SIAH1之间的相互作用并发现SIAH1介导了TRB3的泛素化修饰和降解,为TRB3蛋白的功能研究提供了新的线索。     

CHIP controls the sensitivity of transforming growth factor-beta signaling by modulating the basal level of Smad3 through ubiquitin-mediated degradation

Transforming growth factor-beta (TGF-beta) signaling is critical in a variety of biological processes such as cell proliferation, differentiation, and apoptosis. TGF-beta signaling is mediated by a group of proteins including TGF-beta receptors and Smads. It is known that different cells can exhibit different sensitivities to TGF-beta. Several molecular mechanisms, such as the differential expression of the receptor levels, have been suggested as contributing to these differences. Here, we report evidence for a novel mechanism of regulating TGF-beta sensitivity that depends on the role of CHIP (carboxyl terminus of Hsc70-interacting protein) in regulating the basal level of Smad3 via the ubiquitin-dependent degradation pathway. First, using a luciferase assay we found that overexpression of CHIP inhibited TGF-beta signaling, whereas silencing CHIP expression by small interfering RNAs led to increased TGF-beta signaling sensitivity. Second, based on the results of cell proliferation assays and JunB expression, we found that TGF-beta signaling could be abolished by stably overexpressing CHIP. Third, in those cell lines with stably expressed CHIP, we observed that the Smad3 protein level was dramatically decreased. Finally, we demonstrated that CHIP served as a U-box dependent E3 ligase that can directly mediate ubiquitination and degradation of Smad3 and that this action of CHIP was independent of TGF-beta signaling. Taken together, these findings suggest that CHIP can modulate the sensitivity of the TGF-beta signaling by controlling the basal level of Smad3 through ubiquitin-mediated degradation.     

Atrophin-1-interacting protein 4/human Itch is a ubiquitin E3 ligase for human enhancer of filamentation 1 in transforming growth factor-beta signaling pathways

Atrophin-1-interacting protein 4 (AIP4) is the human homolog of the mouse Itch protein (hItch), an E3 ligase for Notch and JunB. Human enhancer of filamentation 1 (HEF1) has been implicated in signaling pathways such as those mediated by integrin, T cell receptor, and B cell receptor and functions as a multidomain docking protein. Recent studies suggest that HEF1 is also involved in the transforming growth factor-beta (TGF-beta) signaling pathways, by interacting with Smad3, a key signal transducer downstream of the TGF-beta type I receptor. The interaction of Smad3 with HEF1 induces HEF1 proteasomal degradation, which was further enhanced by TGF-beta stimulation. The detailed molecular mechanisms of HEF1 degradation regulated by Smad3 were poorly understood. Here we report our studies that demonstrate the function of AIP4 as an ubiquitin E3 ligase for HEF1. AIP4 forms a complex with both Smad3 and HEF1 through its WW domains in a TGF-beta-independent manner and regulates HEF1 ubiquitination and degradation, which can be enhanced by TGF-beta stimulation. These findings reveal a new mechanism for Smad3-regulated proteasomal degradation events and also broaden the network of cross-talk between the TGF-beta signaling pathway and those involving HEF1 and AIP4.     

Human papillomavirus type 5 E6 oncoprotein represses the transforming growth factor beta signaling pathway by binding to SMAD3

Mechanisms of cellular transformation associated with human papillomavirus type 5 (HPV5), which is responsible for skin carcinomas in epidermodysplasia verruciformis (EV) patients, are poorly understood. Using a yeast two-hybrid screening and molecular and cellular biology experiments, we found that HPV5 oncoprotein E6 interacts with SMAD3, a key component in the transforming growth factor beta1 (TGF-beta1) signaling pathway. HPV5 E6 inhibits SMAD3 transactivation by destabilizing the SMAD3/SMAD4 complex and inducing the degradation of both proteins. Interestingly, the E6 protein of nononcogenic EV HPV9 failed to interact with SMAD3, suggesting that downregulation of the TGF-beta1 signaling pathway could be a determinant in HPV5 skin carcinogenesis.     

Anaphase-promoting complex/cyclosome-cdh1 mediates the ubiquitination and degradation of TRB3

We have recently demonstrated that TRB3, a novel endoplasmic reticulum (ER) stress-inducible protein, is induced by CHOP and ATF4 to regulate their function and ER stress-induced cell death; however, the regulation of TRB3 function has not been well characterized. Here we demonstrate that TRB3 is an unstable protein regulated by the ubiquitin-proteasome system. The carboxyl-terminal domain of TRB3 is necessary for protein degradation, and in this region, we found the typical D-box motif, which is a critical sequence for the anaphase-promoting complex/cyclosome (APC/C) dependent proteolysis. TRB3 proteins were stabilized by deletion of its D-box motif and interacted with APC/C coactivator proteins, Cdc20 and Cdh1. The expression level of TRB3 protein is down-regulated by over-expression of Cdh1 but not by that of Cdc20. In addition, knockdown of Cdh1 enhanced the endogenous TRB3 expression level and suppressed its ubiquitination level. These results suggest that APC/C^Cdh1 is involved in ubiquitination and down-regulating the stability of TRB3 protein.     

SIAH ubiquitin ligases regulate breast cancer cell migration and invasion independent of the oxygen status

Seven-in-absentia homolog (SIAH) proteins are evolutionary conserved RING type E3 ubiquitin ligases responsible for the degradation of key molecules regulating DNA damage response, hypoxic adaptation, apoptosis, angiogenesis, and cell proliferation. Many studies suggest a tumorigenic role for SIAH2. In breast cancer patients SIAH2 expression levels correlate with cancer aggressiveness and overall patient survival. In addition, SIAH inhibition reduced metastasis in melanoma. The role of SIAH1 in breast cancer is still ambiguous; both tumorigenic and tumor suppressive functions have been reported. Other studies categorized SIAH ligases as either pro- or antimigratory, while the significance for metastasis is largely unknown. Here, we re-evaluated the effects of SIAH1 and SIAH2 depletion in breast cancer cell lines, focusing on migration and invasion. We successfully knocked down SIAH1 and SIAH2 in several breast cancer cell lines. In luminal type MCF7 cells, this led to stabilization of the SIAH substrate Prolyl Hydroxylase Domain protein 3 (PHD3) and reduced Hypoxia-Inducible Factor 1α (HIF1α) protein levels. Both the knockdown of SIAH1 or SIAH2 led to increased apoptosis and reduced proliferation, with comparable effects. These results point to a tumor promoting role for SIAH1 in breast cancer similar to SIAH2. In addition, depletion of SIAH1 or SIAH2 also led to decreased cell migration and invasion in breast cancer cells. SIAH knockdown also controlled microtubule dynamics by markedly decreasing the protein levels of stathmin, most likely via p27^Kip1. Collectively, these results suggest that both SIAH ligases promote a migratory cancer cell phenotype and could contribute to metastasis in breast cancer.     

Polarity of response to transforming growth factor-beta1 in proximal tubular epithelial cells is regulated by beta-catenin

Transforming growth factor-beta1 (TGF-beta1)-mediated loss of proximal tubular epithelial cell-cell interaction is regulated in a polarized fashion. The aim of this study was to further explore the polarity of the TGF-beta1 response and to determine the significance of R-Smad-beta-catenin association previously demonstrated to accompany adherens junction disassembly. Smad3 signaling response to TGF-beta1 was assessed by activity of the Smad3-responsive reporter gene construct (SBE)(4)-Lux and by immunoblotting for phospho-Smad proteins. Similar results were obtained with both methods. Apical application of TGF-beta1 led to increased Smad3 signaling compared with basolateral stimulation. Association of Smad proteins with beta-catenin was greater following basolateral TGFbeta-1 stimulation, as was the expression of cytoplasmic Triton-soluble beta-catenin. Inhibition of beta-catenin expression by small interfering RNA augmented Smad3 signaling. Lithium chloride, a GSK-3 inhibitor, increased expression of beta-catenin and attenuated TGF-beta1-dependent Smad3 signaling. Lithium chloride did not influence degradation of Smad3 but resulted in decreased nuclear translocation. Smad2 activation as assessed by Western blot analysis and activity of the Smad2-responsive reporter constructs ARE/MF1 was also greater following apical as compared with basolateral TGFbeta-1 stimulation, suggesting that this is a generally applicable mechanism for the regulation of TGF-beta1-dependent R-Smads. Caco-2 cells are a colonic carcinoma cell line, with known resistance to the anti-proliferative effects of TGF-beta1 and increased expression of beta-catenin. We used this cell line to address the general applicability of our observations. Inhibition of beta-catenin in this cell line by small interfering RNA resulted in increased TGF-beta1-dependent Smad3 phosphorylation and restoration of TGF-beta1 anti-proliferative effects.     

Regulation of Sprouty2 stability by mammalian Seven-in-Absentia homolog 2

Mammalian Sprouty (Spry) gene expression is rapidly induced upon activation of the FGF receptor signaling pathway in multiple cell types including cells of mesenchymal and epithelial origin. Spry2 inhibits FGF-dependent ERK activation and thus Spry acts as a feedback inhibitor of FGF-mediated proliferation. In addition, Spry2 interacts with the ring-finger-containing E3 ubiquitin ligase, c-Cbl, in a manner that is dependent upon phosphorylation of Tyr55 of Spry2. This interaction results in the poly-ubiquitination and subsequent degradation of Spry2 by the proteasome. Here, we describe the identification of another E3 ubiquitin ligase, human Seven-in-Absentia homolog-2 (SIAH2), as a Spry2 interacting protein. We show by yeast two-hybrid analysis that the N-terminal domain of Spry2 and the ring finger domain of SIAH2 mediated this interaction. Co-expression of SIAH2 resulted in proteasomal degradation of Spry1, 2, and to a lesser extent Spry4. The related E3 ubiquitin-ligase, SIAH1, had little effect on Spry2 protein stability when co-expressed. Unlike c-Cbl-mediated degradation of Spry2, SIAH2-mediated degradation was independent of phosphorylation of Spry2 on Tyr55. Spry2 was also phosphorylated on Tyr227, and phosphorylation of this residue was also dispensable for SIAH2-mediated degradation of Spry2. Finally, co-expression of SIAH2 with Spry2 resulted in a rescue of FGF2-mediated ERK phosphorylation. These data suggest a novel mechanism whereby Spry2 stability is regulated in a manner that is independent of tyrosine phosphorylation, and provides an addition level of control of Spry2 protein levels.     

Loss of TRB3 Alters Dynamics of MLK3-JNK Signaling and Inhibits Cytokine-activated Pancreatic Beta Cell Death

Disabling cellular defense mechanisms is essential for induction of apoptosis. We have previously shown that cytokine-mediated activation of the MAP3K MLK3 stabilizes TRB3 protein levels to inhibit AKT and compromise beta cell survival. Here, we show that genetic deletion of TRB3 results in basal activation of AKT, preserves mitochondrial integrity, and confers resistance against cytokine-induced pancreatic beta cell death. Mechanistically, we find that TRB3 stabilizes MLK3, most likely by suppressing AKT-directed phosphorylation, ubiquitination, and proteasomal degradation of MLK3. Accordingly, TRB3^−/− islets show a decrease in both the amplitude and duration of cytokine-stimulated MLK3 induction and JNK activation. It is well known that JNK signaling is facilitated by a feed forward loop of sequential kinase phosphorylation and is reinforced by a mutual stabilization of the module components. The failure of TRB3^−/− islets to mount an optimal JNK activation response, coupled with the ability of TRB3 to engage and maintain steady state levels of MLK3, recasts TRB3 as an integral functional component of the JNK module in pancreatic beta cells.     

Differential expression of transforming growth factor-beta receptors I and II and activation of Smad 3 in keloid fibroblasts

Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extracellular matrix, especially collagen. However, the molecular mechanisms regulating this pathologic collagen deposition still remain to be elucidated. A previous study by this group demonstrated that transforming growth factor (TGF)-beta1 and -beta2 ligands were expressed at greater levels in keloid fibroblasts when compared with normal human dermal fibroblasts (NHDFs), suggesting that TGF-beta may play a fibrosis-promoting role in keloid pathogenesis.To explore the biomolecular mechanisms of TGF-beta in keloid formation, the authors first compared the expression levels of the type I and type II TGF-beta receptors in keloid fibroblasts and NHDFs. Next, they investigated the phosphorylation of Smad 3, an intracellular TGF-beta signaling molecule, in keloid fibroblasts and NHDFs. Finally, they examined the regulation of TGF-beta receptor II by TGF-beta1, TGF-beta2, and TGF-beta3 ligands.Our findings demonstrated an increased expression of TGF-beta receptors (types I and II) and increased phosphorylation of Smad 3 in keloid fibroblasts relative to NHDFs. These data support a possible role of TGF-beta and its receptors as fibrosis-inducing growth factors in keloids. In addition, all three isoforms of recombinant human TGF-beta proteins could further stimulate the expression of TGF-beta receptor II in both keloids and NHDFs. Taken together, these results substantiate the hypothesis that the elevated levels of TGF-beta ligands and receptors present in keloids may support increased signaling and a potential role for TGF-beta in keloid pathogenesis.     

Glycogen synthase kinase 3beta modulates synphilin-1 ubiquitylation and cellular inclusion formation by SIAH: implications for proteasomal function and Lewy body formation

alpha-Synuclein is known to play a major role in the pathogenesis of Parkinson disease. We previously identified synphilin-1 as an alpha-synuclein-interacting protein and more recently found that synphilin-1 also interacts with the E3 ubiquitin ligases SIAH-1 and SIAH-2. SIAH proteins ubiquitylate synphilin-1 and promote its degradation through the ubiquitin proteasome system. Inability of the proteasome to degrade synphilin-1 promotes the formation of ubiquitylated inclusion bodies. We now show that synphilin-1 is phosphorylated by GSK3beta within amino acids 550-659 and that this phosphorylation is significantly decreased by pharmacological inhibition of GSK3beta and suppression of GSK3beta expression by small interfering RNA duplex. Mutation analysis showed that Ser556 is a major GSK3beta phosphorylation site in synphilin-1. GSK3beta co-immunoprecipitated with synphilin-1, and protein 14-3-3, an activator of GSK3beta activity, increased synphilin-1 phosphorylation. GSK3beta decreased the in vitro and in vivo ubiquitylation of synphilin-1 as well as its degradation promoted by SIAH. Pharmacological inhibition and small interfering RNA suppression of GSK3beta greatly increased ubiquitylation and inclusion body formation by SIAH. Additionally, synphilin-1 S556A mutant, which is less phosphorylated by GSK3beta, formed more inclusion bodies than wild type synphilin-1. Inhibition of GSK3beta in primary neuronal cultures decreased the levels of endogenous synphilin-1, indicating that synphilin-1 is a physiologic substrate of GSK3beta. Using GFPu as a reporter to measure proteasome function in vivo, we found that synphilin-1 S556A is more efficient in inhibiting the proteasome than wild type synphilin-1, raising the possibility that the degree of synphilin-1 phosphorylation may regulate the proteasome function. Activation of GSK3beta during endoplasmic reticulum stress and the specific phosphorylation of synphilin-1 by GSK3beta place synphilin-1 as a possible mediator of endoplasmic reticulum stress and proteasomal dysfunction observed in Parkinson disease.