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Murine protein serine/threonine kinase 38 stimulates TGF-beta signaling in a kinase-dependent manner via direct phosphorylation of Smad proteins.

ABSTRACT: The present study demonstrated that murine protein serine/threonine kinase 38 (MPK38) coimmunoprecipitates with Smad proteins (Smad2, -3, -4, and -7) and that this association is mediated by the catalytic kinase domain of MPK38. The association between MPK38 and Smad2, -3, and -4 was significantly increased by TGF-? or ASK1 signals, whereas these signals decreased association of MPK38 with Smad7. MPK38 stimulated TGF-?-induced transcription required for TGF-?-mediated biological functions, such as apoptosis and cell growth arrest, in a kinase-dependent manner. Knockdown of endogenous MPK38 showed an opposite effect, inhibiting TGF-? signaling. MPK38-mediated phosphorylation of Smad proteins (Ser(245) of Smad2, Ser(204) of Smad3, Ser(343) of Smad4, and Thr(96) of Smad7) was also found to be crucial to the positive regulation of TGF-? signaling induced by MPK38. In addition, MPK38 enhanced nuclear translocation of Smad3, as well as redistribution of Smad7 from the nucleus to the cytoplasm, in response to TGF-?. Together, these results indicate that MPK38 functions as a stimulator of TGF-? signaling through direct interaction with and phosphorylation of Smad proteins.

SUBMITTER: Seong HA

PROVIDER: S-EPMC2945587 | BioStudies | 2010-01-01T00:00:00Z

REPOSITORIES: biostudies

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Smad proteins differentially regulate obesity-induced glucose and lipid abnormalities and inflammation via class-specific control of AMPK-related kinase MPK38/MELK activity.

Project description:Smad proteins have been implicated in metabolic processes, but little is known about how they regulate metabolism. Because Smad 2, 3, 4, and 7 have previously been shown to interact with murine protein serine-threonine kinase 38 (MPK38), an AMP-activated protein kinase (AMPK)-related kinase that has been implicated in obesity-associated metabolic defects, we investigated whether Smad proteins regulate metabolic processes via MPK38. Smads2/3/4 increased, but Smad7 decreased, MPK38-mediated apoptosis signal-regulating kinase-1 (ASK1)/transforming growth factor-? (TGF-?)/p53 signaling. However, MPK38-mediated phosphorylation-defective Smad mutants (Smad2 S245A, Smad3 S204A, Smad4 S343A, and Smad7 T96A) had no such effect. In addition, Smads2/3/4 increased, but Smad7 decreased, the stability of MPK38. Consistent with this, Smads2/3/4 attenuated complex formation between MPK38 and its negative regulator thioredoxin (Trx), whereas Smad7 increased this complex formation. However, an opposite effect was observed on complex formation between MPK38 and its positive regulator zinc-finger-like protein 9 (ZPR9). When Smads were overexpressed in high-fat diet (HFD)-fed obese mice using an adenoviral delivery system, Smads2/3/4 improved, but Smad7 worsened, obesity-associated metabolic parameters and inflammation in a MPK38 phosphorylation-dependent manner. These findings suggest that Smad proteins have class-specific impacts on obesity-associated metabolism by differentially regulating MPK38 activity in diet-induced obese mice.

2018-01-01 | S-EPMC5920110 | BioStudies

Inhibition of transforming growth factor-beta1-induced signaling and epithelial-to-mesenchymal transition by the Smad-binding peptide aptamer Trx-SARA.

Project description:Overexpression of the inhibitory Smad, Smad7, is used frequently to implicate the Smad pathway in cellular responses to transforming growth factor beta (TGF-beta) signaling; however, Smad7 regulates several other proteins, including Cdc42, p38MAPK, and beta-catenin. We report an alternative approach for more specifically disrupting Smad-dependent signaling using a peptide aptamer, Trx-SARA, which comprises a rigid scaffold, the Escherichia coli thioredoxin A protein (Trx), displaying a constrained 56-amino acid Smad-binding motif from the Smad anchor for receptor activation (SARA) protein. Trx-SARA bound specifically to Smad2 and Smad3 and inhibited both TGF-beta-induced reporter gene expression and epithelial-to-mesenchymal transition in NMuMG murine mammary epithelial cells. In contrast to Smad7, Trx-SARA had no effect on the Smad2 or 3 phosphorylation levels induced by TGF-beta1. Trx-SARA was primarily localized to the nucleus and perturbed the normal cytoplasmic localization of Smad2 and 3 to a nuclear localization in the absence of TGF-beta1, consistent with reduced Smad nuclear export. The key mode of action of Trx-SARA was to reduce the level of Smad2 and Smad3 in complex with Smad4 after TGF-beta1 stimulation, a mechanism of action consistent with the preferential binding of SARA to monomeric Smad protein and Trx-SARA-mediated disruption of active Smad complexes.

2006-01-01 | S-EPMC1556379 | BioStudies

Deficient Smad7 expression: a putative molecular defect in scleroderma.

Project description:Scleroderma is a chronic systemic disease that leads to fibrosis of affected organs. Transforming growth factor (TGF) beta has been implicated in the pathogenesis of scleroderma. Smad proteins are signaling transducers downstream from TGF-beta receptors. Three families of Smads have been identified: (i) receptor-regulated Smad2 and -3 (R-Smads); (ii) common partner Smad4 (Co-Smad); and (iii) inhibitory Smad6 and -7 (I-Smads, part of a negative feedback loop). We have investigated the signaling components for the TGF-beta pathway and TGF-beta activity in scleroderma lesions in vivo and in scleroderma fibroblasts in vitro. Basal level and TGF-beta-inducible expression of Smad7 are selectively decreased, whereas Smad3 expression is increased both in scleroderma skin and in explanted scleroderma fibroblasts in culture. TGF-beta signaling events, including phosphorylation of Smad2 and -3, and transcription of the PAI-1 gene are increased in scleroderma fibroblasts, relative to normal fibroblasts. In vitro adenoviral gene transfer with Smad7 restores normal TGF-beta signaling in scleroderma fibroblasts. These results suggest that alterations in the Smad pathway, including marked Smad7 deficiency and Smad3 up-regulation, may be responsible for TGF-beta hyperresponsiveness observed in scleroderma.

| S-EPMC122622 | BioStudies

Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice.

Project description:Overexpression of transforming growth factor beta (TGF-beta) has been shown to play pathogenic roles in progression of renal fibrosis, and the severity of tubulointerstitial fibrosis correlates better with renal function than the severity of glomerulosclerosis. Smad proteins are signaling transducers downstream from TGF-beta receptors. Three families of Smad proteins have been identified: receptor-regulated Smad2 and Smad3, common partner Smad4, and inhibitory Smad7 (part of a negative-feedback loop). We investigated Smad-mediated TGF-beta signaling pathway and regulatory mechanisms of inhibitory Smad7 in unilateral ureteral obstruction (UUO) kidneys in mice, a model of progressive tubulointerstitial fibrosis. Compared with sham-operated kidneys, the level of Smad7 protein, but not mRNA, decreased progressively in UUO kidneys, whereas immunoreactivity for nuclear phosphorylated Smad2 and Smad3 and renal fibrosis were inversely increased. Furthermore, we demonstrated that both the degradation and ubiquitination activity of Smad7 protein were increased markedly in UUO kidneys compared with sham-operated ones. We also found that both Smurf1 and Smurf2 (Smad ubiquitination regulatory factors), which are E3 ubiquitin ligases for Smad7, were increased and that they interacted with Smad7 in UUO kidneys. Our results suggest that the reduction of Smad7 protein resulting from enhanced ubiquitin-dependent degradation plays a pathogenic role in progression of tubulointerstitial fibrosis.

2004-01-01 | S-EPMC423256 | BioStudies

TGF-?/Smad signaling in renal fibrosis.

Project description:TGF-? (transforming growth factor-?) is well identified as a central mediator in renal fibrosis. TGF-? initiates canonical and non-canonical pathways to exert multiple biological effects. Among them, Smad signaling is recognized as a major pathway of TGF-? signaling in progressive renal fibrosis. During fibrogenesis, Smad3 is highly activated, which is associated with the down-regulation of an inhibitory Smad7 via an ubiquitin E3-ligases-dependent degradation mechanism. The equilibrium shift between Smad3 and Smad7 leads to accumulation and activation of myofibroblasts, overproduction of ECM (extracellular matrix), and reduction in ECM degradation in the diseased kidney. Therefore, overexpression of Smad7 has been shown to be a therapeutic agent for renal fibrosis in various models of kidney diseases. In contrast, another downstream effecter of TGF-?/Smad signaling pathway, Smad2, exerts its renal protective role by counter-regulating the Smad3. Furthermore, recent studies demonstrated that Smad3 mediates renal fibrosis by down-regulating miR-29 and miR-200 but up-regulating miR-21 and miR-192. Thus, overexpression of miR-29 and miR-200 or down-regulation of miR-21 and miR-192 is capable of attenuating Smad3-mediated renal fibrosis in various mouse models of chronic kidney diseases (CKD). Taken together, TGF-?/Smad signaling plays an important role in renal fibrosis. Targeting TGF-?/Smad3 signaling may represent a specific and effective therapy for CKD associated with renal fibrosis.

2015-01-01 | S-EPMC4365692 | BioStudies

The effect of transforming growth factor-beta1 on nasopharyngeal carcinoma cells: insensitive to cell growth but functional to TGF-beta/Smad pathway.

Project description:OBJECTIVES: This study explored the response of nasopharyngeal carcinoma cells to TGF-beta1-induced growth suppression and investigated the roles of the TGF-beta/Smad signaling pathway in nasopharyngeal carcinoma cells. METHODS: The cells of nasopharyngeal carcinoma cell line CNE2 were treated with TGF-beta1. The growth responses of CNE2 cells were analyzed by MTT assay. The mRNA expression and protein subcellular localization of the TGF-beta/Smad signaling components in the CNE2 were determined by real time RT-PCR and immunocytochemical analysis. RESULTS: We found that the growth of CNE2 cells was not suppressed by TGF-beta1. The signaling proteins TbetaRII, Smad 7 were expressed normally, while Smad2, Smad3, and Smad4 increased significantly at the mRNA level. TGF-beta type II receptor and Smad7 had no change compared to the normal nasopharyngeal epithelial cells. In addition, Smad2 was phosphorylated to pSmad2, and the activated pSmad2 translocated into the nucleus from the cytoplasm, while the inhibitory Smad-Smad7 translocated from the nucleus to the cytoplasm after TGF-beta1 stimulation. CONCLUSION: The results suggested that CNE2 cells are not sensitive to growth suppression by TGF-beta1, but the TGF-beta/Smad signaling transduction is functional. Further work is needed to address a more detailed spectrum of the TGF-beta/Smad signaling pathway in CNE2 cells.

2010-01-01 | S-EPMC2865451 | BioStudies

Rhubarb Protect Against Tubulointerstitial Fibrosis by Inhibiting TGF-?/Smad Pathway and Improving Abnormal Metabolome in Chronic Kidney Disease.

Project description:Tubulointerstitial fibrosis is the final common pathway for all kidney diseases leading to chronic kidney disease (CKD). TGF-?/Smad signaling pathway plays a key role in renal fibrosis. Previous studies have revealed that rhubarb extracts attenuated the increase of transforming growth factor-? 1 (TGF-?1) in CKD rats. To gain an in-depth insight into the mechanism of the anti-fibrotic activities of the rhubarb extracts, we investigated the influence of rhubarb extracts on TGF-?/Smad signaling pathway and the influence on metabolome in a rat model of CKD with adenine-induced chronic tubulointerstitial nephropathy. Male Sprague-Dawley rats were divided into four groups, including control, CKD, CKD + petroleum ether extract, CKD + ethyl acetate extract, and CKD + n-butanol extract groups. Kidneys harvested on the week three were evaluated for renal fibrosis, the expression of proteins in TGF-?/Smad signaling pathway and metabolomic study. We found rhubarb extracts suppressed TGF-?/Smad3-mediated renal fibrosis by reducing the TGF-?1, transforming growth factor-? receptor I (TGF-? RI), transforming growth factor-? receptor II (TGF-? RII), Smad2, p-Smad2, Smad3, p-Smad3, and Smad4, meanwhile increased Smad7. In addition, rhubarb extracts mitigated renal injury and dysfunction, and either fully or partially reversed the abnormalities of tissue metabolites. Thus, rebalancing the disorder of TGF-?/Smad signaling and metabolic dysfunction by treatment with rhubarb extracts may represent as an effective therapy for CKD associated with fibrosis.

2018-01-01 | S-EPMC6146043 | BioStudies

The kinase activity of the Ser/Thr kinase BUB1 promotes TGF-? signaling.

Project description:Transforming growth factor-? (TGF-?) signaling regulates cell proliferation and differentiation, which contributes to development and disease. Upon binding TGF-?, the type I receptor (TGFBRI) binds TGFBRII, leading to the activation of the transcription factors SMAD2 and SMAD3. Using an RNA interference screen of the human kinome and a live-cell reporter for TGFBR activity, we identified the kinase BUB1 (budding uninhibited by benzimidazoles-1) as a key mediator of TGF-? signaling. BUB1 interacted with TGFBRI in the presence of TGF-? and promoted the heterodimerization of TGFBRI and TGFBRII. Additionally, BUB1 interacted with TGFBRII, suggesting the formation of a ternary complex. Knocking down BUB1 prevented the recruitment of SMAD3 to the receptor complex, the phosphorylation of SMAD2 and SMAD3 and their interaction with SMAD4, SMAD-dependent transcription, and TGF-?-mediated changes in cellular phenotype including epithelial-mesenchymal transition (EMT), migration, and invasion. Knockdown of BUB1 also impaired noncanonical TGF-? signaling mediated by the kinases AKT and p38 MAPK (mitogen-activated protein kinase). The ability of BUB1 to promote TGF-? signaling depended on the kinase activity of BUB1. A small-molecule inhibitor of the kinase activity of BUB1 (2OH-BNPP1) and a kinase-deficient mutant of BUB1 suppressed TGF-? signaling and formation of the ternary complex in various normal and cancer cell lines. 2OH-BNPP1 administration to mice bearing lung carcinoma xenografts reduced the amount of phosphorylated SMAD2 in tumor tissue. These findings indicated that BUB1 functions as a kinase in the TGF-? pathway in a role beyond its established function in cell cycle regulation and chromosome cohesion.

2015-01-01 | S-EPMC4440544 | BioStudies

Requirements of transcription factor Smad-dependent and -independent TGF-? signaling to control discrete T-cell functions.

Project description:TGF-? modulates immune response by suppressing non-regulatory T (Treg) function and promoting Treg function. The question of whether TGF-? achieves distinct effects on non-Treg and Treg cells through discrete signaling pathways remains outstanding. In this study, we investigated the requirements of Smad-dependent and -independent TGF-? signaling for T-cell function. Smad2 and Smad3 double deficiency in T cells led to lethal inflammatory disorder in mice. Non-Treg cells were spontaneously activated and produced effector cytokines in vivo on deletion of both Smad2 and Smad3. In addition, TGF-? failed to suppress T helper differentiation efficiently and to promote induced Treg generation of non-Treg cells lacking both Smad2 and Smad3, suggesting that Smad-dependent signaling is obligatory to mediate TGF-? function in non-Treg cells. Unexpectedly, however, the development, homeostasis, and function of Treg cells remained intact in the absence of Smad2 and Smad3, suggesting that the Smad-independent pathway is important for Treg function. Indeed, Treg-specific deletion of TGF-?-activated kinase 1 led to failed Treg homeostasis and lethal immune disorder in mice. Therefore, Smad-dependent and -independent TGF-? signaling discretely controls non-Treg and Treg function to modulate immune tolerance and immune homeostasis.

1000-01-01 | S-EPMC3271895 | BioStudies

Selective targeting of activating and inhibitory Smads by distinct WWP2 ubiquitin ligase isoforms differentially modulates TGF? signalling and EMT.

Project description:Ubiquitin-dependent mechanisms have emerged as essential regulatory elements controlling cellular levels of Smads and TGF?-dependent biological outputs such as epithelial-mesenchymal transition (EMT). In this study, we identify a HECT E3 ubiquitin ligase known as WWP2 (Full-length WWP2-FL), together with two WWP2 isoforms (N-terminal, WWP2-N; C-terminal WWP2-C), as novel Smad-binding partners. We show that WWP2-FL interacts exclusively with Smad2, Smad3 and Smad7 in the TGF? pathway. Interestingly, the WWP2-N isoform interacts with Smad2 and Smad3, whereas WWP2-C interacts only with Smad7. In addition, WWP2-FL and WWP2-C have a preference for Smad7 based on protein turnover and ubiquitination studies. Unexpectedly, we also find that WWP2-N, which lacks the HECT ubiquitin ligase domain, can also interact with WWP2-FL in a TGF?-regulated manner and activate endogenous WWP2 ubiquitin ligase activity causing degradation of unstimulated Smad2 and Smad3. Consistent with our protein interaction data, overexpression and knockdown approaches reveal that WWP2 isoforms differentially modulate TGF?-dependent transcription and EMT. Finally, we show that selective disruption of WWP2 interactions with inhibitory Smad7 can stabilise Smad7 protein levels and prevent TGF?-induced EMT. Collectively, our data suggest that WWP2-N can stimulate WWP2-FL leading to increased activity against unstimulated Smad2 and Smad3, and that Smad7 is a preferred substrate for WWP2-FL and WWP2-C following prolonged TGF? stimulation. Significantly, this is the first report of an interdependent biological role for distinct HECT E3 ubiquitin ligase isoforms, and highlights an entirely novel regulatory paradigm that selectively limits the level of inhibitory and activating Smads.

| S-EPMC4073228 | BioStudies

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