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Kai Lin - One of the best experts on this subject based on the ideXlab platform.
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competition between ski and creb binding Protein for binding to Smad Proteins in transforming growth factor β signaling
Journal of Biological Chemistry, 2007Co-Authors: Weijun Chen, Suvana S. Lam, Hema Srinath, Celia A. Schiffer, William E. Royer, Kai LinAbstract:Abstract The family of Smad Proteins mediates transforming growth factor-β (TGF-β) signaling in cell growth and differentiation. Smads 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. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad Protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad·Smad4 heterotrimer to form a pentamer. CBP-(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 competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-β signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad Protein activity is delicately balanced by Ski and CBP in the TGF-β pathway.
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Competition between Ski and CREB-binding Protein for binding to Smad Proteins in transforming growth factor-beta signaling.
The Journal of biological chemistry, 2007Co-Authors: Weijun Chen, Suvana S. Lam, Hema Srinath, Celia A. Schiffer, William E. Royer, Kai LinAbstract:Abstract The family of Smad Proteins mediates transforming growth factor-β (TGF-β) signaling in cell growth and differentiation. Smads 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. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad Protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad·Smad4 heterotrimer to form a pentamer. CBP-(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 competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-β signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad Protein activity is delicately balanced by Ski and CBP in the TGF-β pathway.
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Structural Basis of Heteromeric Smad Protein Assembly in TGF-β Signaling
Molecular cell, 2004Co-Authors: B.m. Chacko, Bin Y. Qin, Ashutosh Tiwari, Genbin Shi, Suvana S. Lam, Lawrence J. Hayward, Mark P. De Caestecker, Kai LinAbstract:The formation of Protein complexes between phosphorylated R-Smads and Smad4 is a central event in the TGF-β signaling pathway. We have determined the crystal structure of two R-Smad/Smad4 complexes, Smad3/Smad4 to 2.5 A, and Smad2/Smad4 to 2.7 A. Both complexes are heterotrimers, comprising two phosphorylated R-Smad subunits and one Smad4 subunit, a finding that was corroborated by isothermal titration calorimetry and mutational studies. Preferential formation of the R-Smad/Smad4 heterotrimer over the R-Smad homotrimer is largely enthalpy driven, contributed by the unique presence of strong electrostatic interactions within the heterotrimeric interfaces. The study supports a common mechanism of Smad Protein assembly in TGF-β superfamily signaling.
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Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation
Nature Structural & Molecular Biology, 2003Co-Authors: Bin Y Qin, Suvana S. Lam, John J Correia, Hema Srinath, Cheng Liu, Rachel Delston, Rik Derynck, Kai LinAbstract:IRF-3, a member of the interferon regulatory factor (IRF) family of transcription factors, functions as a molecular switch for antiviral activity. IRF-3 uses an autoinhibitory mechanism to suppress its transactivation potential in uninfected cells, and virus infection induces phosphorylation and activation of IRF-3 to initiate the antiviral responses. The crystal structure of the IRF-3 transactivation domain reveals a unique autoinhibitory mechanism, whereby the IRF association domain and the flanking autoinhibitory elements condense to form a hydrophobic core. The structure suggests that phosphorylation reorganizes the autoinhibitory elements, leading to unmasking of a hydrophobic active site and realignment of the DNA binding domain for transcriptional activation. IRF-3 exhibits marked structural and surface electrostatic potential similarity to the MH2 domain of the Smad Protein family and the FHA domain, suggesting a common molecular mechanism of action among this superfamily of signaling mediators.
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the l3 loop and c terminal phosphorylation jointly define Smad Protein trimerization
Nature Structural & Molecular Biology, 2001Co-Authors: B.m. Chacko, Suvana S. Lam, Bin Y Qin, John J Correia, M P De Caestecker, Kai LinAbstract:Smad Proteins mediate the transforming growth factor beta responses. C-terminal phosphorylation of R-Smads leads to the recruitment of Smad4 and the formation of active signaling complexes. We investigated the mechanism of phosphorylation-induced Smad complex formation with an activating pseudo-phosphorylated Smad3. Pseudo-phosphorylated Smad3 has a greater propensity to homotrimerize, and recruits Smad4 to form a heterotrimer containing two Smad3 and one Smad4. The trimeric interaction is mediated through conserved interfaces to which tumorigenic mutations map. Furthermore, a conserved Arg residue within the L3 loop, located near the C-terminal phosphorylation sites of the neighboring subunit, is essential for trimerization. We propose that the phosphorylated C-terminal residues interact with the L3 loop of the neighboring subunit to stabilize the trimer interaction.
Catherine E. Campbell - One of the best experts on this subject based on the ideXlab platform.
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correction phosphoproteomic analysis reveals Smad Protein family activation following rift valley fever virus infection
PLOS ONE, 2018Co-Authors: Cynthia De La Fuente, Chelsea Pinkham, Deemah Dabbagh, Brett Beitzel, Aura R. Garrison, Gustavo Palacios, Kimberley Alex Hodge, Emanuel F. Petricoin, Connie S. Schmaljohn, Catherine E. CampbellAbstract:Rift Valley fever virus (RVFV) infects both ruminants and humans leading to a wide variance of pathologies dependent on host background and age. Utilizing a targeted reverse phase Protein array (RPPA) to define changes in signaling cascades after in vitro infection of human cells with virulent and attenuated RVFV strains, we observed high phosphorylation of Smad transcription factors. This evolutionarily conserved family is phosphorylated by and transduces the activation of TGF-β superfamily receptors. Moreover, we observed that phosphorylation of Smad Proteins required active RVFV replication and loss of NSs impaired this activation, further corroborating the RPPA results. Gene promoter analysis of transcripts altered after RVFV infection identified 913 genes that contained a Smad-response element. Functional annotation of these potential Smad-regulated genes clustered in axonal guidance, hepatic fibrosis and cell signaling pathways involved in cellular adhesion/migration, calcium influx, and cytoskeletal reorganization. Furthermore, chromatin immunoprecipitation confirmed the presence of a Smad complex on the interleukin 1 receptor type 2 (IL1R2) promoter, which acts as a decoy receptor for IL-1 activation.
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Phosphoproteomic analysis reveals Smad Protein family activation following Rift Valley fever virus infection.
PloS one, 2018Co-Authors: Cynthia De La Fuente, Chelsea Pinkham, Deemah Dabbagh, Brett Beitzel, Aura R. Garrison, Gustavo Palacios, Kimberley Alex Hodge, Emanuel F. Petricoin, Connie S. Schmaljohn, Catherine E. CampbellAbstract:Rift Valley fever virus (RVFV) infects both ruminants and humans leading to a wide variance of pathologies dependent on host background and age. Utilizing a targeted reverse phase Protein array (RPPA) to define changes in signaling cascades after in vitro infection of human cells with virulent and attenuated RVFV strains, we observed high phosphorylation of Smad transcription factors. This evolutionarily conserved family is phosphorylated by and transduces the activation of TGF-β superfamily receptors. Moreover, we observed that phosphorylation of Smad Proteins required active RVFV replication and loss of NSs impaired this activation, further corroborating the RPPA results. Gene promoter analysis of transcripts altered after RVFV infection identified 913 genes that contained a Smad-response element. Functional annotation of these potential Smad-regulated genes clustered in axonal guidance, hepatic fibrosis and cell signaling pathways involved in cellular adhesion/migration, calcium influx, and cytoskeletal reorganization. Furthermore, chromatin immunoprecipitation confirmed the presence of a Smad complex on the interleukin 1 receptor type 2 (IL1R2) promoter, which acts as a decoy receptor for IL-1 activation.
Suvana S. Lam - One of the best experts on this subject based on the ideXlab platform.
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competition between ski and creb binding Protein for binding to Smad Proteins in transforming growth factor β signaling
Journal of Biological Chemistry, 2007Co-Authors: Weijun Chen, Suvana S. Lam, Hema Srinath, Celia A. Schiffer, William E. Royer, Kai LinAbstract:Abstract The family of Smad Proteins mediates transforming growth factor-β (TGF-β) signaling in cell growth and differentiation. Smads 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. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad Protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad·Smad4 heterotrimer to form a pentamer. CBP-(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 competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-β signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad Protein activity is delicately balanced by Ski and CBP in the TGF-β pathway.
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Competition between Ski and CREB-binding Protein for binding to Smad Proteins in transforming growth factor-beta signaling.
The Journal of biological chemistry, 2007Co-Authors: Weijun Chen, Suvana S. Lam, Hema Srinath, Celia A. Schiffer, William E. Royer, Kai LinAbstract:Abstract The family of Smad Proteins mediates transforming growth factor-β (TGF-β) signaling in cell growth and differentiation. Smads 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. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad Protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad·Smad4 heterotrimer to form a pentamer. CBP-(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 competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-β signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad Protein activity is delicately balanced by Ski and CBP in the TGF-β pathway.
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Structural Basis of Heteromeric Smad Protein Assembly in TGF-β Signaling
Molecular cell, 2004Co-Authors: B.m. Chacko, Bin Y. Qin, Ashutosh Tiwari, Genbin Shi, Suvana S. Lam, Lawrence J. Hayward, Mark P. De Caestecker, Kai LinAbstract:The formation of Protein complexes between phosphorylated R-Smads and Smad4 is a central event in the TGF-β signaling pathway. We have determined the crystal structure of two R-Smad/Smad4 complexes, Smad3/Smad4 to 2.5 A, and Smad2/Smad4 to 2.7 A. Both complexes are heterotrimers, comprising two phosphorylated R-Smad subunits and one Smad4 subunit, a finding that was corroborated by isothermal titration calorimetry and mutational studies. Preferential formation of the R-Smad/Smad4 heterotrimer over the R-Smad homotrimer is largely enthalpy driven, contributed by the unique presence of strong electrostatic interactions within the heterotrimeric interfaces. The study supports a common mechanism of Smad Protein assembly in TGF-β superfamily signaling.
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Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation
Nature Structural & Molecular Biology, 2003Co-Authors: Bin Y Qin, Suvana S. Lam, John J Correia, Hema Srinath, Cheng Liu, Rachel Delston, Rik Derynck, Kai LinAbstract:IRF-3, a member of the interferon regulatory factor (IRF) family of transcription factors, functions as a molecular switch for antiviral activity. IRF-3 uses an autoinhibitory mechanism to suppress its transactivation potential in uninfected cells, and virus infection induces phosphorylation and activation of IRF-3 to initiate the antiviral responses. The crystal structure of the IRF-3 transactivation domain reveals a unique autoinhibitory mechanism, whereby the IRF association domain and the flanking autoinhibitory elements condense to form a hydrophobic core. The structure suggests that phosphorylation reorganizes the autoinhibitory elements, leading to unmasking of a hydrophobic active site and realignment of the DNA binding domain for transcriptional activation. IRF-3 exhibits marked structural and surface electrostatic potential similarity to the MH2 domain of the Smad Protein family and the FHA domain, suggesting a common molecular mechanism of action among this superfamily of signaling mediators.
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the l3 loop and c terminal phosphorylation jointly define Smad Protein trimerization
Nature Structural & Molecular Biology, 2001Co-Authors: B.m. Chacko, Suvana S. Lam, Bin Y Qin, John J Correia, M P De Caestecker, Kai LinAbstract:Smad Proteins mediate the transforming growth factor beta responses. C-terminal phosphorylation of R-Smads leads to the recruitment of Smad4 and the formation of active signaling complexes. We investigated the mechanism of phosphorylation-induced Smad complex formation with an activating pseudo-phosphorylated Smad3. Pseudo-phosphorylated Smad3 has a greater propensity to homotrimerize, and recruits Smad4 to form a heterotrimer containing two Smad3 and one Smad4. The trimeric interaction is mediated through conserved interfaces to which tumorigenic mutations map. Furthermore, a conserved Arg residue within the L3 loop, located near the C-terminal phosphorylation sites of the neighboring subunit, is essential for trimerization. We propose that the phosphorylated C-terminal residues interact with the L3 loop of the neighboring subunit to stabilize the trimer interaction.
Cynthia De La Fuente - One of the best experts on this subject based on the ideXlab platform.
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correction phosphoproteomic analysis reveals Smad Protein family activation following rift valley fever virus infection
PLOS ONE, 2018Co-Authors: Cynthia De La Fuente, Chelsea Pinkham, Deemah Dabbagh, Brett Beitzel, Aura R. Garrison, Gustavo Palacios, Kimberley Alex Hodge, Emanuel F. Petricoin, Connie S. Schmaljohn, Catherine E. CampbellAbstract:Rift Valley fever virus (RVFV) infects both ruminants and humans leading to a wide variance of pathologies dependent on host background and age. Utilizing a targeted reverse phase Protein array (RPPA) to define changes in signaling cascades after in vitro infection of human cells with virulent and attenuated RVFV strains, we observed high phosphorylation of Smad transcription factors. This evolutionarily conserved family is phosphorylated by and transduces the activation of TGF-β superfamily receptors. Moreover, we observed that phosphorylation of Smad Proteins required active RVFV replication and loss of NSs impaired this activation, further corroborating the RPPA results. Gene promoter analysis of transcripts altered after RVFV infection identified 913 genes that contained a Smad-response element. Functional annotation of these potential Smad-regulated genes clustered in axonal guidance, hepatic fibrosis and cell signaling pathways involved in cellular adhesion/migration, calcium influx, and cytoskeletal reorganization. Furthermore, chromatin immunoprecipitation confirmed the presence of a Smad complex on the interleukin 1 receptor type 2 (IL1R2) promoter, which acts as a decoy receptor for IL-1 activation.
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Phosphoproteomic analysis reveals Smad Protein family activation following Rift Valley fever virus infection.
PloS one, 2018Co-Authors: Cynthia De La Fuente, Chelsea Pinkham, Deemah Dabbagh, Brett Beitzel, Aura R. Garrison, Gustavo Palacios, Kimberley Alex Hodge, Emanuel F. Petricoin, Connie S. Schmaljohn, Catherine E. CampbellAbstract:Rift Valley fever virus (RVFV) infects both ruminants and humans leading to a wide variance of pathologies dependent on host background and age. Utilizing a targeted reverse phase Protein array (RPPA) to define changes in signaling cascades after in vitro infection of human cells with virulent and attenuated RVFV strains, we observed high phosphorylation of Smad transcription factors. This evolutionarily conserved family is phosphorylated by and transduces the activation of TGF-β superfamily receptors. Moreover, we observed that phosphorylation of Smad Proteins required active RVFV replication and loss of NSs impaired this activation, further corroborating the RPPA results. Gene promoter analysis of transcripts altered after RVFV infection identified 913 genes that contained a Smad-response element. Functional annotation of these potential Smad-regulated genes clustered in axonal guidance, hepatic fibrosis and cell signaling pathways involved in cellular adhesion/migration, calcium influx, and cytoskeletal reorganization. Furthermore, chromatin immunoprecipitation confirmed the presence of a Smad complex on the interleukin 1 receptor type 2 (IL1R2) promoter, which acts as a decoy receptor for IL-1 activation.
Liliana Attisano - One of the best experts on this subject based on the ideXlab platform.
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Regulation of the TGFβ signalling pathway by ubiquitin-mediated degradation
Oncogene, 2004Co-Authors: Luisa Izzi, Liliana AttisanoAbstract:The transforming growth factor- β (TGF β ) superfamily controls a plethora of biological responses, and alterations in its signalling pathway are associated with a range of human diseases, including cancer. TGF β superfamily ligands signal through a heteromeric complex of Ser/Thr kinase receptors that propagate the signal to the Smad family of intracellular Proteins. The ubiquitin-mediated proteasomal degradation pathway is an evolutionary conserved cascade that tightly regulates TGF β superfamily signalling. Both the size of the Smad pool in unstimulated cells and Smad Protein levels subsequent to the activation of the pathway are controlled by ubiquitination. E3 ligases are components of the ubiquitin-degradation complex that specifically recognize targeted Proteins and the E3 ligases, Smad ubiquitination-related factor 1 (Smurf1), Smurf2 and SCF/Roc1 have been implicated in Smad degradation. The Smurfs are of particular importance to TGF β signalling, as Smads also function as adapters that recruit the Smurfs to various pathway components including the TGF β receptor complex and the transcriptional repressor, SnoN, and thereby regulate the degradation of these Smad-associating Proteins. Thus, by controlling the level of Smads as well as positive and negative regulators of the pathway, Smurfs provide for complex and fine control of signalling output. Finally, growing evidence demonstrates that ubiquitination and proteasomal degradation is also implicated in the turnover of tumor-derived Smad mutants and may thus contribute to disease progression.
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Mutations in the tumor suppressors Smad2 and Smad4 inactivate transforming growth factor beta signaling by targeting Smads to the ubiquitin-proteasome pathway.
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Jing Xu, Liliana AttisanoAbstract:Biological signals for transforming growth factor β (TGF-β) are transduced through transmembrane serine/threonine kinase receptors that signal to a family of intracellular mediators known as Smads. Smad2 and Smad4 are important for transcriptional and antiproliferative responses to TGF-β, and their inactivation in human cancers indicates that they are tumor suppressors. A missense mutation at a conserved arginine residue in the amino-terminal MH1 domain of both Smad2 and Smad4 has been identified in tumors from patients with colorectal and pancreatic cancers, respectively. However, the mechanism whereby this mutation interferes with Smad activity is uncertain. Here we show that these mutations do not disrupt activation of Smads, including receptor-mediated phosphorylation of Smad2, Smad2/Smad4 heteromeric complex formation, and Smad nuclear translocation. In contrast, we demonstrate that the mutant Smads are degraded rapidly in comparison with their wild-type counterparts. We show that this decrease in Smad Protein stability occurs through induction of Smad ubiquitination by pathways involving the UbcH5 family of ubiquitin ligases. These studies thus reveal a mechanism for tumorigenesis whereby genetic defects in Smads induce their degradation through the ubiquitin-mediated pathway.