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Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

Hough C, Radu M, Doré JJ - PLoS ONE (2012)

Bottom Line: TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells.By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation.In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription.

View Article: PubMed Central - PubMed

Affiliation: BioMedical Sciences, Memorial University, St. John's, Newfoundland, Canada.

ABSTRACT
The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-β/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-β-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

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Nuclear Smad levels are controlled by the proteasome and activated Erk.(A) AKR-2B fibroblasts were treated for 3 h with TGF-β (2 ng/ml) with or without MG132 (10 µM), 30 minutes prior to TGF-β addition. Nuclear and cytoplasmic fractions were isolated and probed for smad2 linker region phosphorylation (245/250/255), or receptor phosphorylation (465/467). Linker phosphorylation blots were stripped and reprobed for total smad2 as a loading control, while receptor phosphorylated blots were stripped and reprobed for GAPDH to monitor the presence of cytoplasmic protein in the nuclear fraction. (B) Photomicrographs of NIH 3T3 fibroblasts treated with TGF-β (2 ng/ml) for 3 h with or without MG132 (10 µM) added 30 minutes prior to TGF-β treatment. Cells were incubated with phospho-smad2 (S245/250/255) linker antibody and specific immune complexes detected using Rhodamine X conjugated secondary antibody. (C) Cell lysates from AKR-2B fibroblasts pulsed for 10 minutes with TGF-β (2 ng/ml) with or without U0126 (10 µM) were probed for receptor phosphorylated smad2 and smad3. Blots were stripped and reprobed for β-actin as a loading control. (D) The density of phospho-smad2 and 3 bands for each time point relative to its β-actin control were determined. The mean values for each time point (n = 3 for smad 2, n = 4 for smad 3) are displayed with the solid line representing the curve for TGF-β+U0126 and the dotted line representing TGF-β treatment.
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pone-0042513-g005: Nuclear Smad levels are controlled by the proteasome and activated Erk.(A) AKR-2B fibroblasts were treated for 3 h with TGF-β (2 ng/ml) with or without MG132 (10 µM), 30 minutes prior to TGF-β addition. Nuclear and cytoplasmic fractions were isolated and probed for smad2 linker region phosphorylation (245/250/255), or receptor phosphorylation (465/467). Linker phosphorylation blots were stripped and reprobed for total smad2 as a loading control, while receptor phosphorylated blots were stripped and reprobed for GAPDH to monitor the presence of cytoplasmic protein in the nuclear fraction. (B) Photomicrographs of NIH 3T3 fibroblasts treated with TGF-β (2 ng/ml) for 3 h with or without MG132 (10 µM) added 30 minutes prior to TGF-β treatment. Cells were incubated with phospho-smad2 (S245/250/255) linker antibody and specific immune complexes detected using Rhodamine X conjugated secondary antibody. (C) Cell lysates from AKR-2B fibroblasts pulsed for 10 minutes with TGF-β (2 ng/ml) with or without U0126 (10 µM) were probed for receptor phosphorylated smad2 and smad3. Blots were stripped and reprobed for β-actin as a loading control. (D) The density of phospho-smad2 and 3 bands for each time point relative to its β-actin control were determined. The mean values for each time point (n = 3 for smad 2, n = 4 for smad 3) are displayed with the solid line representing the curve for TGF-β+U0126 and the dotted line representing TGF-β treatment.

Mentions: Although these results indicate a direct cross-talk between smad2 and Erk, in light of the controversy between the Erk and smad linker region phosphorylation, we wished to examine their spatial relationship. Since smad2 is a transcription factor that translocates quickly into the nucleus following receptor mediated phosphorylation, we fractionated TGF-β treated fibroblasts into cytoplasmic and nuclear fractions and examined smad2 phosphorylation. Linker phosphorylation was seen primarily in the nuclear fraction (Figure 5A). Similarly, receptor phosphorylated smad2 was primarily in the nucleus, with small amounts in the cytoplasmic fraction, following TGF-β treatment. Total smad2 was present in both the cytoplasm and the nucleus. Since our concern was cytoplasmic protein contamination of the nuclear fractions, both cytoplasmic and nuclear fractions were probed for GAPDH to demonstrate purity of nuclear fractions relative to cytoplasmic protein contamination (Figure 5A).


Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

Hough C, Radu M, Doré JJ - PLoS ONE (2012)

Nuclear Smad levels are controlled by the proteasome and activated Erk.(A) AKR-2B fibroblasts were treated for 3 h with TGF-β (2 ng/ml) with or without MG132 (10 µM), 30 minutes prior to TGF-β addition. Nuclear and cytoplasmic fractions were isolated and probed for smad2 linker region phosphorylation (245/250/255), or receptor phosphorylation (465/467). Linker phosphorylation blots were stripped and reprobed for total smad2 as a loading control, while receptor phosphorylated blots were stripped and reprobed for GAPDH to monitor the presence of cytoplasmic protein in the nuclear fraction. (B) Photomicrographs of NIH 3T3 fibroblasts treated with TGF-β (2 ng/ml) for 3 h with or without MG132 (10 µM) added 30 minutes prior to TGF-β treatment. Cells were incubated with phospho-smad2 (S245/250/255) linker antibody and specific immune complexes detected using Rhodamine X conjugated secondary antibody. (C) Cell lysates from AKR-2B fibroblasts pulsed for 10 minutes with TGF-β (2 ng/ml) with or without U0126 (10 µM) were probed for receptor phosphorylated smad2 and smad3. Blots were stripped and reprobed for β-actin as a loading control. (D) The density of phospho-smad2 and 3 bands for each time point relative to its β-actin control were determined. The mean values for each time point (n = 3 for smad 2, n = 4 for smad 3) are displayed with the solid line representing the curve for TGF-β+U0126 and the dotted line representing TGF-β treatment.
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Related In: Results  -  Collection

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pone-0042513-g005: Nuclear Smad levels are controlled by the proteasome and activated Erk.(A) AKR-2B fibroblasts were treated for 3 h with TGF-β (2 ng/ml) with or without MG132 (10 µM), 30 minutes prior to TGF-β addition. Nuclear and cytoplasmic fractions were isolated and probed for smad2 linker region phosphorylation (245/250/255), or receptor phosphorylation (465/467). Linker phosphorylation blots were stripped and reprobed for total smad2 as a loading control, while receptor phosphorylated blots were stripped and reprobed for GAPDH to monitor the presence of cytoplasmic protein in the nuclear fraction. (B) Photomicrographs of NIH 3T3 fibroblasts treated with TGF-β (2 ng/ml) for 3 h with or without MG132 (10 µM) added 30 minutes prior to TGF-β treatment. Cells were incubated with phospho-smad2 (S245/250/255) linker antibody and specific immune complexes detected using Rhodamine X conjugated secondary antibody. (C) Cell lysates from AKR-2B fibroblasts pulsed for 10 minutes with TGF-β (2 ng/ml) with or without U0126 (10 µM) were probed for receptor phosphorylated smad2 and smad3. Blots were stripped and reprobed for β-actin as a loading control. (D) The density of phospho-smad2 and 3 bands for each time point relative to its β-actin control were determined. The mean values for each time point (n = 3 for smad 2, n = 4 for smad 3) are displayed with the solid line representing the curve for TGF-β+U0126 and the dotted line representing TGF-β treatment.
Mentions: Although these results indicate a direct cross-talk between smad2 and Erk, in light of the controversy between the Erk and smad linker region phosphorylation, we wished to examine their spatial relationship. Since smad2 is a transcription factor that translocates quickly into the nucleus following receptor mediated phosphorylation, we fractionated TGF-β treated fibroblasts into cytoplasmic and nuclear fractions and examined smad2 phosphorylation. Linker phosphorylation was seen primarily in the nuclear fraction (Figure 5A). Similarly, receptor phosphorylated smad2 was primarily in the nucleus, with small amounts in the cytoplasmic fraction, following TGF-β treatment. Total smad2 was present in both the cytoplasm and the nucleus. Since our concern was cytoplasmic protein contamination of the nuclear fractions, both cytoplasmic and nuclear fractions were probed for GAPDH to demonstrate purity of nuclear fractions relative to cytoplasmic protein contamination (Figure 5A).

Bottom Line: TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells.By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation.In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription.

View Article: PubMed Central - PubMed

Affiliation: BioMedical Sciences, Memorial University, St. John's, Newfoundland, Canada.

ABSTRACT
The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-β/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-β-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

Show MeSH
Related in: MedlinePlus