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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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TGF-β directs Erk phosphorylation of Smad2 linker region.(A) Western blots from AKR-2B fibroblast cell lysates treated with TGF-β (2 ng/ml) for the indicated time periods with or without U0126 (10 µM) for 120 min. Blots were probed for smad2 phosphorylated within the linker region at S245, 250 and 255, striped and reprobed for total smad2 to demonstrate each loading. (B) Receptor mediated phosphorylation of smad2 was also determined in the same samples using antibodies specific to C-terminal Ser 465/467. The blots were stripped and reprobed for total smad2 to demonstrate similar loading of all samples. Representative western-blots are shown with each time course performed in triplicate with consistent results. (C) Western blot and relative quantification of smad2 linker region phosphorylation in AKR-2B fibroblasts treated for 30 min. with or without TGF-β (2 ng/ml) or EGF (50 ng/ml). P-Erk blots are also shown to indicate Erk activation. Intensity of P-smad2 linker region band was determined and expressed graphically as fold increase (using total Erk band intensity as the loading control) relative to untreated control values for each experiment. The mean values of three independent experiments are shown (±SEM). Letters above each column indicate the different statistically significant (P>0.05) groupings. (D) Representative western blots showing phosphorylation of smad2 linker region of AKR-2B fibroblasts treated for 120 min. with or without TGF-β (2 ng/ml) and/or inhibitors U0126 or LY364947. Blots were stripped and reprobed for total smad 2 as a loading control.
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pone-0042513-g004: TGF-β directs Erk phosphorylation of Smad2 linker region.(A) Western blots from AKR-2B fibroblast cell lysates treated with TGF-β (2 ng/ml) for the indicated time periods with or without U0126 (10 µM) for 120 min. Blots were probed for smad2 phosphorylated within the linker region at S245, 250 and 255, striped and reprobed for total smad2 to demonstrate each loading. (B) Receptor mediated phosphorylation of smad2 was also determined in the same samples using antibodies specific to C-terminal Ser 465/467. The blots were stripped and reprobed for total smad2 to demonstrate similar loading of all samples. Representative western-blots are shown with each time course performed in triplicate with consistent results. (C) Western blot and relative quantification of smad2 linker region phosphorylation in AKR-2B fibroblasts treated for 30 min. with or without TGF-β (2 ng/ml) or EGF (50 ng/ml). P-Erk blots are also shown to indicate Erk activation. Intensity of P-smad2 linker region band was determined and expressed graphically as fold increase (using total Erk band intensity as the loading control) relative to untreated control values for each experiment. The mean values of three independent experiments are shown (±SEM). Letters above each column indicate the different statistically significant (P>0.05) groupings. (D) Representative western blots showing phosphorylation of smad2 linker region of AKR-2B fibroblasts treated for 120 min. with or without TGF-β (2 ng/ml) and/or inhibitors U0126 or LY364947. Blots were stripped and reprobed for total smad 2 as a loading control.

Mentions: Having shown a direct TGF-β/Erk signaling pathway, our next goal was to show an association existed between Erk and smad signaling, both under the direct control of TGF-β. AKR-2B fibroblasts were treated with TGF-β and probed for phosphorylation of smad2 at the Erk phosphorylation sites (Figure 4A; Ser 245, 250, and 255). We observed a temporal increase in smad2 linker region phosphorylation that was dependant on MEK activitivation of Erk, as treatment with U0216 abolished linker region phosphorylation (Figure 4A,C). Similarly, inhibition of TGF-β receptor kinase activity (LY364947) blocked smad2 linker phosphorylation (Figure 4C). Smad2 phosphorylation showed prominent C-terminal phosphorylation (Ser465/467) at all times tested, independent of MEK activity (Figure 4B; TGF-β+U0126). The linker phosphorylation seen at the 30 min. time point (Figure 4A) was not significantly above background levels (0 min.). However, rapid activation of Erk by EGF did result in significant smad2 linker phosphorylation (Figure 4D). Together this data indicates TGF-β stimulation causes phosphorylation of smad 2 via two distinct mechanisms, within the linker region, through Erk, and at the C-terminus, by TGF-β receptors.


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

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

TGF-β directs Erk phosphorylation of Smad2 linker region.(A) Western blots from AKR-2B fibroblast cell lysates treated with TGF-β (2 ng/ml) for the indicated time periods with or without U0126 (10 µM) for 120 min. Blots were probed for smad2 phosphorylated within the linker region at S245, 250 and 255, striped and reprobed for total smad2 to demonstrate each loading. (B) Receptor mediated phosphorylation of smad2 was also determined in the same samples using antibodies specific to C-terminal Ser 465/467. The blots were stripped and reprobed for total smad2 to demonstrate similar loading of all samples. Representative western-blots are shown with each time course performed in triplicate with consistent results. (C) Western blot and relative quantification of smad2 linker region phosphorylation in AKR-2B fibroblasts treated for 30 min. with or without TGF-β (2 ng/ml) or EGF (50 ng/ml). P-Erk blots are also shown to indicate Erk activation. Intensity of P-smad2 linker region band was determined and expressed graphically as fold increase (using total Erk band intensity as the loading control) relative to untreated control values for each experiment. The mean values of three independent experiments are shown (±SEM). Letters above each column indicate the different statistically significant (P>0.05) groupings. (D) Representative western blots showing phosphorylation of smad2 linker region of AKR-2B fibroblasts treated for 120 min. with or without TGF-β (2 ng/ml) and/or inhibitors U0126 or LY364947. Blots were stripped and reprobed for total smad 2 as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412844&req=5

pone-0042513-g004: TGF-β directs Erk phosphorylation of Smad2 linker region.(A) Western blots from AKR-2B fibroblast cell lysates treated with TGF-β (2 ng/ml) for the indicated time periods with or without U0126 (10 µM) for 120 min. Blots were probed for smad2 phosphorylated within the linker region at S245, 250 and 255, striped and reprobed for total smad2 to demonstrate each loading. (B) Receptor mediated phosphorylation of smad2 was also determined in the same samples using antibodies specific to C-terminal Ser 465/467. The blots were stripped and reprobed for total smad2 to demonstrate similar loading of all samples. Representative western-blots are shown with each time course performed in triplicate with consistent results. (C) Western blot and relative quantification of smad2 linker region phosphorylation in AKR-2B fibroblasts treated for 30 min. with or without TGF-β (2 ng/ml) or EGF (50 ng/ml). P-Erk blots are also shown to indicate Erk activation. Intensity of P-smad2 linker region band was determined and expressed graphically as fold increase (using total Erk band intensity as the loading control) relative to untreated control values for each experiment. The mean values of three independent experiments are shown (±SEM). Letters above each column indicate the different statistically significant (P>0.05) groupings. (D) Representative western blots showing phosphorylation of smad2 linker region of AKR-2B fibroblasts treated for 120 min. with or without TGF-β (2 ng/ml) and/or inhibitors U0126 or LY364947. Blots were stripped and reprobed for total smad 2 as a loading control.
Mentions: Having shown a direct TGF-β/Erk signaling pathway, our next goal was to show an association existed between Erk and smad signaling, both under the direct control of TGF-β. AKR-2B fibroblasts were treated with TGF-β and probed for phosphorylation of smad2 at the Erk phosphorylation sites (Figure 4A; Ser 245, 250, and 255). We observed a temporal increase in smad2 linker region phosphorylation that was dependant on MEK activitivation of Erk, as treatment with U0216 abolished linker region phosphorylation (Figure 4A,C). Similarly, inhibition of TGF-β receptor kinase activity (LY364947) blocked smad2 linker phosphorylation (Figure 4C). Smad2 phosphorylation showed prominent C-terminal phosphorylation (Ser465/467) at all times tested, independent of MEK activity (Figure 4B; TGF-β+U0126). The linker phosphorylation seen at the 30 min. time point (Figure 4A) was not significantly above background levels (0 min.). However, rapid activation of Erk by EGF did result in significant smad2 linker phosphorylation (Figure 4D). Together this data indicates TGF-β stimulation causes phosphorylation of smad 2 via two distinct mechanisms, within the linker region, through Erk, and at the C-terminus, by TGF-β receptors.

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