Limits...
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.

Show MeSH

Related in: MedlinePlus

Erk activation requires activation of Pak2 and c-Raf.(A) AKR-2B fibroblasts were infected at an MOI of 1∶125 with adenovirus containing either dominant-negative PAK2 (Ad-EGFPdnPAK2) or Ad-EGFP as a negative control. Cells were treated with TGF-β (2 ng/ml) for 2 h prior to lysis. Cell lysates were then probed for phospho-Erk, and phospho-c-Raf (Ser338). The phospho-Erk1/2, and the stripped/reprobed total Erk loading control blot was obtained using the lower molecular mass portion of the same blot as that for P-cRaf. The same cell lysates were analyzed on a separate blot for PAK2 to confirm expression. (B) Cell lysates were analyzed for phospho-Erk and total Erk levels, in MEF cells that contained the Pak2 gene with flanking flox sites and a MEF cell line derived from this parental line in which Cre recombinase had been used to excise the Pak2 gene, following treatment with or without TGF-β for 2 h.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3412844&req=5

pone-0042513-g003: Erk activation requires activation of Pak2 and c-Raf.(A) AKR-2B fibroblasts were infected at an MOI of 1∶125 with adenovirus containing either dominant-negative PAK2 (Ad-EGFPdnPAK2) or Ad-EGFP as a negative control. Cells were treated with TGF-β (2 ng/ml) for 2 h prior to lysis. Cell lysates were then probed for phospho-Erk, and phospho-c-Raf (Ser338). The phospho-Erk1/2, and the stripped/reprobed total Erk loading control blot was obtained using the lower molecular mass portion of the same blot as that for P-cRaf. The same cell lysates were analyzed on a separate blot for PAK2 to confirm expression. (B) Cell lysates were analyzed for phospho-Erk and total Erk levels, in MEF cells that contained the Pak2 gene with flanking flox sites and a MEF cell line derived from this parental line in which Cre recombinase had been used to excise the Pak2 gene, following treatment with or without TGF-β for 2 h.

Mentions: Since inhibition of Ras farnysylation did not appear to effect TGF-β induced Erk activation, and previous data had shown Pak to be downstream of PI3K and phosphorylate Raf [34], [35], we wanted to determine where Pak2 acted within this signaling pathway. Initially we assessed S338 phosphorylation of c-Raf in fibroblasts expressing a dn-Pak2. When fibroblasts were treated with TGF-β, both c-Raf and Erk phosphorylation was dramatically reduced (Figure 3A). Additionally, a dramatic reduction in phosphorylation of Erk (Figure 3B) was seen when Pak2 was knocked out in MEFs. The low levels of Erk activation in both dnPak2 expressing fibroblasts and Pak2 KO-MEFs, suggests Erk activation can occur via Ras, albeit at a greatly reduced amount. This data is consistent with the hypothesis that TGF-β induced phosphorylation of Erk primarily follows the pathway of PI3K/Pak2/c-Raf/MEK/Erk, with a secondary contribution of Ras, similarly to that described for PDGF [21].


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

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

Erk activation requires activation of Pak2 and c-Raf.(A) AKR-2B fibroblasts were infected at an MOI of 1∶125 with adenovirus containing either dominant-negative PAK2 (Ad-EGFPdnPAK2) or Ad-EGFP as a negative control. Cells were treated with TGF-β (2 ng/ml) for 2 h prior to lysis. Cell lysates were then probed for phospho-Erk, and phospho-c-Raf (Ser338). The phospho-Erk1/2, and the stripped/reprobed total Erk loading control blot was obtained using the lower molecular mass portion of the same blot as that for P-cRaf. The same cell lysates were analyzed on a separate blot for PAK2 to confirm expression. (B) Cell lysates were analyzed for phospho-Erk and total Erk levels, in MEF cells that contained the Pak2 gene with flanking flox sites and a MEF cell line derived from this parental line in which Cre recombinase had been used to excise the Pak2 gene, following treatment with or without TGF-β for 2 h.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412844&req=5

pone-0042513-g003: Erk activation requires activation of Pak2 and c-Raf.(A) AKR-2B fibroblasts were infected at an MOI of 1∶125 with adenovirus containing either dominant-negative PAK2 (Ad-EGFPdnPAK2) or Ad-EGFP as a negative control. Cells were treated with TGF-β (2 ng/ml) for 2 h prior to lysis. Cell lysates were then probed for phospho-Erk, and phospho-c-Raf (Ser338). The phospho-Erk1/2, and the stripped/reprobed total Erk loading control blot was obtained using the lower molecular mass portion of the same blot as that for P-cRaf. The same cell lysates were analyzed on a separate blot for PAK2 to confirm expression. (B) Cell lysates were analyzed for phospho-Erk and total Erk levels, in MEF cells that contained the Pak2 gene with flanking flox sites and a MEF cell line derived from this parental line in which Cre recombinase had been used to excise the Pak2 gene, following treatment with or without TGF-β for 2 h.
Mentions: Since inhibition of Ras farnysylation did not appear to effect TGF-β induced Erk activation, and previous data had shown Pak to be downstream of PI3K and phosphorylate Raf [34], [35], we wanted to determine where Pak2 acted within this signaling pathway. Initially we assessed S338 phosphorylation of c-Raf in fibroblasts expressing a dn-Pak2. When fibroblasts were treated with TGF-β, both c-Raf and Erk phosphorylation was dramatically reduced (Figure 3A). Additionally, a dramatic reduction in phosphorylation of Erk (Figure 3B) was seen when Pak2 was knocked out in MEFs. The low levels of Erk activation in both dnPak2 expressing fibroblasts and Pak2 KO-MEFs, suggests Erk activation can occur via Ras, albeit at a greatly reduced amount. This data is consistent with the hypothesis that TGF-β induced phosphorylation of Erk primarily follows the pathway of PI3K/Pak2/c-Raf/MEK/Erk, with a secondary contribution of Ras, similarly to that described for PDGF [21].

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