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Raf-independent, PP2A-dependent MEK activation in response to ERK silencing.

ABSTRACT: Biological roles of ERK and MEK in signal transduction have been controversial. The aim of the current study was to determine the role of ERK1/2 in signaling through the ERK-MAPK cascade by using RNAi methodology. Transient transfection of erk1 or erk2 siRNA decreased the respective protein level to 3-8% in human lung fibroblasts. Interestingly, individual ERK isoform silencing resulted in a 2-fold reciprocal increase in phosphorylation of the alternate ERK isoform, with no change in respective total protein expression. Moreover, MEK was hyperphosphorylated as a result of combined ERK1 and ERK2 silencing, but was unaffected in individual ERK1 or ERK2 silenced cells. This hyperactivation of MEK was not due to activation of Raf family members, but rather was associated with PP2A downregulation. These data highlight the existence of a feedback loop in normal cells whereby ERK silencing is associated with decreased PP2A activity and consequent MEK activation.

SUBMITTER: Bae D

PROVIDER: S-EPMC2748906 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Raf-independent, PP2A-dependent MEK activation in response to ERK silencing.

Bae Dongsoon D Ceryak Susan S

Biochemical and biophysical research communications 20090522 4

Biological roles of ERK and MEK in signal transduction have been controversial. The aim of the current study was to determine the role of ERK1/2 in signaling through the ERK-MAPK cascade by using RNAi methodology. Transient transfection of erk1 or erk2 siRNA decreased the respective protein level to 3-8% in human lung fibroblasts. Interestingly, individual ERK isoform silencing resulted in a 2-fold reciprocal increase in phosphorylation of the alternate ERK isoform, with no change in respective ...[more]

PMID: 19465001

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Project description:Mutations in polycystins are a cause of polycystic liver disease. In polycystin-2 (PC2)-defective mice, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent activation of the Rat Sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen signal-regulated kinase-extracellular signal-regulated kinase (ERK) 1/2 pathway stimulates the growth of liver cysts. To test the hypothesis that sorafenib, a Raf inhibitor used for the treatment of liver and kidney cancers, inhibits liver cyst growth in PC2-defective mice, we treated PC2 (i.e., Pkd2(flox/-) :pCxCreER(TM) [Pkd2cKO]) mice with sorafenib-tosylate for 8 weeks (20-60 mg/kg/day). Sorafenib caused an unexpected increase in liver cyst area, cell proliferation (Ki67), and expression of phosphorylated ERK (pERK) compared with Pkd2cKO mice treated with vehicle. When given to epithelial cells isolated from liver cysts of Pkd2cKO mice (Pkd2cKO-cells), sorafenib progressively stimulated pERK1/2 and cell proliferation [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and bromodeoxyuridine assay (MTS)] at doses between 0.001 and 1 ?M; however, both pERK1/2 and cell proliferation significantly decreased at the dose of 10 ?M. Raf kinase activity assay showed that whereas B-Raf is inhibited by sorafenib in both wild-type (WT) and Pkd2cKO cells, Raf-1 is inhibited in WT cells but is significantly stimulated in Pkd2cKO cells. In Pkd2cKO cells pretreated with the PKA inhibitor 14-22 amide, myristolated (1 ?M) and in mice treated with octreotide in combination with sorafenib, the paradoxical activation of Raf/ERK1/2 was abolished, and cyst growth was inhibited.In PC2-defective cells, sorafenib inhibits B-Raf but paradoxically activates Raf-1, resulting in increased ERK1/2 phosphorylation, cell proliferation, and cyst growth in vivo. These effects are consistent with the ability of Raf inhibitors to transactivate Raf-1 when a PKA-activated Ras promotes Raf-1/B-Raf heterodimerization, and are inhibited by interfering with cAMP/PKA signaling both in vitro and in vivo, as shown by the reduction of liver cysts in mice treated with combined octreotide and sorafenib.

Dataset Information

Raf-independent, PP2A-dependent MEK activation in response to ERK silencing.

Publications

Raf-independent, PP2A-dependent MEK activation in response to ERK silencing.

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