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Ribosomal protein S6 kinase (RSK)-2 as a central effector molecule in RON receptor tyrosine kinase mediated epithelial to mesenchymal transition induced by macrophage-stimulating protein.

Ma Q, Guin S, Padhye SS, Zhou YQ, Zhang RW, Wang MH - Mol. Cancer (2011)

Bottom Line: These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-β1, an EMT-inducing cytokine.Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT.Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cancer Biology at State Key Laboratory for Diagnosis & Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University College of Medicine, Hangzhou 310003, China.

ABSTRACT

Background: Epithelial to mesenchymal transition (EMT) occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP) has been implicated in cellular EMT program; however, the major signaling determinant(s) responsible for MSP-induced EMT is unknown.

Results: The study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-β1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration.

Conclusions: MSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.

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MSP-induced RSK2 dissociation with Erk1/2 and their phosphorylation in M-RON cells. A) MSP-induced dissociation of RSK2 from Erk1/2 in intact cells: M-RON cells (3 × 106 cells/dish) were incubated in DMEM containing 1% FBS overnight and then stimulated for 30 min with MSP (2 nM), TGF-β1 (5 ng/ml), or both in the presence or absence of 5 μM of U0126. Cellular proteins (250 μg/sample) from cell lysates were subjected to immunoprecipitation with rabbit IgG antibody specific to Erk1/2. Proteins in anti-Erk1/2 immunocomplex were subjected to Western blot analysis using antibodies specific to RSK1 or RSK2. Membranes were also reprobed with IgG antibody to Erk1/2 as the loading control. B) and C) MSP-induced RSK2 phosphorylation and its correlation with Erk1/2 activation: M-RON cells (3 × 106 cells/dish) in DMEM with 1% FBS were stimulated with MSP, TGF-β1, or both for various times. Cellular proteins (50 μg/sample) from cell lysates were subjected to Western blot analysis. Phosphorylation of RSK2 and Erk1/2 was detected by individual antibodies specific to phospho-RSK2 Ser380 or Erk1/2 T202/204, respectively. RSK2 and Erk1/2 detected by their corresponding regular antibodies were used as the loading control.
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Figure 1: MSP-induced RSK2 dissociation with Erk1/2 and their phosphorylation in M-RON cells. A) MSP-induced dissociation of RSK2 from Erk1/2 in intact cells: M-RON cells (3 × 106 cells/dish) were incubated in DMEM containing 1% FBS overnight and then stimulated for 30 min with MSP (2 nM), TGF-β1 (5 ng/ml), or both in the presence or absence of 5 μM of U0126. Cellular proteins (250 μg/sample) from cell lysates were subjected to immunoprecipitation with rabbit IgG antibody specific to Erk1/2. Proteins in anti-Erk1/2 immunocomplex were subjected to Western blot analysis using antibodies specific to RSK1 or RSK2. Membranes were also reprobed with IgG antibody to Erk1/2 as the loading control. B) and C) MSP-induced RSK2 phosphorylation and its correlation with Erk1/2 activation: M-RON cells (3 × 106 cells/dish) in DMEM with 1% FBS were stimulated with MSP, TGF-β1, or both for various times. Cellular proteins (50 μg/sample) from cell lysates were subjected to Western blot analysis. Phosphorylation of RSK2 and Erk1/2 was detected by individual antibodies specific to phospho-RSK2 Ser380 or Erk1/2 T202/204, respectively. RSK2 and Erk1/2 detected by their corresponding regular antibodies were used as the loading control.

Mentions: RSK isoforms such as RSK1 or RSK2 associate with Erk1/2 in quiescent cells [21]. Dissociation between RSK and Erk1/2 requires phosphorylation [21]. To determine which RSK isoform(s) is regulated by MSP, M-RON cells were stimulated in the presence or absence of U0126, an inhibitor that blocks RSK dissociation with Erk1/2 [39]. TGF-β1 was used as the control. RSK isoforms associated with Erk1/2 were determined by anti-Erk1/2 mAb immunoprecipitation followed by Western blot analysis using anti-RSK1 or RSK2 antibody. As shown in Figure 1A, RSK2 but not RSK1 was spontaneously associated with Erk1/2 in M-RON cells cultured in DMEM containing 1% FBS. In contrast, interaction between RSK1 and Erk1/2 was not observed. It should be pointed out that RSK1 was expressed in M-RON cells (data not shown); however, Erk1/2 was not detected in anti-RSK1 immunoprecipitation. After MSP stimulation, RSK2-Erk1/2 complex dissociated. TGF-1β also induced RSK2-Erk1/2 dissociation although its effect was moderate. However, in cells treated with U0126, MSP or MSP plus TGF-β1-induced dissociation of RSK2-Erk1/2 complex was blocked. Similar results were observed when immunoprecipitation was performed using anti-RSK2 mAb (data not shown). Taken together, these results suggested that MSP is capable of regulating RSK2 interaction with Erk1/2 and TGF-β1 exerts a similar effect. MSP-induced dissociation could be the first step in regulating RSK2 activity.


Ribosomal protein S6 kinase (RSK)-2 as a central effector molecule in RON receptor tyrosine kinase mediated epithelial to mesenchymal transition induced by macrophage-stimulating protein.

Ma Q, Guin S, Padhye SS, Zhou YQ, Zhang RW, Wang MH - Mol. Cancer (2011)

MSP-induced RSK2 dissociation with Erk1/2 and their phosphorylation in M-RON cells. A) MSP-induced dissociation of RSK2 from Erk1/2 in intact cells: M-RON cells (3 × 106 cells/dish) were incubated in DMEM containing 1% FBS overnight and then stimulated for 30 min with MSP (2 nM), TGF-β1 (5 ng/ml), or both in the presence or absence of 5 μM of U0126. Cellular proteins (250 μg/sample) from cell lysates were subjected to immunoprecipitation with rabbit IgG antibody specific to Erk1/2. Proteins in anti-Erk1/2 immunocomplex were subjected to Western blot analysis using antibodies specific to RSK1 or RSK2. Membranes were also reprobed with IgG antibody to Erk1/2 as the loading control. B) and C) MSP-induced RSK2 phosphorylation and its correlation with Erk1/2 activation: M-RON cells (3 × 106 cells/dish) in DMEM with 1% FBS were stimulated with MSP, TGF-β1, or both for various times. Cellular proteins (50 μg/sample) from cell lysates were subjected to Western blot analysis. Phosphorylation of RSK2 and Erk1/2 was detected by individual antibodies specific to phospho-RSK2 Ser380 or Erk1/2 T202/204, respectively. RSK2 and Erk1/2 detected by their corresponding regular antibodies were used as the loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: MSP-induced RSK2 dissociation with Erk1/2 and their phosphorylation in M-RON cells. A) MSP-induced dissociation of RSK2 from Erk1/2 in intact cells: M-RON cells (3 × 106 cells/dish) were incubated in DMEM containing 1% FBS overnight and then stimulated for 30 min with MSP (2 nM), TGF-β1 (5 ng/ml), or both in the presence or absence of 5 μM of U0126. Cellular proteins (250 μg/sample) from cell lysates were subjected to immunoprecipitation with rabbit IgG antibody specific to Erk1/2. Proteins in anti-Erk1/2 immunocomplex were subjected to Western blot analysis using antibodies specific to RSK1 or RSK2. Membranes were also reprobed with IgG antibody to Erk1/2 as the loading control. B) and C) MSP-induced RSK2 phosphorylation and its correlation with Erk1/2 activation: M-RON cells (3 × 106 cells/dish) in DMEM with 1% FBS were stimulated with MSP, TGF-β1, or both for various times. Cellular proteins (50 μg/sample) from cell lysates were subjected to Western blot analysis. Phosphorylation of RSK2 and Erk1/2 was detected by individual antibodies specific to phospho-RSK2 Ser380 or Erk1/2 T202/204, respectively. RSK2 and Erk1/2 detected by their corresponding regular antibodies were used as the loading control.
Mentions: RSK isoforms such as RSK1 or RSK2 associate with Erk1/2 in quiescent cells [21]. Dissociation between RSK and Erk1/2 requires phosphorylation [21]. To determine which RSK isoform(s) is regulated by MSP, M-RON cells were stimulated in the presence or absence of U0126, an inhibitor that blocks RSK dissociation with Erk1/2 [39]. TGF-β1 was used as the control. RSK isoforms associated with Erk1/2 were determined by anti-Erk1/2 mAb immunoprecipitation followed by Western blot analysis using anti-RSK1 or RSK2 antibody. As shown in Figure 1A, RSK2 but not RSK1 was spontaneously associated with Erk1/2 in M-RON cells cultured in DMEM containing 1% FBS. In contrast, interaction between RSK1 and Erk1/2 was not observed. It should be pointed out that RSK1 was expressed in M-RON cells (data not shown); however, Erk1/2 was not detected in anti-RSK1 immunoprecipitation. After MSP stimulation, RSK2-Erk1/2 complex dissociated. TGF-1β also induced RSK2-Erk1/2 dissociation although its effect was moderate. However, in cells treated with U0126, MSP or MSP plus TGF-β1-induced dissociation of RSK2-Erk1/2 complex was blocked. Similar results were observed when immunoprecipitation was performed using anti-RSK2 mAb (data not shown). Taken together, these results suggested that MSP is capable of regulating RSK2 interaction with Erk1/2 and TGF-β1 exerts a similar effect. MSP-induced dissociation could be the first step in regulating RSK2 activity.

Bottom Line: These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-β1, an EMT-inducing cytokine.Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT.Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cancer Biology at State Key Laboratory for Diagnosis & Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University College of Medicine, Hangzhou 310003, China.

ABSTRACT

Background: Epithelial to mesenchymal transition (EMT) occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP) has been implicated in cellular EMT program; however, the major signaling determinant(s) responsible for MSP-induced EMT is unknown.

Results: The study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-β1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration.

Conclusions: MSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.

Show MeSH
Related in: MedlinePlus