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Computational and Biochemical Discovery of RSK2 as a Novel Target for Epigallocatechin Gallate (EGCG).

Chen H, Yao K, Chang X, Shim JH, Kim HG, Malakhova M, Kim DJ, Bode AM, Dong Z - PLoS ONE (2015)

Bottom Line: Protein interaction with EGCG is a critical step for mediating the effects of EGCG on the regulation of various key molecules involved in signal transduction.RSK2 includes two kinase catalytic domains in the N-terminal (NTD) and the C-terminal (CTD) and RSK2 full activation requires phosphorylation of both terminals.In RSK2+/+ and RSK2-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2.

View Article: PubMed Central - PubMed

Affiliation: The Hormel Institute, University of Minnesota, 801 16th Ave NE, Austin, MN, 55912, United States of America.

ABSTRACT
The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). Protein interaction with EGCG is a critical step for mediating the effects of EGCG on the regulation of various key molecules involved in signal transduction. By using computational docking screening methods for protein identification, we identified a serine/threonine kinase, 90-kDa ribosomal S6 kinase (RSK2), as a novel molecular target of EGCG. RSK2 includes two kinase catalytic domains in the N-terminal (NTD) and the C-terminal (CTD) and RSK2 full activation requires phosphorylation of both terminals. The computer prediction was confirmed by an in vitro kinase assay in which EGCG inhibited RSK2 activity in a dose-dependent manner. Pull-down assay results showed that EGCG could bind with RSK2 at both kinase catalytic domains in vitro and ex vivo. Furthermore, results of an ATP competition assay and a computer-docking model showed that EGCG binds with RSK2 in an ATP-dependent manner. In RSK2+/+ and RSK2-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2. EGCG also suppressed epidermal growth factor-induced neoplastic cell transformation by inhibiting phosphorylation of histone H3 at Ser10. Overall, these results indicate that RSK2 is a novel molecular target of EGCG.

No MeSH data available.


Related in: MedlinePlus

EGCG suppresses EGF-induced colony formation mediated through RSK2 in JB6 Cl41 cells.(A) EGCG suppresses EGF-induced anchorage-independent growth of JB6 Cl41 cells. JB6 Cl41 cells (8 × 103/mL) were exposed to different doses of EGCG in 1 mL of 0.3% BME agar containing 10% FBS with or without 10 ng/mL EGF. Each dose was repeated in triplicate wells. The cultures were maintained in a 37°C, 5% CO2 incubator for 10 days and then colonies were counted using a microscope and the Image-Pro PLUS (vs. 4) computer software program. Data are shown as mean values ± S.D. obtained from triplicate experiments. Differences were evaluated using the Student's t test and the asterisks (*, **) indicate avsignificant inhibitory effect of EGCG on colony formation (p < 0.05; p < 0.01, respectively). (B) EGCG inhibits histone H3 phosphorylation at Ser10. JB6 Cl41cells (5 X 105) were seeded into 10-cm dishes in 5% FBS/MEM and cultured until cells reached 80–90% confluence. The cells were starved for 24 h in 0.1% FBS/MEM. Cells were treated with the indicated dose of EGCG or kaempferol (as positive control) for 1 h and then stimulated with EGF (10 ng/mL) for 15 min and subsequently harvested. Histone proteins were extracted as described in Materials and Methods. The phosphorylation of histone H3 (Ser10) was detected by Western blot. Equal protein loading and transfer were confirmed by stripping and incubating the same membrane with an antibody against total histone H3. Each assay was performed three times and similar results were obtained. Representative blots are shown.
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pone.0130049.g005: EGCG suppresses EGF-induced colony formation mediated through RSK2 in JB6 Cl41 cells.(A) EGCG suppresses EGF-induced anchorage-independent growth of JB6 Cl41 cells. JB6 Cl41 cells (8 × 103/mL) were exposed to different doses of EGCG in 1 mL of 0.3% BME agar containing 10% FBS with or without 10 ng/mL EGF. Each dose was repeated in triplicate wells. The cultures were maintained in a 37°C, 5% CO2 incubator for 10 days and then colonies were counted using a microscope and the Image-Pro PLUS (vs. 4) computer software program. Data are shown as mean values ± S.D. obtained from triplicate experiments. Differences were evaluated using the Student's t test and the asterisks (*, **) indicate avsignificant inhibitory effect of EGCG on colony formation (p < 0.05; p < 0.01, respectively). (B) EGCG inhibits histone H3 phosphorylation at Ser10. JB6 Cl41cells (5 X 105) were seeded into 10-cm dishes in 5% FBS/MEM and cultured until cells reached 80–90% confluence. The cells were starved for 24 h in 0.1% FBS/MEM. Cells were treated with the indicated dose of EGCG or kaempferol (as positive control) for 1 h and then stimulated with EGF (10 ng/mL) for 15 min and subsequently harvested. Histone proteins were extracted as described in Materials and Methods. The phosphorylation of histone H3 (Ser10) was detected by Western blot. Equal protein loading and transfer were confirmed by stripping and incubating the same membrane with an antibody against total histone H3. Each assay was performed three times and similar results were obtained. Representative blots are shown.

Mentions: We previously reported that RSK2 promoted anchorage-independent cell growth induced by EGF or TPA [18]. Phosphorylated and total RSK protein levels were increased in a time-dependent manner with EGF or TPA treatment, suggesting that the tumor promoters EGF and TPA activated the ERKs/RSK signaling pathway. As indicated above, RSK2 plays an important role in the inhibition of cell proliferation by EGCG. Therefore, we examined the effect of increasing doses of EGCG (up to 40 μM) on EGF-stimulated colony formation. JB6 Cl41 cells were stimulated for 10 days with EGF (10 ng/mL) with or without different doses of EGCG. The results showed that EGF alone significantly increased colony formation (Fig 5Aa-b). Notably, EGCG inhibited colony formation by 15% at 10 μM (Fig 5Ac) but dramatically inhibited colony formation by 85% at 20 μM (Fig 5Ad-f). Kaempferol, an RSK2 NTD inhibitor, was used as a positive control (Fig 5Ag-h) [28, 32]. Similar results were also observed for the effect of EGCG or kaempferol on colony size (S2 Fig). Thus, EGCG significantly inhibited both the size and number of EGF-induced colonies. We also previously reported that RSK2 is a kinase for EGF-induced histone H3 phosphorylation [32]. Knockout of RSK2 (RSK2-/-) totally abolished the phosphorylation of histone H3 at Ser10. Histone H3 phosphorylation at Ser10 by RSK2 is critical for neoplastic cell transformation induced by EGF or TPA [33]. We examined the effect of EGCG on EGF-induced histone H3 phosphorylation at Ser10 and total histone H3 levels in JB6 Cl41 cells. Results indicated that phosphorylated histone H3 (Ser10) was increased by EGF treatment (Fig 5B, lane 2) and was significantly inhibited by treatment with EGCG at 20 μM (Fig 5B, lane 3). Kaempferol was used as a positive control. Overall, these results revealed that EGCG suppresses EGF-induced cell transformation mediated through RSK2.


Computational and Biochemical Discovery of RSK2 as a Novel Target for Epigallocatechin Gallate (EGCG).

Chen H, Yao K, Chang X, Shim JH, Kim HG, Malakhova M, Kim DJ, Bode AM, Dong Z - PLoS ONE (2015)

EGCG suppresses EGF-induced colony formation mediated through RSK2 in JB6 Cl41 cells.(A) EGCG suppresses EGF-induced anchorage-independent growth of JB6 Cl41 cells. JB6 Cl41 cells (8 × 103/mL) were exposed to different doses of EGCG in 1 mL of 0.3% BME agar containing 10% FBS with or without 10 ng/mL EGF. Each dose was repeated in triplicate wells. The cultures were maintained in a 37°C, 5% CO2 incubator for 10 days and then colonies were counted using a microscope and the Image-Pro PLUS (vs. 4) computer software program. Data are shown as mean values ± S.D. obtained from triplicate experiments. Differences were evaluated using the Student's t test and the asterisks (*, **) indicate avsignificant inhibitory effect of EGCG on colony formation (p < 0.05; p < 0.01, respectively). (B) EGCG inhibits histone H3 phosphorylation at Ser10. JB6 Cl41cells (5 X 105) were seeded into 10-cm dishes in 5% FBS/MEM and cultured until cells reached 80–90% confluence. The cells were starved for 24 h in 0.1% FBS/MEM. Cells were treated with the indicated dose of EGCG or kaempferol (as positive control) for 1 h and then stimulated with EGF (10 ng/mL) for 15 min and subsequently harvested. Histone proteins were extracted as described in Materials and Methods. The phosphorylation of histone H3 (Ser10) was detected by Western blot. Equal protein loading and transfer were confirmed by stripping and incubating the same membrane with an antibody against total histone H3. Each assay was performed three times and similar results were obtained. Representative blots are shown.
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Related In: Results  -  Collection

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Show All Figures
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pone.0130049.g005: EGCG suppresses EGF-induced colony formation mediated through RSK2 in JB6 Cl41 cells.(A) EGCG suppresses EGF-induced anchorage-independent growth of JB6 Cl41 cells. JB6 Cl41 cells (8 × 103/mL) were exposed to different doses of EGCG in 1 mL of 0.3% BME agar containing 10% FBS with or without 10 ng/mL EGF. Each dose was repeated in triplicate wells. The cultures were maintained in a 37°C, 5% CO2 incubator for 10 days and then colonies were counted using a microscope and the Image-Pro PLUS (vs. 4) computer software program. Data are shown as mean values ± S.D. obtained from triplicate experiments. Differences were evaluated using the Student's t test and the asterisks (*, **) indicate avsignificant inhibitory effect of EGCG on colony formation (p < 0.05; p < 0.01, respectively). (B) EGCG inhibits histone H3 phosphorylation at Ser10. JB6 Cl41cells (5 X 105) were seeded into 10-cm dishes in 5% FBS/MEM and cultured until cells reached 80–90% confluence. The cells were starved for 24 h in 0.1% FBS/MEM. Cells were treated with the indicated dose of EGCG or kaempferol (as positive control) for 1 h and then stimulated with EGF (10 ng/mL) for 15 min and subsequently harvested. Histone proteins were extracted as described in Materials and Methods. The phosphorylation of histone H3 (Ser10) was detected by Western blot. Equal protein loading and transfer were confirmed by stripping and incubating the same membrane with an antibody against total histone H3. Each assay was performed three times and similar results were obtained. Representative blots are shown.
Mentions: We previously reported that RSK2 promoted anchorage-independent cell growth induced by EGF or TPA [18]. Phosphorylated and total RSK protein levels were increased in a time-dependent manner with EGF or TPA treatment, suggesting that the tumor promoters EGF and TPA activated the ERKs/RSK signaling pathway. As indicated above, RSK2 plays an important role in the inhibition of cell proliferation by EGCG. Therefore, we examined the effect of increasing doses of EGCG (up to 40 μM) on EGF-stimulated colony formation. JB6 Cl41 cells were stimulated for 10 days with EGF (10 ng/mL) with or without different doses of EGCG. The results showed that EGF alone significantly increased colony formation (Fig 5Aa-b). Notably, EGCG inhibited colony formation by 15% at 10 μM (Fig 5Ac) but dramatically inhibited colony formation by 85% at 20 μM (Fig 5Ad-f). Kaempferol, an RSK2 NTD inhibitor, was used as a positive control (Fig 5Ag-h) [28, 32]. Similar results were also observed for the effect of EGCG or kaempferol on colony size (S2 Fig). Thus, EGCG significantly inhibited both the size and number of EGF-induced colonies. We also previously reported that RSK2 is a kinase for EGF-induced histone H3 phosphorylation [32]. Knockout of RSK2 (RSK2-/-) totally abolished the phosphorylation of histone H3 at Ser10. Histone H3 phosphorylation at Ser10 by RSK2 is critical for neoplastic cell transformation induced by EGF or TPA [33]. We examined the effect of EGCG on EGF-induced histone H3 phosphorylation at Ser10 and total histone H3 levels in JB6 Cl41 cells. Results indicated that phosphorylated histone H3 (Ser10) was increased by EGF treatment (Fig 5B, lane 2) and was significantly inhibited by treatment with EGCG at 20 μM (Fig 5B, lane 3). Kaempferol was used as a positive control. Overall, these results revealed that EGCG suppresses EGF-induced cell transformation mediated through RSK2.

Bottom Line: Protein interaction with EGCG is a critical step for mediating the effects of EGCG on the regulation of various key molecules involved in signal transduction.RSK2 includes two kinase catalytic domains in the N-terminal (NTD) and the C-terminal (CTD) and RSK2 full activation requires phosphorylation of both terminals.In RSK2+/+ and RSK2-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2.

View Article: PubMed Central - PubMed

Affiliation: The Hormel Institute, University of Minnesota, 801 16th Ave NE, Austin, MN, 55912, United States of America.

ABSTRACT
The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). Protein interaction with EGCG is a critical step for mediating the effects of EGCG on the regulation of various key molecules involved in signal transduction. By using computational docking screening methods for protein identification, we identified a serine/threonine kinase, 90-kDa ribosomal S6 kinase (RSK2), as a novel molecular target of EGCG. RSK2 includes two kinase catalytic domains in the N-terminal (NTD) and the C-terminal (CTD) and RSK2 full activation requires phosphorylation of both terminals. The computer prediction was confirmed by an in vitro kinase assay in which EGCG inhibited RSK2 activity in a dose-dependent manner. Pull-down assay results showed that EGCG could bind with RSK2 at both kinase catalytic domains in vitro and ex vivo. Furthermore, results of an ATP competition assay and a computer-docking model showed that EGCG binds with RSK2 in an ATP-dependent manner. In RSK2+/+ and RSK2-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2. EGCG also suppressed epidermal growth factor-induced neoplastic cell transformation by inhibiting phosphorylation of histone H3 at Ser10. Overall, these results indicate that RSK2 is a novel molecular target of EGCG.

No MeSH data available.


Related in: MedlinePlus